Quimica AC

Physiological Reviews
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The American Physiological Society Vol. 47, No. 4, October 1967
Carbonic Anhydrase: Chemistry,

Physiology, and Inhibition
THOMAS H. MAREN
Department of Phurmacology and Therapeutics, University of Florida
College of Medicine, Gainesville, Florida; and Mount Desert
Island Biological Laboratory, Salisbury Cove, Maine
1. Introduction: Scope and Definitions. ................................... 597

II. Chemistry ........................................................... 600
1. Outline of Methods .............................................. 602
A. Manometric methods. .................................... 602
B. pH methods. ............................................ 602
C. Histochemical methods. ................................... 603
D. Recent advances: whole-cell methods. ....................... 604
2. Kinetics of the Uncatalyzed Reaction. .............................. 605
3. Chemical and Physical Properties of Carbonic Anhydrases. ............ 607
A. Primate red cell carbonic anhydrase. ........................ 607
B. Bovine red cell carbonic anhydrase (BCA) ................... 611
C. Bovine and human lens carbonic anhydrase .................. 612
D. Dog red cell and renal carbonic anhydrase ................... 613
E. Other vertebrate carbonic anhydrases ....................... 613
F. Plant carbonic anhydrase. ................................. 614
G. Other receptors possibly related to carbonic anhydrase. ........ 6 14
H. General chemical and physical properties of carbonic anhydrase:
relation to its molecular mechanism. ........................ 615
4. Kinetics of the Catalyzed Reaction. ................................ 624
A. Normal substrates. ....................................... 624
B. Other substrates. .......................................... 627
5. Inhibitors : Structure and Activity. ................................. 629
A. Inorganic anions and cations. .............................. 629
B. Organic inhibitors. ....................................... 630
C. Miscellaneous inhibitors, synthetic and natural. ............... 636
6. Kinetics of Inhibition and Binding of Sulfonamides to Carbonic Anhy-
drase.......................................................... 637
A. Inhibition of the hydration reaction. ........................ 637
B. Inhibition of the dehydration reaction. ...................... 638
C. Physical binding. ......................................... 639
7. Sulfonamide Inhibitors-General and Specific Pharmacology. .......... 640
A. Sulfanilamide ............................................ 641
B. Acetazolamide (Diamox) .................................. 642
595
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Copyright © 1967 American Physiological Society. All rights reserved.
596 T. H. MAREN Volume 47
C. Methazolamide (Neptazane) ............................... 650

D. CL11,366 ............................................... 653
E. Ethoxzolamide (Cardrase) ................................. 656
F. Miscellaneous other inhibitors. ............................. 658
G. Thiazide diuretics. ....................................... 661
H. Pharmacology of three representative inhibitors correlated with
their enzyme and in vivo effects. ........................... 662
III. Distribution and Physiology. .......................................... 664
1. Distribution and General Significance in Plants and Invertebrates. ....... 664
2. Distribution and Significance in Vertebrates Other than Mammals. ...... 666
A. Fish .................................................... 666
B. Amphibians. ............................................ 666
C. Reptiles. ................................................ 669
D. Birds. .................................................. 669
3. Distribution in Mammals. ........................................ 669
A. Normal: molar equivalent of activity. ....................... 669
B. Abnormal states: Zn concentration and deficiency. ............ 673
C. Tissues and fluids lacking carbonic anhydrase. ................ 674
4. Development in Birds and Mammals. .............................. 675
A. Red cells. ............................................... 675
B. Eye ..................................................... 676
C. Kidney. ................................................. 676
D. Stomach. ............................................... 677
E. Pancreas. ............................................... 677
F. Lung. .................................................. 677
G. Liver. .................................................. 677
H. Allantoic membranes. ..................................... 678
5. Function of Carbonic Anhydrase in Organ Systems and Effects of In-
hibition........................................................ 678
A. Respiration and red cells. ................................. 678
B. Kidney ................................................. 690
c. The eye. ................................................ 709
D. Cerebrospinal fluid and brain. ............................. 723
E. Stomach ................................................ 732
F. Pancreas. ............................................... 738
G. Liver and biliary system. .................................. 742
H. Salivary glands. .......................................... 747
I. Sweat glands; taste. ...................................... 749
J
Reproductive system. ..................................... 749
Ii.
Avian salt gland. ......................................... 753
L. Rectal gland. ............................................ 754
M. Alkalinegland ........................................... 754
N. Gel ..................................................... 755
6. Swim bladder. ........................................... 756
P. Intestine ................................................. 758
Q
Innerear ................................................ 758
R: Calcification ............................................. 759
s. Sickling in red cells. ...................................... 760
T. Adrenal and thyroid glands. ............................... 761
u. Teratological effects. ...................................... 762
v. Bacteria. ................................................ 763
IV. General Summary and Conclusions. .................................... 763
1. Chemical ... ?. .................................................. 764
2. Pharmacological ................................................. 764
3. Physiological. ................................................... 765

O:tober 1967 CARBONIC ANHYDRASE 597
I. INTRODUCTION: SCOPE AND DEFINITIONS
The title of this review at once reveals the writer’s intent. The reason for such
unfashionable scope is his belief and interest in unifying these diverse aspects of
the subject. In fact, they are not truly diverse and it would be less meaningful to
attempt to treat them separately, at least in the context of biological implication.
The main theme of this review is that in the carbonic anhydrase system, it is
often possible to apply the chemical data to physiological and pharmacological
events. The chief attraction of work in this field is the opportunity to use this
enzyme system as a model for such connections, as they may be made in the future
for other systems.
For introductory purposes, carbonic anhydrase is defined as a catalyst in the
interconversion between CO2 and H&O3 , or any of its ionic species. It will appear
that this may be but one example of the general potential of this protein for acid-
base catalysis.
This review includes illustrative material from plants and invertebrates, as
well as vertebrates; the distribution is uneven, reflecting the writer’s experience in
vertebrate pharmacology. Since this is the 19th review of one type or another on
this subject, exhaustive documentation is not a primary goal. Table 1 summarizes
previous reviews. The early history of CO* equilibria, as it applies to physiology,
culminated in the discovery of carbonic anhydrase by Meldrum and Roughton in
1932 (350). That era, which included the pioneering work of Henriques, Faurholt,
Stadie, and many others, was reviewed lucidly by Roughton in 1935 (444). Mel-
drum died at age 26, the year after his discovery; Roughton has continued with
major contributions down to the present (246). This review, in addition to the
theme discussed above, emphasizes research of the past 12 years since the review
of Berliner and Orloff (37), which has a rather similar point of view.
It was necessary to decide how to deal with the very large amount of clinical
material on carbonic anhydrase inhibition. This was made relatively easy by the
fact that almost all the clinical work is an extension of the physiological and does
not define any new principles. And it appears that the clinical and human data
are nearly always less quantitative than the animal data for at least two reasons:
in man, full dose-response curves are usually not done, and the drug is seldomgiven
intravenously. Thus the clinical data have not been included, except in the few
caseswhere they provide unique basic information; an example is the effect of
acetazolamide on composition of sweat.
It is necessary to avo’id many depths in the various and diverse subjects that
come under survey. Many of these have been and will be again, themselves, ap-
propriate for individual full review -for example, enzyme kinetics and renal
acidification. Yet in avoiding the depths, the writer is quite aware of the peril of
the shoals. The attempt is to make coherent connections between the chemical
and the physiological, while sighting the unsolved and more sophisticated problems
in each area and relegating them to the referencesand to the future.
The following terms and definitions are used:
CA Carbonic anhydrase. Any substance that catalyzes the reversible reac-
tion Hz0 + CO* S H&03. In the presence of suitable buffers,

Author and Reference Comment
Roughton (444) 1935 A vital link between the early fundamental era in CO*
physiology and carbonic anhydrase discovered by the
author and Meldrum in 1932. Contains many important
measurements and poses questions that have been par-
tially answered in the ensuing 30 years, particularly
those relating the chemistry of CO2 hydration to the
events of the respiratory cycle.
Roughton (445) 1943 Eight very active years of work reviewed; many new kinetic
measurements; data on the carbamino compounds; the
discovery of the sulfonamides as specific inhibitors; Zn
as a constituent of the enzyme. Concerned only with red
cells.
Davenport (104) 1946 Carbonic anhydrase in tissues other than blood. Particular
emphasis on kidney, stomach, and pancreas. Work on the
latter two organs inconclusive due to lack of a powerful
inhibitor for in vivo work. Attempts to apply kinetic
measurements to the gastric problem.
Van Goor (502) 1948 An exhaustive review of all aspects of the problem except
kinetic measurements and constants. Distribution par-
ticularly well covered. Not very critical: contains the
curious error of stating that kidney contains no carbonic
anhydrase.
Roughton and Clark 1951 A short review, emphasizing chemical and kinetic proper-
ww ties; also discusses the vexing activator problem.
Gibian (175) 1954 A thorough and comprehensive review. Contains refer-
ences to important articles in some lesser known Euro-
pean journals.
Waygood (569) 1955 A brief account of early purification and analytical meth-
ods. Contains a useful summary of data on plant enzyme.
Berliner and Orloff 1956 An excellent critical essay emphasizing the physiological
(37) results of specific inhibition in various organs.
Hunter and Lowry 1956 An early and penetrating attempt to correlate the physi-
(223) ological actions of acetazolamide with its quantitative
inhibition of the enzyme.
Maetz (388) 1956 The role of carbonic anhydrase in the teleost, emphasizing
eye, gill, and swim bladder.
Edsall and Wyman 1958 The most complete modern treatment of the chemistry of
W) CO* and H#Oa in biological systems.
Becker (22) 1959 An important summary of early work largely by the author
on the role of carbonic anhydrase in aqueous humor for-
mation and the effect of inhibitors. Relations to other
secretory systems are presented.
Kern (240) 1960 A critical and synthesizing review of the kinetics of the un-
catalyzed reaction. Essential for physical constants and
their meaning.
Davis (116) 1961 Chemistry of carbonic anhydrase, including mechanism of
inhibition.
Ludwig (300) 1961 An attempt to give the physiological basis for a variety of
possible therapeutic applications of acetazolamide.
Bar (17) 1963 The best treatment of relations between structure and ac-
tivity and the general chemistry of the drugs.
Davis (117) 1963 Methods of measuring the enzyme.
Polya and Wirtz 1965 Summary of chemistry of the enzyme and distribution,
(411, 412) particularly in invertebrates.
598

October 1967 CARBONIC ANHYDRASE 599
the overall reaction is OH- + CO2 G= HCOa-, or even 2 OH- +
co, s co,= + H20. The fundamental change is from a nonpolar
gas with the linear structure 0=4&O to an acid or its conjugate
bases with the coplanar configuration (140) :
O-H
I
C
A
0 O-H
First-order rate constant for CO2 + H30 -D H&03, the hydration
reaction.
First-order rate constant for the reverse or dehydration reaction.
k-1 [CO21
Equilibrium constant = - = -
kl [H2COa] ’
Second-order rate constant for CO3 + OH- + HCO3-.

Rate constant for HCO3- + CO3 + OH-.
The true ionization constant of H&03.
= [H+l [HCQ-I
lI32CQl
K a’1 The observed ionization constant of H&03.
= [H+l [HCQ-I
cc021
The ionization constant of HC03-.
[H+] [CO,-1
= [HC03-]
V UUO Velocity of the uncatalyzed hydration reaction, kl [Cog].

V’ UUO Velocity of the uncatalyzed dehydration reaction, k-1 [H&o&
IGo l WI
Kn The overall Michaelis dissociation constant Km = -
[EC021 l For-
mally, this implies

knowledge of the individual rate constants in
E+ CO2eECO2 + P but these have not been rigorously evalu-
ated (116) and not yet studied for pure enzyme. The matter is not
discussed here.
K’m The Michaelis constant, as above, but for H&O3 l
V [COel in “itro
V oat
Ip8x l
Velocity of the catalyzed hydration reaction

K,+ccQl l
V’ oat The similar expression for dehydration from H3COa .

V maxand Catalyzed rate at infinite [CO,] or
Knax @&CO& Turnover number X moles E.
VB and The potential velocity of the enzymic reaction in vivo. It is calculated
V’E as Vcat (or V’,,) using in V~VO E and substrate.
PI; E Molar concentration of carbonic anhydrase; weight of carbonic anhydrase.
Turnover number
(T.O.N.) &,,l[E] or L&,/E, depending on units of Y,,.
V mh Velocity after inhibition of carbonic anhydrase. If inhibition is com-

plete, and no other process is involved, it is equal to Vu,, .

Vobs The observed velocity of all the processes involving CO* in vivo. The
specific meaning is defined for each physiological situation.
Vobe oat The observed catalyzed velocity in viva. It is important to note that
this is not the true Vg, but represents the overall observed velocity
of enzymedependent steps in vivo. Its magnitude is determined
by completely inhibiting the enzyme.
CI601 Molar concentration of drug to inhibit 50% of enzyme activity.
e.u. A unit of carbonic anhydrase; the amount of enzyme that doubles the
hydration or dehydration rate in the particular system used. Usually
defined here as in reference 324.
KI or &!?I The dissociation constant between enzyme and inhibitor =
[concn free enzyme] [concn free inhibitor]
[enzyme-inhibitor complex] l
Concentration of free inhibitor. Also written [lfw.]

Total or original concentration of inhibitor.
Concentration of free enzyme.
Total or original concentration of enzyme.
Concentration of enzyme-inhibitor complex.
i Fractional inhibition
EII
= - = -
[IfI
Pa [a + G
II. CHEMISTRY
Before reviewing the individual types of chemical reactions in this section, it

appears useful to discussgeneral characteristics of the chemistry of CO2 , carbonic
anhydrase, and the inhibitors.
Two features, at least, set this system off from those of other enzymes: the
reaction is inorganic, and it has a most significant rate without enzyme. There are
alsopeculiar complications : the substrate is a gas, and a number of different molecu-
lar speciesrelated to CO2 and Hz0 are involved.
Although the matter is well treated in earlier reviews (37, 116, 140, 240, 445),
it is essentialto set down again the equilibria involved. In terms of the Henderson-
Hasselbalch rearrangement and using the symbols given above:
WCQ-I
pH =
pKal + log [Hz CO81
[CO,-1
pH = PKaz + log [HCIa-]
The seven speciesinvolved in thesereactions (including OH- and HZO) attain

virtually instant equilibria, with the all-important exception of CO2 (e+ la). As
indicated in section I, and more fully discussedin reference 140, CO2 and H&O3

are structurally distinct and it is not surprising that their interconversion is time
dependent. The kinetics of this interconversion are treated in section II, 2 below.
At equilibrium, the molar concentration of CO2 is 400 times that of H&O3
(see section II, 2). The unusual situation thus exists in which acid-base equilibria
are profoundly affected by “dilution” of a relatively strong acid with a nonionic
substance--in thiscase its anhydride. The true pK, designated pK,, , for carbonic
acid is about 3.6, but when “diluted” as shown by the transition from equation I
to equation la, it appears to be approximately 6.2, designated p&t 1 (37, 240). The
implications of this unique chemical system for physiological purposeshave been
widely discussed,particularly in the reviews listed in Table 1; in summary, CO2 ,
the body’s chief metabolite, provides an almost inexhaustible store of buffering
power.
The molecular mechanism by which CO2 is converted to HCOa at physiologi-
cal pH is not known with certainty. As is shown in equation 7, kinetic data for the
uncatalyzed reaction are consistent with either of two separate or coexisting mecha-
nisms:
followed by ionization
H&O8 s HCOa- (3 a )
or
co2 + OH- s HCOs- (4)
It is not possibleto say whether equation 3 or 4 is catalyzed by carbonic anhy-

drase. Speculations on the molecular enzymic mechanism (114, 128, 241, 292,471)
favor equation 4. In brief, these studies suggestthat carbonic anhydrase contains a
site for HCOa- rather than for H&03 . Enzyme-bound OH- then reacts with CO2 ,
and at physiological pH the conversion may be directly CO2 e HC03 (section
II, 4). Resolution of this problem is not essentialfor the main theme of this review
since the initial and final physiological reactants are the same in any case. At
physiological pH the rate constants of equation 3 are applicable to equation 4, since
[OH-] is negligible and the ionization of H&03 to HCO, may be readily taken
into account.
The nature of the reaction between carbonic anhydrase and aromatic sul-
fonamides is also of compelling interest in several fields of chemistry. The reaction
is characterized by amazing specificity, high reactivity, and complete reversibility.
A large body of quantitative data is available, from the varied areas of physical
binding, kinetics, spectral analysis, X-ray crystallography, and organic structural
variations. The precise molecular event is not yet understood and may be compli-
cated by the fact that kinetics of drug combination are probably different at the
hydration and the dehydration site. Nevertheless, a reasonably coherent pattern
may be presented, and there is vigorous current activity in the several areas men-
tioned (88, 283, 491, 527; Table 4).

1. Outline of Methods
The recent review on this subject by Davis (117) makes it possibleto reduce
this section to a brief critical outline of the types of methods used for determination
of carbonic anhydrase and its inhibitors. A few papers published sinceDavis’ review
are mentioned individually.
All methods depend on either the evolution or absorption of CO2 or on a pH
change. It is obvious that a wide variety of physical techniques and chemical
principles is applicable.
A. Manometric methods
These were developed by Meldrum and Roughton (350) at the time they
discovered the enzyme and were refined in later years by Roughton and colleagues.
The basic reaction is evolution of CO2 from NaHCOs--buffer mixtures or uptake
of CO2 by buffer solutions. A bibliography and description are given in reference
117. These methods have yielded a great deal of useful information. In recent
years, however, their usehas lessened,for at least three reasons:rapid and vigorous
shaking has frequently damaged the enzyme; the methods are somewhat cumber-
some; and they are not suitable for rapid flow measurements.An inherent ad-
vantage of the manometric principle, however, is its potential for extreme sensitiv-
ity. Using the Cartesian diver, it has been possibleto detect carbonic anhydrase in
single red cells, glia, and epithelia of choroid plexus ( 171).
B. pH methods
This encompassesa wide variety of methods, including simple indicator

changes, automatic electrometric recording, the use of the pH stat, and their
application to rapid and stop-flow systems(117). Essentially any physical or chemi-
cal system that can sensea pH change can be used; for example, the generation of
H+ drives the oxidation of reduced pyridine nucleotide in the interconversion of
ethanol and acetaldehyde catalyzed by liver alcohol dehydrogenase (E. Cutolo,
cited in ref. 117).
The simplestsystemis the titration of a slightly alkaline buffer by gaseousCO2 ,
introduced by Philpot and Philpot (396). This has been adapted by the author
(3 18, 324) for determination of inhibitors; the simplified micromethod (3 18) has
been particularly useful. We cannot agree with Davis (117) that these methods are
difficult, time consuming, or demand custom equipment. A test tube with an inlet
port and capillary outflow port is useful (3 18); one need only add a bucket of ice,
a CO2 cylinder, and common reagents. From 20 to 30 determinations may be done
in an hour with an accuracy of & 10%. As is indicated below, these methods are
also suitable for kinetic work. A recent paper shows excellent quantitative data
using the pH stat for both hydration and dehydration reactions (195). Excellent
kinetic data are obtained from rapid calorimetric stop-flow techniques (173, 211,
241).

C. Histochemical methods
Hausler’s modification (203) of Kurata’s original method (266) for the histo-
chemical demonstration of carbonic anhydrase resulted in a number of publica-
tions on the cellular distribution of the enzyme, some of which are included in
section III. The contro,e&i nature of this method warrants a discussionat this
point _
In this method (203), thin sectionsof tissueare floated on an HCO,- medium
containing Co++. The immediate precipitation of ,basiccobalt saltsis prevented by
adding SO?= to the medium and by adjusting its pH to about 7.4. On exposure to
the atmosphere CO2 derived from the dehydration of HCOS- is lost from the
medium and its pl-! U-.:,‘zasesslowly. The assumption underlying the histochemical
reaction is that the rate of dehydration of HCOa- in areas of the section that con-
tain the enzyme greatly exceeds the uncatalyzed rate in the medium. This gen-
erates high COS’ concentrations, localized to enzymic tissue sites, which effect
selective precipitation of CoCO3 at thesesites.The precipitated cobalt is then made
visible as the black CoS by dipping the section into (NH&S.
A number of workers have shown that these black precipitates are localized in
tissue areas such as the renal tubular epithelium (203) and the glial elements of
brain (262) where, from physiological and biochemical studies, the enzyme can
be expected to occur. Conversely, structures such as the cornea (261) and rat
adrenal gland (368), which do not contain the enzyme, as determined biochemi-
cally, do not give a positive histochemical reaction. Also, electrophoretically
purified enzyme gives a positive histochemical reaction when the paper or gel
containing the electrophoretic bands is incubated in the medium (225). Denatura-
tion of enzyme by boiling destroys the histochemical reaction (203). Proof of
specificity of the reaction for carbonic anhydrase is believed to have been es-
tablished by Hausler (203), who showed that the presenceof acetazolamide in the
medium (4 mu) inhibits the reaction completely.
On the other hand, the specificity of this reaction has been challenged by
Fand et al. (151) and Diamantstein et al. (129), who found that insulin and chon-
droitin sulfate gave positive reactions both in vivo (islets of pancreas, mamillae of
egg shells) and in vitro (pure substances). Similarly, carboxypeptidase A reacts
positively (368). This suggestsa Co++-protein interaction not specific for carbonic
anhydrase. Further evidence for such an interaction is that treatment of tissuesby
certain fixatives prior to the incubation reduces the biochemically measured
enzymic activity by as much as 90 %, but sectionsfrom thesetissues,on incubation,
show a histochemical reaction similar to that found after mild acetone fixation
according to Hgusler’s method (368). Muther (367) has further shown that the
appearance of the turbidity that develops in the incubation medium on standing is
delayed by the addition of acetazolamide. This effect of acetazolamide occurs both
in the absenceand presenceof tissue sectionsin the medium, which suggeststhat
its inhibitory action on the histochemical reaction can be mediated by complexa-
tion of the cobalt, when present in the same concentrations (l-5 mu).
From the kinetic considerations elsewhere in this review (II, 2) it is evident
that if the histochemical method is truly a manifestation of carbonic anhydrase

604 T. H. MAREN Vohme 47
activity, it must be shown that the procedure is run more rapidly than the un-
catalyzed rate. For the dehydration reaction, the pH is critical in calculating this
rate. The implication in Hausler’s (203) and Hansson’s( 198) methods is that at
the surface and at the critical areas of the section, evolution of CO2 markedly
elevates the pH, slowing the uncatalyzed rate. .This pH is not known, but if it were
7.4, the half-time for dehydration is 3 min; if pH were 8.4, it is 30 min. In the
Hansson method ( 198) sectionsfloat for only 2-10 min, so that it is reasonable to
supposethat enzyme activity is involved. Hansson ( 198) was also the first to show
inhibition of the reaction at low (0.3 PM) concentrations of acetazolamide, ruling
out complexation of cobalt present ( 1.3 mu). The drug at 20 mg/kg given 30 min
before autopsy also inhibited staining. It is anticipated that the application of this
new method will permit recognition of carbonic anhydrase in specific secretory
sites, as well as in places (i.e. skin) where in the chemical method the mass of
tissue may obscure enzyme in a relatively few critical cells.
The nature of the cobalt deposit in the incubated tissuesections is at present
unknown. Hansson (198), using a phosphate-bicarbonate medium quite different
from that of Hausler, studied the uptake of 6oCoand 32Pinto sectionsduring incu-
bation and found the tissue content of both isotopesincreased. From this he con-
cluded that the deposit was a basic cobalt phosphate salt. By analogy, the deposit
in Hausler’s method may be a cobalt bicarbonate complex.
Hansson (197) has demonstrated the enzyme in red cells by fluorescent anti-
bodies; it would be interesting if this could be applied to tissues.
D. Recent advances: whole-cell methods
A new method (524) based on an early suggestionby Keilin and Mann (237)
is important because it applies to intact red cells. The pH change of methemo-
globin is studied spectrophotometrically during hydration or dehydration of CO2
within the cell. Kinetics of inhibition were studied, taking into account the iso-
enzymes in the human erythrocytes. Measurements were also made on the neonatal
red cell (524).
Carbonic anhydrase activity in intact red cells can be studied by the method
of Jacobs and Stewart (227), which depends on the hemolysis of cells suspendedin
NH&l-NaHC03 solution. Because the enzyme concentration is the same as in
vivo, the method permits an approach to the relation between catalyzed and un-
catalyzed rates in the physiological situation. The observed rates are such that
enzyme inhibition at the 99 % range can be accurately assessed, whereas, in the
usual dilute cell-free system, quantitative estimatesof inhibition are limited to the
intermediate ranges centering on 50 % (340).
An important recent advance, presagedby Roughton 25 years ago (445), is the
rapid thermal measure of CO2 hydration in intact red cells. Two significant con-
clusionsare at hand from the preliminary note by Kernohan and Roughton (246) :
enzyme activity in red cells is the sameas that of lysates of equivalent Fe content,
and magnification of the uncatalyzed rate by intact red cells is about 25,000.

2. Kinetics of the Uncataly~ed Reaction
A remarkably incisive review by Kern (240), as well as the treatment by Edsall

and Wyman (140), makes it possible to treat this section in outline form, giving
only the constants essential for physiological work and some data from the past
few years.
The first-order hydration rate constant (eq. 3) is given by:
0-d [Cod
kl I- w21 (5)
dt /
The value is independent of pH. Kern’s best figure from the older literature is
0.03 set-l at 25 C. Ho and Sturtevant (211) and Gibbons and Edsall (173) recently
published values of 0.0358 set-1 and 0.0375 set-l (25 C), respectively, and reviewed
work of the last few years. Very few values are available for 37 C. Pinsent et al.
(399) gave 0 062 for 38 C and an Arrhenius plot giving a 30-fold increase from 0 to
38 C. Maren (320) gave 0.0434 set-l at 37 C, as well as an Arrhenius plot for
l-37 C. In this range the rate constant increased 17-fold. This 37 C value is used
for calculation of the physiological rates in the kidney and pancreas (section III, 5),
and it should be recognized that it is somewhat low compared to the other values
cited. The physiological argument would not suffer if the value were as high as
0.06 set-?
The dehydration rate constant k, 1 for equation 3 has been tabulated by Kern
from data of 10 investigators using widely different svstems. The values lie between
8 and 15 se@, corrected to 18 C. Two recent and ‘critical studies agree at about
15 set-1 at 25 C (173,211). The value of 83 set- l at 37 C has been obtained recently
(92), but it is felt that this needs confirmation in view of the discrepancy with data
just cited for a & of 2.
The second-order rate constant for the hydration reaction that predominates
at pH > 10 (eq. 4) is given by:
d [CO21
k2 1- [OH] l-CO21 (6)
dt /
Kern has reviewed this work and gives as the best value for 25 C 8500 set-1M-1.
k-2 is 2 X lo-* set- 1. A mechanism involving the species CO:! OH- as intermediate
l
in equation 4 has been proposed, to best explain certain kinetic measurements (98).
Use of the terms kz and k-2 are rarely necessary in physiological work (422)
and are not considered further here.
Equation 3 and the two rate constants taken from the careful work of Gibbons
and Edsall (173) yield the all-important equilibrium constant for 25 C.
13.7
c-s 370
0.037
This is a lower value than is generally given. However, Gibbons and Edsall (173)
point out that this does fit the relation between the equilibrium constants K,, and
K atI . The first of these, the true first ionization constant of carbonic acid, is 2.7 X

lo-’ (pa, = 3.57). The second, or observed constant, is 6.8 X lo-1 (p&p1 =
6.17). An independent estimate of K is then given by the relation
Ku*
K =-- 1 =4-00
K a’1
The data accumulated by Kern (240) suggestthat k-1 and kl increase with tem-
perature in the same way, and both have a Qio of about 2. Tentatively, then, we
say that K is the same at 37 C as at 25 C.
Recent workers (173, 211) accept the schemeof Eigen et al. (142) as a tenta-
tive way of viewing the overall equilibria at pH < 8.
(7)
IL ksl
k 1a =+ Hz0 + CO2
The advantage of this schemeis that it encompassesboth H&OS and HCO,- in

direct equilibria with CO2 , in contrast to the classicalview of equations3 and 3a.
There is no present evidence to distinguish between the pathways represented at
the right and at the left of this diagram. In terms of the conventions used in this
paper :
k12and ka are very fast compared to the other rate constants (142) and are not
considered further, except to note that their ratio is equal to’the true ionization
constant Ko, . It is of passinginterest to compare the constantsin the SOShydration
systemwith those of the far slower CO2 hydration (142), but to note that those for
klz and hzl above are comparable to their SO2 analogues.
Roughton and Booth (447) investigated the effect of buffers and ionic strength
on kr and k-1 . Phosphate had a mild catalytic effect, which has been confirmed in
one recent study (173) but not another (211). Although this effect is frequently
cited, it is small at best; in one study (173) kr was 0.0375 set-1 at zero buffer con-
centration and 0.047 sec.1 at the highest phosphate concentration used, 30 mu.
Imidazole and barbital buffers had no effect, nor did 100 mu NaCl (173). KC1 and
NaHC03 (140 mu) had no effect on kl (my own observations). Roughton and
Booth (447) also investigated other anions, some of which (sulfite, selenite, and
tellurate) had a remarkable catalytic effect. Kiese and Hastings (248) confirmed
many of these findings and added that Brs and HOBr were relatively strong cata-
lysts. The various buffers and ions tested were as active on k-1 as on kl There are
a number of interesting other points in reference 248, such as the testing of the
conventional catalysts Pt, Pd, charcoal, and WOa*. All were negative.
The most recent and perhaps most sophisticated study of inorganic anion
catalysis of CO2 + Hz0 is that of Dennard and Williams (125a). Their main
findings are : KC1 and COS’ (and its acids) had no effect; phosphate and other
common anions had little or no effect. The best catalysts are oxyanions of lower
oxidation state of nonmetals that have at least one lone pair of electrons; examples

are SOa-, SeO2,, AsOz, and CIO-. There was no correlation between activity
and pK, of the conjugate acid. Several mechanismsare proposed, the most ac-
ceptable (but not conclusive) is attack of the anion on CO2 to form a complex to
which water is added. Organic basessuch as pyridine were inactive; imidazole had
weak activity that was increased by addition of metals. This paper also contains a
useful treatment of overall observed rate constants in the manometric system, as a
function of pH.
3. Chemical and Physical Properties of Carbonic Anhydrases
With a few interesting exceptions, the work on carbonic anhydrase derives

from red cells of the various species.This is a concentrated and abundant source
of enzyme; in mammals there are about l-2 g of carbonic anhydrase per liter of
red cells (236, 394, 460). These and other early observations (249, 350, 459) es-
tablished the following cardinal facts: red cell carbonic anhydrases are Zn-con-
taining proteins of about 30,000 mol wt with 1 mole metal strongly bound per
mole protein. They are relatively stable, water soluble, and retain activity over a
pH range of 6-10, with maximum activity at pH 8, and have isoelectric points
varying from about 5.5 to 7.5 depending on the enzyme type.
After an interval of almost 20 years, this subject has been reactivated and
revolutionized by the current chemical work at Uppsala and Goteborg (291, 293-
295, 310, 380, 476), Harvard (126, 172, 174, 43 l-433), and Marseilles (274-276,
429). The main isolation work has been on human red cell carbonic anhydrase
(HCA) but additional measurementsof a fundamental nature, which probably
apply to all species,have been made (292, 294) on the bovine enzyme (BCA).
These recent developments are of enough scope and importance to provide ma-
terial for a separate review; the emphasishere is on aspectsof probable interest in
physiology and pharmacology. A principal advance, on which all three groups
agree, is the isolation, purification, and kinetic characterization of at least two
separatecarbonic anhydrases from human blood. For this reason, earlier work on
unseparated HCA is not reviewed; those studies did at the time show certain
kinetic anomalies (114, 128) that are almost certainly due to the mixture of en-
zymes.
In this section the isolation and characterization of the enzymes are first
reviewed according to speciesand tissues.Then general chemical and physical
properties of mammalian carbonic anhydrase are discussed.
A. Primate red cell carbonic anhydrase
Separation of three forms of human red cell carbonic anhydrase (HCA) from
an EtOH-CHCl3 hemolysate of red cells was achieved by Nyman (380), using
ion-exchange chromatography and zone electrophoresis. Similar studies were
under way at the sametime by Derrien and Laurent et al. (126, 275), using EtOH-
CHC13extracts chromatographed on Amberlite CG 50. Both groups found striking

similarities between two of their three fractions. Clearly each of the three fractions
were the same in the Swedish and French laboratories (275). Rickli and Edsall
(431), starting from the EtOH-CHCl, extract, found similar components using
starch gel electrophoresis. Table 2 shows the properties of the fractions as they were
found in the three laboratories, using their original nomenclatures. Later Rickli
et al. (432) and Gibbons (172) were particularly concerned about preparation
artifacts and avoided the EtOH-CHCl, step in some preparations. Their best
method involved gel filtration of red cell hemolysate on Sephadex-G-75. This
effectively separates the hemoglobin from carbonic anhydrase. The active enzyme
fractions are pooled, lypholized, and chromatographed on hydroxyapatite columns.
With this procedure, only two fractions are generally found. In this laboratory,
Byvoet and Gotti (60) have also found but two fractions when they took up hemoly-
sate on diethylaminoetkyl (DEAE) cellulose columns, followed by a second run
on DEAE-cellulose with tris buffer gradient elution.
Armstrong et al. (8) review existing procedures and describe their newly
evolved method, which is relatively simple, rapid, suited to large-scale work, and,
most important, gentle. Red cell hemolysate at pH 8.7 is run onto DEAE-Sepha-
dex-A-50. Hemoglobin is selectively adsorbed, and carbonic anhydrase is eluted
TABLE 2. Properties of human red cell carbonic anlzydrases

--
I
A B C A B C A B C
1 I -- 1 I I I
Rickli et al. (431, 432),* Laurent (276),
Nyman (380) Armstron et al. (8), and Raynaud (429) *
Gib %ons (172)
-
II III V Sub I I II x2 Xl Y
_- -~
70 Total CA, by wt. 4 86 10 75 13 4 30 16
Mol. wt. X 10-3t 34 34 27.2 27.8 25 29.1 31.6
Sedimentation constant, 3.01 2.8 2.7 3.23 3.30
s20, 9 set X 1013
Partial specific vol., ml/g 0.74 0.740
2% % 0.2c 0.2c 0.2( 0.22 0.22
Electrophoretic mobility at -1.1 -1 .ot -0.5
pH 7, 106 cm*/v set
Isoelectric point 5.7
Relative specific activity 1 1 5 1 1 15
K, , 25 C, pH 7.0, mM 3
K’wa 9 25 C, pH 7.0, mM 32
V,,,&EO] X 10m3 se@ 17
VLax/[Eb] X 10m3 set-l 25
[EJ/e.u. X log M (329) 80 2
* Ref. 8 has modified and refined some of the earlier physical data from that laboratory
(432). Both refs. 8 and 429 contain additional physical data such as intrinsic viscosity, Scher-
aga-Mandelkorn coefficients, and optical rotatory dispersion. tDone by a variety of
methods, including sedimentation equilibrium, diffusion, osmotic pressure, amino acid com-
position. The standard error of the physical methods is about 4% (8). Authors’ most reliable
data are listed; original papers should be consulted.

October 1967 CARBONIC ANHYDFUSE 609
in 90 % yield. The crude enzyme is precipitated with ammonium sulfate and the
separation into pure fractions C and B is done by elution from DEAE with tris
buffer [tris(hydroxymethyl)amino methane] 0.05 ~-0.012 M at pH 8.7. Fraction A
is obtained when buffer is increased to 0.1 M.
Table 2 summarizes work on these various human red cell fractions and
includes kinetic data discussedin section II, 4. The vertical columns are arranged
so that for each of the three studies cited, the analagous fractions are in the same
position. It seemsclear, for example, that Nyman’s V = Rickli’s II = Laurent’s
Y. These workers have agreed to call the fractions A, B, and C, in the order of
these columns (432). The extensive findings of the Marseilles (429) and Harvard
(8) groups have been brought up to date, and the literature is reviewed in the
references cited. Additional physical constants and molecular dimensions, not
shown in Table 2, are given and discussed.With respect to both physical and ac-
tivity data, there is generally good agreement among the different workers; Arm-
strong et al. (8) have given particular attention to the sedimentation constant
(&J and feel that their new and lower values are well justified and of some
significance. The value for intrinsic viscosity of HCA-B, 2.75 ml/g, (8, 429) is of
particular physiological interest, since it indicates that the protein is compact and
not far from spherical.
The status of HCA-A has not been determined with certainty. It is possible
that it is a preparation artifact of B. Laurent et al. (277) have confirmed, in greater
detail, the important fact that the amino acid composition of HCA-A and HCA-B
is identical (381). Other properties linking it with B include immunological
identity (158, 528) and similar affinities to the sulfonamides (section II, 5). How-
ever, becauseof differences in electrophoretic mobility (60, 276, 380,432), Laurent
et al. (277) believe there may be differences in tertiary structure that might be
revealed by studies of its hydrodynamic properties, already available for B and C
(8, 429).
Table 2 showsthat B and C are similar but certainly not identical. The chief
differences shown here are in electrophoretic mobility and activity. It is also shown
(section II, 5) that B and C differ in susceptibility to inhibition by sulfonamides.
The two proteins may also be distinguished immunologically (158, 354, 528). The
amino acid composition of B and C is given by Laurent et al. (274), Nyman and
Lindskog (381), and Armstrong et al. (8). There is close agreement among the
French, Swedish, and American workers. B differs from C in amino acid composi-
tion; B has 2 and C has J methionine groups, and there are differences in the con-
tent of alanine, leucine, lysine, and serine. B and C are alike in lacking a disulfide
bridge and reactive nitrogen terminal amino group (341, 381, 432) ; the latter
appears to be acetylated (342). The human and canine, but not the bovine, red
cell enzymes contain cysteine (8, 60, 274,43 1,432), one residue per mole.
We may reasonably expect the full amino acid sequenceto be published by
the Marseilles, Gothenburg, and Harvard groups shortly. Meanwhile the carboxy
terminal sequenceof HCA-B has been reported by the first two of these teams
(34la, 382). It is:
Leu-(Gly, Lys)-Arg-Thr-Val-Arg-Ala-Ser-Phe

The data strongly suggest a single polypeptide chain. The carboxy-terminal amino
acid for HCA-C and bovine B (which have similar kinetics, see II, 3C), however, is
lysine (341, 382), and the sequence of the 20 terminal amino acids of HCA-C is
quite different from that of HCA-B (382).
An interesting difference between B and C is that zinc dissociates (half-time)
from B in l-2 days but from C in 5-10 days when dialyzed against 1, lo-phenan-
throline (295). Chemical properties of the purified human red cell enzymes, in-
cluding the important matter of metal binding (295), which appear to be general
characteristics of most mammalian carbonic anhydrases, are considered at the end
of this section.
Duff and Coleman (136) found that red cells of Mzcaca mulatta contain two en-
zymes almost indistinguishable from HCA-B and HCA-C on the basis of amino
acid composition, physical measurements, and specific activity. Were it not for the
difference in methionine residues (for human B, 2 and monkey B, 1; for human C,
1 and monkey C, 2) it would be tempting to say that we are dealing with proteins
of the same structure. This relationship of isoenzymes appears characteristic for
certain primate red cells only; as is shown below, it does not necessarily extend to
other mammalian red cells or to enzyme in tissues.
The distribution of B and C in various species and individuals is significant
from genetic, evolutionary, chemical, and physiological aspects. This has been
explored chiefly in connection with the recently discovered esterase activity of
human and other red cell carbonic anhydrases (481, 482). The occurrence of B
and C among primates is considered here; chemical aspects of esterase and other
substrate interaction are reserved for section II, 4.
Tashian found that both B and C contained the ability to split the acetate and
butyrate esters of a- and fl-naphthol (481, 482). He used zone electrophoresis of
hemolysates or purified red cells followed by histochemical staining directly on the
gel, usually with @-naphthol acetate, which is the most reactive substrate in this
series. With this technique, he and his colleagues have screened variant forms of
the human enzyme, as well as those in 23 primate species. The various forms of B
(identified by electrophoretic mobility) are said to be immunologically identical.
Some species do not have B. Analysis of C is more difficult, since its concentration
is less, but thus far all species show C, and there is little or no quantitative variation.
Thus it appears to be the older and more conservative molecule. It also appears
(II, 4) that it is much more active than B and seems to be the working carbonic
anhydrase.
The proportions of B and C were found to be constant in 10 white individuals
(432). In 1400 adult specimens, including white and colored, from a screening
program in Baltimore, only one variation was observed; absence of B in a young
Negro female (435). Of much interest was the finding that total enzyme activity
was normal; all of this supports the ideas of the preceding paragraph. No relative
of this woman showed the abnormality. These two studies were based on elec-
trophoretic analysis without esterase staining.
Although esterase activity has been useful in this context (481), its relation to
carbonic anhydrase is not certain. It should be noted that the turnover number for

p-nitrophenyl acetate is 10-4 that for CO2 , using HCA-B (8). Using monkey red
cell B, the cobalt enzyme is more active as an esterase than the zinc enzyme (136) ;
this is never the case for hydration or dehydration of CO2 . Guinea pig (480) and
bovine (3 10, 480) red cells have esterase activity, but dog red cells (R. E. Tashian,
personal communication) do not, and human kidney does not (482). Furthermore,
Micheli has made the observation that immunoelectrophoresis does not reveal
any esterase activity in the precipitin lines associated with carbonic anhydrase.
He concluded that if the two activities are located on the same molecule, the active
site is different. It is of interest that carbonic anhydrase activity, in his hands, was
not abolished by the antibody reaction (354). The matter is certainly not settled;
further discussion is deferred to II, 4B.
B. Bovine red cell carbonic anhydrase @CA)
Work prior to 1960 has been reviewed by Lindskog (291), who attacked the
problem anew using DEAE-cellulose column chromatography and zone elec-
trophoresis. The initial step usually was the same as the Uppsala treatment of
human red cells (380) : hemolysis followed by EtOH-CHC13 precipitation of hemo-
globin and recovery of the upper aqueous layer. Two fractions were isolated with
essentially identical properties by the various criteria of Table 2, except for a slight
difference in electrophoretic mobility. The amino acid compositions of the sup-
posedtwo proteins were alike (381; S. Lindskog, personal communication). When
the &OH-CHCla step was omitted, only one form was found. Lieflander deter-
mined the amino acid composition and other chemical properties of bovine red
cell enzyme after column chromatography. His electrophoretic pattern suggests
that the activity belongs to one component only (288). Tappan et al. (480) found
a single specific activity in eluates from gel electrophoresis. It therefore seemsthere
is probably but one bovine red cell carbonic anhydrase, and it was designated B
becauseits electrophoretic properties resemblethose of HCA-B (29 1). Dissociation
of bovine B from zinc also resemblesthat of HCA-B (294).
Bovine carbonic anhydrase has a mol wt of 3 1,000, sedimentation constant .
(Sso,,) of 3.06, partial specific volume of 0.742 ml/g, and electrophoretic mobilitv
of - 2.8 to - 2.3 X 10-b cMe/v-set at pH 7. There is 1 atom of Zn per mole enzyme
(291). These physical constants are closely allied to those for human enzymes
(Table 2). It is shown below that bovine enzyme, like dog enzyme and many
others examined, is kinetically close to HCA-C (Table 3).
A principal difference between BCq and HCA is the lack of cysteine or cystine
in BCA; also BCA has three methionine residues(294) whereas HCA has 2 (form
B) or 1 (form C). End-group analysis showsa lysine terminal -COOH for bovine
enzyme, like HCA-C (382). The amino acid composition of bovine, monkey, and
human red cell enzymes has been tabulated in comparative form ( 136).
Bovine blood has been subjected to inhibition analysis (II, 5) and binding by
sulfonamides (II, 6); results are compatible with the presence of but one enzyme,
akin to HCA-C. Some interesting quantitative data on esteraseactivity of bovine
B are given in reference 3 10.

TABLE 3. Kinetic constants of red cell carbonic anhydrase of various species”
Temp, C HorDt Dog ox Horse Human Dogfish

..
K m , mM 0 H 23 9, 13 1 5 12
(128, 448) (114)
12 H 22 5 12
25 H 35 12
(242 )
37 H 41 50
K’m , mM 0 D 22
(128)
V malt
-= T.O.N. X 0 H 230 50
w 4a
CE1 (128)
lb set-’
12 H 330 70
25 H 450 1ooo
(242 >
H 670 200
D w
(128)
2SJe.u. X 10Q M (Car- H 2.4 2 8 7
bonate buffer) (329) (329)
E 0 , PM total 60 140
(329) (333 1
Ref. except where (247 > (338)

given within table
* Red cell hemolysate or crude extract. t Hydration or dehydration. $ Based on

erroneously low values of [&,I, from Zn content of RBC.
C. Bovine and human lens carbonic anhydrase
Sen et al. (465) separated two components of carbonic anhydrase in partially

purified form from cattle lens. Homogenates were centrifuged and applied to
DEAE-cellulose columns in tris buffer. The eluate showed two peaks of enzyme
activity but further purification by disc or zone electrophoresis was unsuccessful.
A then unexplained aspect of this work was the increase in activity during frac-
tionation procedures.
Wistrand and Rao (528) found precipitins for both HCA-B and HCA-C in
human lens. Of much interest was their observation that enzyme activity of lens
homogenates increasesmarkedly when treated with Tween 80. Their data suggest
that part of the lens enzyme that is immunologically akin to C is membrane bound.
This is probably the explanation for the increase in activity during the procedure
of Sen et al. (465). The phenomenon has not been observed with other tissues,but
should be sought carefully.

Octoh 1967 CARBONIC ANHYDRASE 613
D. Dog red cell and renal carbonic anhydrase
Dog red cell enzyme has been the basisfor a large portion of the inhibition
and kinetic work in my laboratory. Since this is the major species-used
in physiologi-
cal work, its measurement is also of importance in quantitatively assessingthe role
of carbonic anhydrase in respiration and the degree of inhibition that follows vari
ous sulfonamides.
The kinetic data on hydration appeared straightforward (320) and it was
tacitly assumedthat we were dealing with a single enzyme (Table 3). Hemolysates
have been applied to DEAE columns, and the enzyme has been separated from
hemoglobin. Further chromatography followed by gradient elution with tris buffer
yields a main single activity peak. Under identical conditions, human blood shows
two activity peaks [Byvoet and Gotti (60)] in agreement with the work cited above.
Immunologically, both the canine red cell hemolysate and the purified peak
appear related to both HCA-B and HCA-C (528). Electrophoretic mobility is the
sameas that of HCA-B. Kinetics, sulfonamide inhibition, and binding of dog red
cell enzyme are discussedin subsequent sections; data are compatible with pres-
ence of one ccfast” enzyme, like HCA-C.
Byvoet and Gotti also studied the dog renal enzyme, which like its red cell
counterpart appears to be a main single component chromatographically (60). On
the basis of electrophoretic mobility, this component resemblesHCA-B, but ac-
cording to its kinetics and inhibition by the sulfonamides, it is a “fast” enzyme,
like HCA-C (325). By immunoelectrophoresis, however, two components were
found, related to HCA-B and HCA-C (528).
E. Othr vertebrate carbonic anhydrases
Among the other mammals, little is known of the chemical or physical prop-
erties of carbonic anhydrases. Guinea pig red cells appear to have at least two
enzymes with very different levels of activity (480). Some indirect light on the
general chemical phylogeny of carbonic anhydrase is furnished by the observations
that appear throughout this review, indicating that all the known vertebrate
enzymes (except rat liver) are susceptibleto inhibition by the sulfonamides.
Leiner et al. (284) have studied some interesting properties of carbonic an-
hydrase from red cells of duck, turtle, tuna, and shark. A common finding in all
these specieswas that the usual EtOH-CHCla precipitation could not be used,
becauseit denatured the enzyme. (This casts an interesting light on some of the
mammalian data discussed above.) The method finally used successfully was
hemoglobin precipitation with MeOH, CHCla , and CaC12. A partial purification
was done on hydroxyapatite columns. Duck enzyme had 0.14 % Zn, turtle had
0.2 %, and tuna 0.09%. The shark enzyme was not analyzed for Zn. In terms of
our own units (seeTable 16) the duck red cell had about 750 e.u./ml, turtle 200,
tuna 1000, and shark 55.

614 T. H. MAREN vohme 47
F. Plant carbonicanhydrase
Waygood (509) has reviewed work through 1955. Plant enzyme was extracted
from leaf cytoplasm . of spinach, lambs-quarters, and beet. It is absent from root
tissue. The enzyme is notably unstable, but is protected by cysteine. Inhibition by
p-mercurichlorobenzoate and arsenite, as well as polarographic identification of
-CH groups, supports a role for mercapto linkage in plant carbonic anhydrase.
It will be recognized that these properties are quite different from those of verte-
brate enzyme.
Plant carbonic anhydrase was not inhibited by sulfanilamide, but there was
some conflict about the presenceof Zn. Fellner (156) has found that neither ace-
tazolamide nor CL 11,366 (see II, 7 below) at lO-6 M inhibits partially purified
plant enzyme. She found no Zn. This appears a fertile field for future study; results
to date are particularly interesting in the light of Lindskog’s and Wistrand and
Rao’s finding that acetazolamide and related drugs do not bind to the metal-free
(and in this caseinactive) bovine enzyme (292, 527).
G. 0th receptors
possiblyrelatedto carbonicanhydrase
In general, the sole receptor or binding site for aromatic unsubstituted sul-
fonamides in tissueshas been found to be carbonic anhydrase (322). [Certain other
receptors extraneous to this review may be identified for particular compounds,
for example, a transport site in the kidney for acidic compounds and a chloruretic
receptor for thiazide sulfonamides (321).] A n exception has been the finding that
canine red cells bind certain sulfonamides in concentrations as much as 3 times
greater than their carbonic anhydrase content (333). The matter was of some
interest in relation to possibleisoenzymesand also the calculation of inhibition in
vivo. Human (333; current work) and bovine (current work) red cells do not have
this property. A relation to carbonic anhydrase was suspectedbecauseof the same
structural specificity (the substituted sulfonamides did not bind) and similarity in
the binding constant. However, it seemssignificant that certain carbonic anhydrase
inhibitors do not bind to this second receptor-notably methazolamide and
ethoxzolamide. Analysis of the data (Table 1 in ref. 333) suggeststhat the thiadia-
zole sulfonamidesbind to the secondsite, but the other drugs do not. No physiologi-
cal correlation has been found for this secondsite in canine red cells. The matter is
certainly unresolved.
New receptors in cornea and stomach, possibly related to carbonic anhydrase,
but probably subserving a role in chloride transport, are suggestedby Kitahara
et al. (255) and Hogben (219). In these organs, in vitro, the sulfonamidesin very
high (10-” M) concentrations reduce potential associated with Cl- transport.
There is no carbonic anhydrase in cornea (255), and in the in vitro frog gastric
mucosa the secretory rate for H+ is lessthan that of Vuno(219). The matter will be
an interesting one for future study.

27. GeneralchemicalandphysicaljvopertiesOfcarbonicanhydrase:
relationto its molecularmechanism
Comparison of properties of human red cell enzymes (Table 2) and those of

the bovine (294) and monkey (136) protein showsrather striking similarities. For
that reason, much of the work on these enzymes is relevant generally to vertebrate
carbonic anhydrases. The prototype enzyme may be taken as HCA-C, with a high
turnover number (II, 4) and a particular pattern of susceptibility to the sulfona-
mides (II, 5 and 6). Except for HCA-B and rat liver enzyme (III, 5G), all known
vertebrate enzymes sharetheseand probably many other properties to be described.
No exchange of inorganic 6sZnwith zinc of bovine red cell carbonic anhydrase
was observed in vitro over a period of 32 days (499). Extension of these to in vivo
experiments should yield the half-life of circulating enzyme.
Lindskog and Malmstrom (294) found that Zn could be removed from bovine
enzyme by prolonged dialysis against 1, IO-phenanthroline; concomitantly, enzyme
activity declined. The supposed two enzymic components showed different rates
of lossof activity, 50 % decreasein 2 and in 10 days. As indicated above, however,
it is not clear whether in cattle these represent native forms; most of the work was
done on the rapidly dissociating form, which the Uppsala group designatesB. It is
of interest, however, that the human enzymes show the same sort of behavior; B
human fraction half dissociateson dialysis against 1, lo-phenanthroline in 1-2
days, but C takes about 10 days (295).
The lossof activity is dependent on pH; the foregoing experiments were done
at pH 5. At pH 7, both bovine (294) and HCA-B (295) enzymes retain half activity
under these conditions for about 10 days. The dissociation constants for bovine
and human Zn carbonic anhydrases are similar: at pH 5, 10-l” M; at pH 7, 10-r2 M;
and at pH 9, 10-l’ M (295). The approximate dissociation constant of Zn-phenan-
throline is 10-l’ M (294).
An important observation on the apoenzyme of HCA-B was made by Wistrand
and Rao (528), who found that the metal-free protein retained its full antigenicity.
The dissociation of enzyme activity from the antibody reaction is of practical as
well as theoretical interest (354).
Addition of Zn* completely restores activity to the apoenzyme (136, 294,.
295). The metal-free protein is relatively stable in solution. Certain other divalent
ions can also restore activity-notably cobalt and, to a much lesserdegree, nickel
manganese, and iron. The following metalloproteins are inactive: Cu, Cd, Pb,
Hg, and Be. It is interesting that someof thesehave high stability constants (295).
All the alkaline earth metals were also inactive (294). The metalloproteins formed
by addition of Mn, Ni, Cu, Cd, and Hg to apoenzyme prevent the binding of Zn
(88) .
The cobalt enzyme was originally stated to have 45 % the activity of the native
molecule, but Lindskog has pointed out that this depends on the conditions of the
assay.Both the turnover number and the K.. of the cobalt enzyme are about one-
fifth that of native Zn protein (bovine), so that at low substrate concentration the

6.16 ‘I’. H. MAREN Volume 47
two enzymes will show equal activity (293). The two metals have the same oxida-
tion state (+2) and bind to the same site. Cu and Co each adds to the metal-free
protein stoichiometrically. The Cu protein is of interest, being inactive; if Zn++ is
added to the Cu protein no activity results, but if Cu++ is removed, Zn++ will
restore activity. It is probably significant that the spectrum of the Cu protein shows
no pH dependence (295). These observations strongly suggest similar binding sites
for the various metals with different conformational effects on the protein. Physical
constants of the different proteins are also given (87, 294-295).
Further work on the properties of carbonic anhydrase utilized the Co enzyme,
because of its visible absorption spectrum. The pH dependence of this spectrum is
compatible with pK, of 7.1 for bovine enzyme (292) and probably the same range
for the human forms (295). It will be noted that this agrees with the isoionic point
for HCA-C but not for HCA-B (434; see below). The work of Kernohan (241) on
the native bovine enzyme suggests that the equilibrium between a protonated and
unprotonated enzyme site operates to provide its role in the reversible catalysis. He
showed that the basic (unprotonated) form is active in the hydration reaction,
whereas the acidic (protonated) form is active in dehydration. From this study,
the bovine enzyme pk’, based on activity appeared to be 6.9, a remarkable agree-
ment with the physical spectral measurement of reference 292.
Further work of Kernohan (242) on bovine enzyme and of Lindskog (293) on
the analogous Co protein is concerned with anionic binding and pH effects as a
means of elucidating the kinetic mechanism and nature of the active site. Both
activity and spectral data are given. Results and implications are remarkably
similar in the two studies, showing that the exchange of Co for Zn does not alter
the catalytic mechanism. The scheme described in the preceding paragraph was
reaffirmed. The data suggest that binding of one proton to a group in the active site
suffices to abolish the CO2 hydration activity. The pK, of enzyme calculated from
these activity data is 6.4; it is of physiological interest that the turnover number
does not change perceptibly between pH 7.1 and 8.8; K, decreases two- to threefold
(293). Anion binding at pH 8-9 reduces the turnover number some sixfold, but
the K.. is constant. Anionic inhibition (order of activity NCO- > I- > Cl-) is
explained as. binding to a site closely linked to the unprotonated form of the active
group; it appears to the reviewer that the same can be said of S- inhibition and
possibly that of the sulfonamides.
Coleman (88) has recently shown binding to HCA-B by azide, cyanate,
sulfide, and cyanide; these ions compete for acetazolamide at the active center.
His data suggest that the active form of the enzyme for hydration is E-Zn-OH,
with a pK, of 8.1. This value, considerably higher than that cited above, was ob-
tained by complexometric titrations with CN-, S-, and acetazolamide. Hydroxyl
ions are released by the anionic inhibitors at pH > 8.1, while protons are released
when the acid form of the inhibitors attacks the enzyme in the form E-Z&H20
at pH about 7.0. It will be recognized that these experiments not only suggest the
active form of enzyme but of inhibitor. The latter would depend on ionization of
the group R--SOzNHs in the range pH 8 to yield the active molecule. It is true
that the pZ?, of this group (for removal of 1 proton) is, for known compounds, in

the range 6.6-10.4 (529). For acetazolamide, Coleman (88) found strong binding
to enzyme only between pH 6 and 8. The pK, is 7.4, but is complicated by the
OH
proton on the CH,C//hT/ group. Further work with other drugs will be of
great interest.
Kernohan (244) has shown, by kinetic measurements, that the association
rate constant of enzyme and sulfonamide increases,and & declines, with increas-
ing pH from 6 to 8. However, care must be taken not to link the overall pK, of
the sulfonamideswith their potency (244), since ionic characteristics are in many
casesconferred by portions of the molecule distant from the active R--SOaH
group. This is evident from the structures and activities shown in Table 4 and is
discussedin section II, 5 below. It will be important to learn more about the equi-
librium between R-SOsNH2 and R-SOZNH- for these drugs.
Zn and Co enzymes bound acetazolamide in molar ratios; but the metal-free
enzyme and metallocarbonic anhydrases other than Zn and Co did not bind (292,
527) until inhibitor concentration reached 1W4 M, about 1000 times greater than
needed for native or Co enzyme (88). The Zn enzyme has a lower dissociation
constant (Table 6) for acetazolamide (527) than the Co enzyme. Acetazolamide
and CL 11,366, as well as CN-, NCO-, and HS-, caused a marked shift in the
visible Co enzyme spectrum (87, 136, 292, 295). These shifts are quite similar
among these varied substancesand suggestthat the sulfonamide may be acting at
or near the Zn. An additional argument to this point is the similarity in inhibition
kinetics between stianilamide and S- (115). Paradoxically, sulfonamides do not
form strong chelates with zinc. The spectral shift for acetazolamide is stable from
pH 5.5 to 10, but above this gradually reverts to the character of original enzyme.
The apparent pK, of enzyme-inhibitor complex by this technique was 11.2 (292),
but the binding and titration data of Coleman (88) suggesta value about 2 units
less.
Denaturation of HCA-B and HCA-C by urea and guanidine HCl occurs at
lower concentrations (l-4 M) than for albumin or ribonuclease. The data suggesta
gradual unfolding with time, which is reversible. Enzyme B is much more stable
than C (139).
Riddiford et al. (433, 434) studied the acid-base equilibria of both human red
cell carbonic anhydrases. The isoionic point of B was 5.85 and of C was 7.3, in
good agreement with the isoelectric point from electrophoresis (Table 2). Other
characteristics were quite similar. Between pH 4.2 and 4.0, the proteins take up 7
protons/mole; analysis of data suggeststhat theseare bound to imidazole groups in
normally masked histidyl residues.Acid denaturation below pH 2 releasesall of
11 histidine residuesand zinc is also releasedat low pH (43 1). Tyrosyl residuesare
alsomasked, and these react (albeit more rapidly for C, explaining its lower stabil-
ity) at pH 13 with subsequentdenaturation of enzyme. The second paper of this
series(434) contains a rich fare of knowledge on the conformation of HCA-B
and HCA-C, correlated with the known amino acid composition. It is suggested
that in someway tyrosine and histidine maintain the conformational stability of
the enzyme. It is perhaps significant that imidazole, as proton donor and acceptor,

T. H. MAREN
m
-
000 0-o 0 00 0
clxx fax R
3
c;”
c ad l
c
h
x
mmQ d- m m
3
z,
I0\/ \/
I0te”
I0\/
0
u2
X
uI2

SEI n H 0 3000 (W
SEI n H 0 7200 (PI9
III. Bicyclic sulfonamides
Diphenylmethane-4,4’-disulfonamide EI 15 D 0 0.08 (239, 518)
DiphenyM-sulfonamide El 20 H 0 0.15 (20)
Diphenyl4,4’-disulfonamide Er 20 H 0 0.08 (20)
Phenoxthin-3-sulfonamide EI 20 H 0 0.12 (20)
Naphthalene-Z-sulfonamide EI 20 H 0 3 (20)
Naphthalene-Z-sulfonamide EI n; e D 0, 15 1; 3 (263)
IV. Esters and amides of
R - CHaO EI 20 H 0 0.1
c&&o EI 20 H 0 0.01
C8HL70 Er 20 H 0 0.005
GzHzaO EI 20 H 0 0.01
Cl 6H680 EI 20 H 0 10
C6H&H20 EI 20 H 0 0.004
NH2 EI 20 H 0 0.25
NH2NH EI 20 H 0 0.7
V. Heterocyclic sulfonamides
(see also ref. 356)
B El e D 0; 10 2;2 (102) same for kid-

s S0,NI-h ney and stomach
all
B Er D 0;15 3;4 (263)

B SEr; EI 2e;3 H 0 10; 1 (356)
B SEI; EI 2;3 H 0 1; 0.1 (356)

r? s SOzNH2

-HN2I
9'0 0 H a 13s a
KO % 0 H s % z3fz3s a '8 CfVfS ml

s~~0%7g!m."lt"up.4q7
(iId> so.0 %O'O L6 H a1 fal z3 !z3s a
(dd :6ZS 'EGG) $0'0 %o'O 0 H 31 !a1 z3!z3s a
‘HNZOS- -SzONbH
OgHz3
(OZS %d) wo $0'0 LG H as la2 z3bvz3 a
(osx fad> Ml'0 %o*o 0 H a01 la1 z3cz3 a
(6az) -bo’ 0 H a z3s a
(9%) to.0 %I'0 0 H 62 z3:z3s 8
R= CHO D SE1 n H 0 0.2 (505)
= nCsHa0 D SE1 n H 0 0.15 (505)
= ClCH&O D SEI n H 0 0.25 ww
= F&CO D SE1 n H 0 0.45 ww
= NH&H&O D SEI n H 0 0.5 (505)
= C&I&O D SE1 n H 0 0.07 (505)
= c&Has02 D SE1 n; 2e H 0 0.08; 0.03 (505; 320)
[CL 11,366] D SEI; EI 5e; 2e H 37 0.05; 0.05 (320, PF)
= 4NHeCaHsS02 D SEI n; e H 0 0.08; 0.04 (505; 529)
[CL 13, 4753
= 4C1CaH&Or D SEI H 0 0.04 (505)
= CaHnOCO B EI 2”o H 0 0.03 (20)
= CtjH6CH2oCo B EI 20 H 0 0.01 (20) n*
D SEI n, e H 0 0.15,0.15 (330) related corn- 0
z
CHa-Y-V
I II pounds in ref. 3
CH8CON=C,s,C-SO~NHr 539
[ methazolamide] D SE1 n, e H 27 0.65, 0.65 (320) izi
D EI 2e H 0, 37 0.05,0.4 (PF)
3
VI. Saluretics, disulfonamides, and relatives w
*
1,3-disulfamylbenzene D SE1 n H 0 21 (PF) 1,4 isomer in
ref. 356 K
1,3-disulfamyM-5-dichlorobenzene D SEI 10e; 2e H 0; 37 0.3; 0.09 (330; PF) 37 C
[dichlorophenamide] value in ref. 320
D EI le; 2e H 0; 37 0.03; 0.03 uw
B SEZ 2 H 0 0.8 (eu
5-Cl-Z-4--disulfamyl-aniline D; D; B SE1 e; 2; 2 H 0 22; 120; 38 (336; 258; 41)

[salamide]
5-Cl-2Adisulfamyl toiuene B EI 20 H 0 0.07
[disulfamide]
D SEI 2 H 0 6 (2W
Chlorothiazide B, D SEI 2; 5e H 0 17; 18 (41; 330)
B, D EI 20; 20e H 0 3; 0.07 (108; PF)
w

Class and Compound Order of Time of H or D8 Temp, C IN M x 10’ Corresponding
Additfont E + I, min# Ref. and Comment
D SEI; EI 2e; 2e H 37 10; 4 (320; PF)

B EI 15 D 0 1 (239)
Hydrochlorothiazide B, D SEI 2, e H 0 230, 210 (41, 330)
1
D EI 5e H 0 ow
D SEI; EI Ze, 2e H 37 56, 59 (320, PF)
Flumethiazide D; D; B SEI n; 2; 2 H 0 123;110;420 (PF; 258; 41)
Hydroflumethiazide D; B SEI 2; 2 H 0 1700; 1700 (258; 41)
Trichloromethiazide B SEr 2; 2 H 0 150; 550 (484; 41)
Cyclopenthiazide B SEI; EI 2; 3 H 0 130; 16 (41; 417)
Benzhydroflumethiazide B SEI 2 H 0 3100 (41)
Chlorthalidon H, B, D SEI 2; 2; ne H 0 2; 3; 2 (417; 41; PF)
D SEI ne H 25 2 PF)
R D EI 3; 2e H 0 0.3; 0.1 (417; PF)
Benzthiazide B, D SEr 2;5e H 0 4; 6 (41; 320)
D SEI le H 37 0.6, 0.3 (326, PF)
D EI 20e; 5e H 0, 37 0.03; 0.03 (PF)
H EI 20e H 5 0.1 (282)
B EI 60 H 0 0.4 WQ)
VII. Quaternary
Ammonium sulfonamide
CH3 +- D SEI; EI 2e H 0 300; 9

I uw
CHI-N-CH2 SOzNHs
I
CH3

Note: (PF) in this and following tables refers to unpublished work from the Pharmacology Dept., University of Florida. * B = bovine;
D= dog; H = human. No distinction is made in this table between hemolysates and crude or partially purified enzyme preparations. t Er
denotes mixing of enzyme and inhibitor (in 1 ml for PF data, and usually 5-10 ml for others) before start of the reaction. For a few drugs,
notably weaker inhibitors (cf. chlorothiazide), the dilution of the incubation mixture by lo-fold increases the 160 by almost this magnitude. 3
This accounts for certain apparent discrepancies in the table between PF and other values. SEI denotes mixing of substrate, enzyme, and inhibi- g-
tor before reaction time is started by buffer. $ A number alone gives contact time in minutes, with no proof for equilibrium. e means that
equilibrium was reached, and is documented in the system studied. Where e is preceded by a number, that is the time in minutes, but not b
necessarily the minimum time for equilibrium. n means substances mixed < 15 set before start of reaction. ne means equilibrium was reached
in < 15 sec. 8 Hydration or dehydration.

may be the active site (292). Clearly, a subtle and elusive structure involving Zn,
and amino acids as H+ exchangers, remains to be deciphered.
To this end, recent sophisticated studies involving X-ray crystallography
(491), protein conformation (8, 39, 87, 136, 371, 440), and model chelates (13 1)
have been brought to bear on the carbonic anhydrase molecule. They are de-
scribed briefly, with the injunction that the original papers must be consulted for
any real understanding of these complex techniques or issues.
Tilander et al. (491) have identified the position of zinc, SH-, and the sul-
fonamide site in HCA-C by X-ray analysis. Zn and SH- are further apart than
10 A. The sulfonamide site is probably less than 5 A from the Zn. These data,
together with the fact that the bovine enzyme lacks SH-, show that this is not a
ligand.
Edsall’s laboratory has made a detailed study of the human enzymes by analy-
sis of optical rotatory dispersion (8, 37 1, 432) and circular dichroism (39). Rosen-
berg’s (440) valuable study on the same topics includes the bovine enzyme. The
results and interpretation are exceedingly complex, and’ &ly a few general com-
ments are appropriate here. Both proteins are compact, nearly spherical, and with
little (< 15 %) right-handed a-helix. Cotton effects were obtained in the region of
260-300 rnp, which are interpreted as revealing tryptophan and tyrosine chains-
the former constant for the different enzymes and the latter varying considerably.
Taken with the work of Riddiford (433, 434), this suggests the possibility of active
sites connected with aromaticity buried in the interior of the molecule.
Rosenberg (440) takes up this important question and concludes that the
ultraviolet Cotton effects are not associated with the catalytic site. His argument is
based largely on comparison of the different enzymes; the Cotton effect from bovine
B is not based on tyrosine, that from human B is. The Zn*-free apoenzymes show
undiminished Cotton effects. The reviewer would add the direct argument that
HCOa- does not perturb the optical rotatory dispersion pattern at any wavelength
studied (8, 87); and acetazolamide only shows an effect at one region in the visible
(87), but not in the ultraviolet region of Cotton effect (8).
Coleman’s (87, 88) studies of HCA-B and HCA-C are of particular impor-
tance, since he compared optical rotatory dispersion effects with visible spectra
(using the Co enzyme) and showed the effects of substrate (HCOa-) and inhibitor
(acetazolamide). Results agree with those of the Malmstrom and Edsall group. His
detailed and thoughtful discussion provides the best current synthesis of the rela-
tion between protein structure, activity, and inhibition. The most interesting new
finding, in the context of this review, is interaction of acetazolamide and HCO~Y
The inhibitor alone yields a shift in spectral absorbance to the red and a perturba-
tion in optical rotatory dispersion in the region of 550 mp. HCOS- addition alone
has the characteristic [like that of other anions (292, 295)] of abolishing the 640.rnp
absorption peak, but does not perturb the optical rotatory dispersion. Acetazola-
mide blocks the effect of HCOS- on the visible spectra, suggesting strongly that
these act at or near the same site; other evidence suggests that both inhibitor
and substrate are involved with the metal, although perhaps not directly bound.

These observations are reinforced by parallel findings in Coleman’s laboratory for

the monkey enzyme (136).
An important new observation by Riepe and Wang (435a) is that CO2
bound to enzyme is not under appreciable strain. They infer that COz(also NZO)
is bound in a “hydrophobic cavity” not coordinated to Zn; but since azide (a
ligand for Zn) displacesCog, the metal is visualized as very closeto or protruding
into the cavity. HCOa-, on the other hand, is seenasdirectly linked to Zn through
its negatively charged oxygen atom.
The precise nature of the active site is not known. Dobry-Duclaux ( 131)
proposesa structure basedon chelation of zinc with the imidazole ring of histidine
and describes experiments in which divalent metal imidazoles appear to react
with COZ. Models of the active site have evolved from leading work in reaction
and inhibition kinetics; these are not primarily concerned with the nature of the
amino acids at the site, but with the catalytic mechanism (88, 114, 128, 241, 283,
292, 435a). There is a consensusthat the enzyme exists in two forms, protonated
and unprotonated; the ‘former may be considered a receptor for HCOS- [and
R-S02NH2 (283)] and the latter for COZ. The models all imply that Zn and Co
confer a special catalytic geometry, or yield a coordination complex, for an active
center for proton transfer. As shown below (II, 4B) such centers can receive not
only CO2 e HCOS-, but a variety of other substrates susceptible to acid-base
catalysis. Since neither the precise nature of the intermediates nor the chemistry
for proton transfer is knowh at the present, the reaction is given in simplest terms:
+H+
HO-Zn-E + substrate + Hz0 q- (H20)-Zn-E + hydrated substrate (7 a 1
-HH+
With respect to CO*, the reaction is similar to the uncatalyzed sequence(& 7).
The role of enzyme is that of rapid separator or exchanger for the infinite source
of HO- and H+ available from water.
4. Kinetics of the Catalyzed Reaction
A. Normal substrates
These reactions have been studied for crude and purified fractions of dog;
bovine, horse, and human red cells. In this treatment the early measurementson
the crude human red cell enzyme (114, 128, 282) give place to data on the pure
carbonic anhydrasesB and C; but it doesseempossibleto retain certain very useful
concepts from the earlier work. As discussedabove, dog and bovine red cells proba-
bly contain one enzyme only, but nothing is known about this situation for the
horse.
Tables 2 and 3 show the data, which require some comment in view of the
fairly wide variability and the important physiological implications. It is felt that
the best figures technically are those of Gibbons (172) on the pure human frac-
tions. Her work showsmeticulous care, a large number of experiments, a rigorously

controlled rapid flow technique, and a pure enzyme source. The bovine data of
Roughton and Booth (448) and DeVoe and Kistiakowsky (128) show rather re-
markable agreement for K, between entirely different techniques. Unfortunately,
[EJ was not estimated in Roughton’s laboratory, so their turnover numbers have
not been reported, except for an approximation (449) appropriately criticized
later (128).
In evaluating the turnover number, attention must be given to the means of
determining [EJ, the molar value of carbonic anhydrase in the system. Maren
(320) computed [EJ from inhibition data, which essentially was a quantitative
titration of enzyme by inhibitors (1J powerful enough so that [&J reacted with
[Eo] without the necessity for excess[I,) to be present. The value found was con-
sistentwith physical data from known molecular weight and the weight of enzyme
in blood (330). This is the basisfor the data of Table 3 on dog and human red cell
hemolysates, aswell askidney tissue; it was used alsofor Kernohan’s recent bovine
data (242). The human fractions B and C were essentially pure chemicals, and
[EJ was derived from weight (172). The other data of Table 3 (bovine, horse)
are based on the Zn content of semipurified enzyme. This introduces an error,
since there is more Zn in red cellsthan can be accounted for by carbonic anhydrase
(330). Moreover, deterioration of the enzyme would dissociate Zn from activity.
Both factors would operate to overestimate [E,] and hence underestimate V,,,/
[E,]. This probably accounts for the low values entered for bovine (128) and horse
enzyme (247). The two studies on human semipurified enzyme, which involved
the samematerial (114,128), report V,,,l[EJ so low (about 2000 set+) that it is
also regarded as an artifact and not entered in Table 3.
It thus appears that all red cell carbonic anhydrases analyzed to date, except
human B, have about the sameturnover number-at 25 C roughly 600,OOO/secor
36 X 106/r&. This is the highest value for any known enzyme. Relative to this,
hU man B is indeed ccslow”-so much that it is barely apparent in an analysis of red
cell hemolysate. The relative rates of B and C in such an in vitro test may be cal-
culated from equation 8, using fo; Vmaxof each enzyme its turnover number X %
total carbonic anhydrase by weight (Table 2). [S] in vitro is 50 mM and KnL is
given in Table 2.
T.O.N. l y. E
Entering these numbers and solving, l?catfor enzyme B is about 4$, and for
C x of the total observed in vitro rate. This explains why the activities of human
and dog red cell hemolysatesappear to be the same (273, 333). Human enzyme B
under these conditions is virtually cCsilent,”perhaps qualitatively no different from
yet undefined receptor in canine red cells (II, 3).
The effect of electrolytes on the catalytic rate has been intensively studied
sincethe first report by Roughton and Booth (448), extending recently to work on
the pure fractions. In general, the effect is inhibitory and K, increaseswith, for
example, phosphate buffer concentration for both hydration and dehydration by

bovine enzyme (128). Increasing ionic strength with neutral salts for human en-
zyme lowers both K, and Vmsx without altering the Michaelis relation; it is sug-
gested that this is due to changes in activity of ionic participants in the reaction,
i.e. the mechanism discussed above of proton attack on bicarbonate ion or OH-
attack on CO2 (114). However, for bovine enzyme, recent work in imidazole
buffers shows that if chloride ion is held constant at 80 11~11,variation in buffer
strength affects neither Vmex nor K,,, . The data also show that very large changes
in [Cl-] reduce but do not abolish activity for both hydration and dehydration
(241). The ISO for HCOa- in the hydration reaction is 60 mu (II, 5). The reviewer
can only suggest, at the ‘present time, that the enzyme contains binding sites for
phosphate and Cl-, as well as HCO~Y For the canine red cell enzyme, our own
data show that 142 mu KC1 lowers enzyme activity 40 % in the barbital buffer sys-
tem (330) at 0 C (60 mu CO2 substrate) but has no effect at 37 C with 3 mu CO* ,
which is closest to physiological conditions. Because &J[.&] is so large even in
the face of these alterations, it is difficult to assess their physiological meaning with
respect to the possibility that carbonic anhydrase in vivo is normally inhibited.
There is no compelling evidence to show any means of regulating carbonic an-
hydrase activity in vivo, except for alterations in CO2 or acid-base equilibria itself,
i.e. alkalinization of urine in hypercapnia (III, 5B).
Kernohan (241) has also restudied the &ect of pH (448), using semipurified
bovine red cell enzyme. As suggested above (II, 3), this is probably a single species
of carbonic anhydrase, which accounts for conformity to Michaelis-Menten kinetics
(128, 241). For hydration, K. was independent of pH from 6.4 to 7.7, but V”*= in-
creased fivefold. For dehydration, K’, and Vk,, could not be assessed individually,
but the rate decreased fivefold over this pH range. As noted in the previous chap-
ter, the data are compatible with activity of a basic group for hydration and an
acidic group for dehydration, the pK on the active site being 6.9. Both in this study
(241) and several others (128, 172), the self-consistency of hydration and dehydra-
tion enzyme kinetics was tested with the Haldane relation for reversible systems,
yielding a pKatl of 6.1, as demanded by equation la.
Maren (320) had found no difference between catalyzed rates in hydration
reactions at a pH of about 6.9 and 7.6. The twofold increase found by Kernohan
over this range (241) was missed.That change, which appears important in terms
of the protein chemistry involved, was obtained with very different buffer and
chloride concentrations. In terms of the physiological implications, however, (see
below) the differences between twofold and no change in rate over this pH range
are trivial.
The effect of temperature on the catalyzed rate (320) contrasts strikingly
with that on the uncatalyzed rate (section II, 2). Table 3 showsthat there is a three-
fold increase in Vmar between 0 and 37 C; the equivalent increase in k1 is 15.fold.
Since Km also increasesat relatively low substrate concentrations, the velocity at
0 C and 37 C is about the same (320). It is of interest that enzyme from a fish living
at 15 C shows the same gradual increment in Vmsxas the dog over the O-37 C
range (338).
The application of these data to physiological events is made in section III, 5
below. It appears that &-J[&] for dog red cell (60) and renal enzyme (325), the

figures used in connection with physiological rates, agree well with data for the
“fast” pure human C enzyme (Table Z), suggesting that some generalizations
about catalyzed rates in vivo may be appropriate. It must also be noted that since
[S] in vivo (about 1 mM) is far lessthan saturation, K, will affect VR to a greater
extent than in vitro. This has some bearing on the relative contribution of human
HCA-B and HCA-C in respiration. However, the physiological exposition shows
that I& calculated for any of the enzymes in vivo is very great, compared to Vunc
or to functional demands.
Extension of kinetic data to physiological events demands identification of the
enzyme in the organ in question, at least in terms of its activity. Table 3 showsthis
in terms of J&J[EJ = turnover number for red cells. Ideally, we should know
Vmax by, for example, Lineweaver-Burk analysis and [E,] by isolation procedures.
This is far from being accomplished for the various organs, although (Table 2) it
has been achieved for the human red cell fractions. For tissue enzyme and other
red cells the term [EJ/ e.u. has proved a useful quantitative guide to activity, with
the same relative meaning as turnover number. [EJ/e.u. is the molar concentra-
tion of enzyme that produces 1 unit of activity in the standard test system. [E,] is
obtained by an analysis of inhibition kinetics, but is independent of the affinity
between enzyme and inhibitor (330). [EJ/ e.u. pi ovides a useful comparison of
the activity of different enzyme sourceswithout the necessityfor rate data directly
involving Vmw or knowledge of [EJ from physical methods.
Table 3 showsthat [E,I/ e.u. and turnover number agree (relation is recipro-
cal) fairly well. Lack of agreement for the bovine enzyme is probably due to
erroneously low values for Vmax/[E,], due to computation of [EJ from Zn molarity
in an impure preparation. It seemslikely on the basisof the [E,]/e.u. value that
bovine carbonic anhydrase is a “fast” enzyme, like dog and human C. Analyses of
[E,]/e.u. values from other tissueshave also been done-dog kidneys yield a value
within twofold of that shown for dog red cells in Table 3 (325). From this, it seems
permissible, for example, to assumethat enzyme sourcesthat have [E,]/e.u. values
in the order of 2 X 10NgM have turnover numbers in the order of 36 X 106min-1.
B. Other substrates
New horizons have appeared in this field by the finding of substrates other
other speciesof CO2 for certain of the red cell carbonic anhydrases. None of these
new substrates are physiological, and the findings are summarized briefly in the
context of the active site. The original observation seemsto have been made by
Schneider and Lieflander (458) for p-nitrophenyl acetate. Tashian’s independent
work (482) has already been mentioned; he showed that primate red cell carbonic
anhydrases have esteraseactivity; substrates are 2-4 carbon aliphatic esters of
naphthol (481). Tissue carbonic anhydrase, notably kidney, lacked esteraseac-
tivity. Other substrates are o-nitrophenyl acetate and ethyl p-nitrophenyl car-
bonate (310); in this study the bovine enzyme was also active. Packer and Stone
(408) carried out kinetic studies on bovine enzyme and p-nitrophenyl acetate; re-
sultsgenerally conformed to those found for hydration of CO2 , including noncom-
petitive inhibition by anions and acetazolamide and increase in activity in the

pH range 6-8. The K1 of acetazolamide was considerably greater than shown for
bovine enzyme in Table 5.
Armstrong et al. (8) used HCA-B in confirming and extending many of these
observations on p-nitrophenyl acetate as substrate. The K, was 5 mu and invariant
with pH; &a= rose with pH. This is reminiscent of hydration kinetics for carbon
dioxide (293) ; it is the hydration rather than dehydration that is the chemical
parallel to hydrolysis of an ester. The turnover number is extremely small, about
80 min-1, almost 10m6 that of CO2 hydration. In this study the KI of acetazolamide
at pH 7 (0.3 PM) is the same as that for hydration of CO2 catalyzed by HCA-B
(Table 6). The noncompetitive kinetics were confirmed. In an extension of this
work, Dr. J. A. Verpoorte finds (personal communication from Harvard Biological
Laboratories) that the K1 of acetazolamide for esterase activity of HCA-C is some
lo- to 40-fold (depending on pH) lower than that for HCA-B, just as noted in Table
6 for COZ hydration. This is a rather strong hint that the same enzymes are cata-
lyzing both reactions.
Packer and Meany (405, 406) made the surprising discovery that human and
bovine red cell carbonic anhydrases reversibly catalyze the hydration of acetalde-
hyde. The hydration system for bovine enzyme obeys Michaelis-Menten kinetics
and has many points of similarity to CO, hydration. Affinity to substrate is low,
Km = 0.65 M. Turnover number is about 50,000 min-I (compare Table 3). pH
effects are of particular interest and suggest that imidazole promotes the transfer
of OH- from zinc to substrate (see II, 3). As for CO2 and p-nitrophenyl acetate, the
active site is unprotonated. Acetazolamide shows noncompetitive inhibition; but
K I = 0.6 PM, about 30 times that for the CO2 system.
The versatility of carbonic anhydrase was further explored by Packer and
Meany (407) using pyridine aldehydes as substrates. These, however, are regarded
as “abnormal” in the sense that the ring nitrogen may constitute an additional
binding site, directly to Zn. The most striking difference between the kinetics of
these substrates and all others thus far examined for hydration is that acetazola-
mide behaves as a competitive inhibitor. The KI is very high, 82 PM, when 2-pyri-
dine aldehyde is the substrate (Km = 14 mM). Turnover number is much lower than
for acetaldehyde, about 4000 min- l. This is of compelling chemical interest; the
authors regard these as distorted systems, compared to that for acetaldehyde. But
we cannot agree that the kinetics of acetaldehyde are a useful model for CO2 hy-
dration in vivo, since quantitative aspects are so very different.
Perhaps the most interesting new substrate is 2-hydroxy-5-nitro-a-toluene-
Ia cB2
sulfonic acid sultone, whose structure is as follows:
OzN \
so2
A
‘0
Lo and Kaiser (298) recognized that such a sulfonate ester, resembling the sul-
fonamides, might be cleaved enzymically. The nitro group had very great ( 104)
labilizing effect. Individual kinetic parameters have not been published, but the
value, turnover number/X, is about 10 --3 that for CO2 hydration. Acetazolamide
inhibits noncompetitively; KI for bovine enzyme is 1W7 M.

This work all suggestsstrongly that carbonic anhydrase can act as a general
acid-base catalyst. Whether any new physiological substratesexist must be left to
the future; the first place to look would appear to be among aldehydes of inter-
mediary metabolism. A number of fascinating new questions thus arise, a few of
which may be mentioned: Can the esteraseand carbonic anhydrase function be
separated, as suggestedby Micheli (354) and Hyyppa et al. (225)? How can the
fact that the cobalt enzyme is more active than native protein for these substrates
(136, 3 10) be explained? What is the significance of the fact that primate red cell
B and C have the sameesteraseactivity (136), a strikingly different relation than
for CO2 or HCOS-? What is the role of the primate red cell B enzyme? What are
the kinetics of inhibition of dimethylamido-ethoxyphosphoryl cyanide (Tabun)
against esteraseor carbonic anhydrase activity (3 lo)?
5. Inhibitors: Structure and Activity
The development of the carbonic anhydrase inhibitors through 1955 has been
described by Berliner and Orloff (37) and discussedin many of the reviews .of Ta-
ble 1. Tabulation of the activities of many of the compounds has been recently
made in two reviews of diuretic drugs (41, 127). Particular attention to structure
and activity is given in reference 17. No attempt is made here to correlate all ac-
tivity data from different laboratories or to put forward any broad scheme relat-
ing structure to activity. The former goal is too exhaustive for our purpose, and the
latter is premature; but in this section representative drugs and their activity are
listed and somegeneral comments are made.
A. Inorganic anions and cations
Van Goor (502) has reviewed and documented this work through 1948. Cya-
nide and sulfide are active in the range of 10-6 M; the effect of azide has been less
well studied (350). Iodide, nitrate, and bromide show 80 % inhibition at 24 mu
(326). The 150of thiocyanate against dog blood and gastric mucosa and against
human blood and gastric mucosa was about 6 X 10m4M at 0 C and pH 6.8 (101).
Sulfide is the only substance of this group to receive attention in recent years;
Davis (115) showed it to be a noncompetitive inhibitor of Kr 3 X 10m7M. Dobry-
Duclaux has used the anion [Fe4S3(N0&)- in what appears to be a significant
study of inhibition kinetics (130) ; its 150is about 10-7 M. Chloride effects (241) and
buffer strength (114) have been considered above. HC03 is 50 % inhibitory to
hydration at 60 mu. This is not due to a pH effect since enzymic hydration rates
in the barbital systemare independent of pH in the range 7-10 (Maren and Wiley,
unpublished observations).
Most metals, including Na, K, Fe, and Co, are without effect; Cu, Ag, Au,
Zn, Hg, and Va are active in the lOa M range but have not been systematically
explored (350).
A number of anionic widizing agents are inhibitory. Permanganate and iodine
are the most active, 150= 5 X 10-s M. The inhibition could be reversed by reduc-
ing agents, if contact between enzyme and inhibitors did not exceed 10 min (249).

630 T. H. MAREN VoLume 47
B. Organic inhibitors
All unsubstituted aromatic sulfonamides are active. The simplest compound,

benzene-sulfonamide, has an 150of.4.6 X 10B7M (263). The structural features
forseen by Mann and Keilin a quarter of a century ago (3 13) are valid : any
substitution of the R-SOSNHZ hydrogens completely’ destroys activity’ (263,
315); aliphatic sulfonamides have but slight activity (263; Maren and Wiley,
unpublished observations); the activity of sulfanilamide may be increased by
substitution on the amino group (263). Thus there is a clear dissociation be-
tween requirements for antibacterial and anti-carbonic-anhydrase activity. As
predicted, none of the antibacterial sulfonamides that followed sulfanilamide had
activity against carbonic anhydrase (263) since all had R-SOSNHS converted to
R-SOgNHR’. This remarkable paper (3 13) also showed that a simple hetero-
cyclic sulfonamide, pyridine-3-sulfonamide, was active. This foreshadowed the
early work on thiophene-2-sulfonamide by Davenport (102) and the major synthetic
and screening advance by Roblin and co-workers (438, 356), who found that
heterocyclic sulfonamides were generally more active than the benezene deriva-
tives, in somecasesby more than lOOO-fold. These two papers described acetazola-
mide and the precursor of ethoxzolamide, benzothiazole=2=sulfonamide,compounds
whose intrinsic activity or affinity for the enzyme is closeto the limit of the method
used for their evaluation (seebelow and section II, 6).
Table 4 describes the quantitative inhibition of red cell carbonic anhydrase
hemolysates and crude fractions by selected drugs from a number of investigators
using different assay procedures. Table 5 gives data for acetazolamide and sul-
fanilamide using a wide variety of procedures and enzyme sources.The 160is re-
corded, and for drugs of 150= 10-8 M and more, this is equivalent to the Kr . For
the more active drugs, the Kl is 2-5 times lessthan the 150(II, 6). No attempt is
made to record all the determinations on a given drug or to include all drugs. Two
papers, for example, have not been represented: one in which the dehydration
technique has been inadequately described and the results showed the drugs to
have much lessactivity than generally reported (344), and a second that usesa
modification of the usual hydration reaction but doesnot discriminate well among
the various drugs, probably becauseexcessiveenzyme was used (535).
Analysis of the data of Tables 4 and 5 showsthat, with a few exceptions, data
from different laboratories and using somewhat diverse procedures can be com-
pared, particularly if hydration (H) and dehydration (D) reactions are kept sepa-
rate and attention is given to the order of addition. The usual order in the standard
assayusing hydration is that substrate (S) is continuously flowing into the reaction
vessel,and enzyme (E) and inhibitor (1) are then added (SEI). It is not predictable
when variations in this order will greatly alter results, except that highly active
drugs are not affected. This is discussedbelow in connection with the EI system of
assay.Other factors, such as speciesof red cell, or degree of enzyme purification, or
buffer system used, do not individually change the results more than about two-
fold. If multiple factors are altered, there may be as much as a fourfold difference.
A conspicuous example is afforded by sulfanilamide as inhibitor of hydration.

TABLE 5. IM of sulfanilamide and acetasolamide against carbonic anhydrase from varied sources and with di&ing techniques
--
Species’ Prep.* Order of Time of H or D’ Corres onding
Temp, C 160, (Mx 10’)’
Addition* E+I,min’ Ref. or e omment
Sulfanilamide B, M C, Hm EI e D 0 9, 10 (263, 102)

vH2 B, M C, Hm EI e D 15, 10 19, 18 (263, 102)
B; D C; Hm EI 20, le; le H 0 lob, 8c; 6bc (20, PF; PF)
H C, Hm EI e, le H ‘1.5, 0 8Op, 18~ (114, PF)
D Hm EI 2e H 37 91b (PW
B C SEI 2e; ne H 0 135c, 130~; 125~ (356, 41; PF)
B Hm SE1 ne H 0 83c (PF)
D Hm SE1 ne H 0; 37 54b, 28~; 57b (320, PF; 320)
Q R Kid. Hm SE1 ne H 0 53c, 35c uw
Acetazolamide B C EI 15 D 0 0.05 (239)

N- B C EI 3, 20, le H 0 0. lc, O.O3b, 0.06~ (356, 20, PF)
PI II YI D Hm EI 2e H 37 0.4b (PW
C-S02NH2
H; D Hm EI 3e; 2e H 5;o 0.08~; O.O5b, 0.03~ (282; PF)
CH,Cr--C\S/
H M;F,H Hm EI D 0 0.09; 0.08, 0.08 (359; 307)
B C SEI 2:2e H 0 0.7c, 0.5~; 0.8~ (356, 41; PF)
D; B Hm SE1 2, 2e; 2e H 0 0.2b; O.l5b, 0.11~ (258; 330)
2-Acetylamino-1,3,4-thiadiazole-5- H Hm SE1 2, 2e H 0 0.2c, 0.3c (417, PF)
sulfonamide D Hm SEI n, ne H 0, 37 0.3b, 0.6b (330, 320)
R Hm SEI 2e H 0 0.26~ (PF)
D Kid. SE1 n, 2e H 0 0.5c, 0.3c ow
R, D St. SEI 2e H 0 0.4c ow

‘B= bovine;M = mouse; D = dog; H = human; F = fish; R = rat. 2Hm= hemolysate (RBC); C = semipurified (RBC); Kid. =
kidney; St. = stomach. 3 Sequence in order given: E = enzyme; S = substrate; 1 = inhibitor. See footnote to Table 4. 4 e d notes
equilibration reached; ne denotes equilibrium reached in < 15 set; n denotes not equilibrated; u denotes unknown; numbers give time in min-
utes. 6H = hydration reaction; D = dehydration reaction. 6 b = barbital buffer; c = carbonate buffer; p = phosphate buffer: listed
for hydration reaction.

632 T. H. MAREN voluT?le47
Against canine hemolysate in the barbital buffer system, which is best for kinetic
work, the 160is 54 X 10-7 M (320). In the carbonate buffer system used for routine
screening, the I50 was 28 X 1OB7M (330)) but 83 X 1W7 M against bovine hemoly-
sate, and 134 X lo-’ M against purified bovine enzyme (unpublished data). All
thesewere done in the samelaboratory on the sameday. The last figure is identical
with data from another laboratory, which used a crude bovine red cell extract (41).
Even these relatively small differences are not necessarilymirrored for other drugs;
for example (seeTable 5), acetazolamide has almost the same activity in the two
buffer systems(330) and against canine and bovine hemolysate (329). It is felt
that Tables 4 and 5, by providing the actual data, yield a fairly unambiguous
guide to the activities of these drugs. Several additional or unresolved matters are
discussedin the following paragraphs, and kinetic aspectsof inhibition in the next
section (II, 6).
An unexplained aspect of the activity data is the different times necessaryfor
equilibration between drug and enzyme. Table 4 shows that there is no relation
between the activity of a drug and the time required for equilibration. This is not
surprising, if the Kr (approximated here by the 150)or affinity is the ratio of associ-
ation and dissociation rates. Conceivably, and in the simplest model, the time of
equilibration reflects the bimolecular rate constant for the forward reaction [&I +
[&,j -+ [EI], w hi ch can be independent of the dissociation constant Kr or KBf.
(This is probably not the sole factor, however, since the purified enzyme fractions
require lesstime for equilibration for the same drug than do hemolysates.) This
appears a fundamental point for future work. A significant start has been made by
Kernohan in his estimation of the rate constant for E, + IOin the caseof acetazola-
mide and benzenesulfonamide. For these drugs these constants stand in direct
relation to their KI ; thus their first-order rate constants for dissociation of [EI]
were about the same, in the face of about 100-fold difference in activity (244).
Several drugs of Class VI in Table 4 (the saluretic disulfonamides and rela-
tives) show, to a very marked extent, an enhancement of activity if drug is pre-
mixed with enzyme before addition to the substrate (EI system). Examples are
chlorothiazide, hydrochlorothiazide, and benzthiazide. Curiously, the enhance-
ment is far lessat 37 C in many cases;it also dependson the volume of the incuba-
tion mixture-the more dilute the lessenhancement. Both these factors obviously
favor dissociation of the enzyme inhibitor complex. Drugs of this type must have
a different balance between association and dissociation rates, not necessarily re-
flected in their Kr . Anomalous kinetic data for benzthiazide have been reported
(282) and are discussedin II, 6 below. The obvious explanation of the increase in
activity in the EI system is that CO2 protects against inhibition, but it is not certain
how this should be interpreted vis civis the mechanism of action of these drugs, par-
ticularly in view of the differences within the same family of compounds. These
facts cannot be taken as an argument that the saluretic effect of hydrochlorothia-
zide, for example, is due to carbonic anhydrase inhibition; even under the most
favorable circumstances of assaythis drug is almost 100 times lessactive than the
“best” inhibitors, which are not chloruretic.
The main purposesof Table 4 are a) to show the very wide range in activity
among these drugs and b) to attempt somegeneralizations about structure and ac-

tivity. c) Additionally, the supplementary Table 6 shows inhibition data for six
drugs on the pure human fractions.
a) The most active drug is probably CL 13,580(160= 1 X 10BgM), although
differences between its activity and those of CL 11,366 and ethoxzolamide are
small. The least active are the “saluretic” hydroflumethiazide (& = 1.7 X lo-4
M) and the simple aliphatic sulfonamides(I60 /V 3 X lOA M). It seemsreasonableto
supposethat the only difference between theseextremes is one of affinity, although
mechanismhas not been explored for very weak inhibitors or for any of the aliphatic
drugs. Before this is done, it would be essential to make certain that activity,
particularly in the case of the aliphatic compounds, is not due to contamination
with a simple aromatic compound present in as little as 0.1% concentration. Since
this has not been done, it cannot even be stated with certainty that the aliphatic
compounds do have intrinsic activity.
The highest activity recorded ( 10egM) may in some casesreflect the limit of
the systemused, rather than the affinity of drug to enzyme. As is shown in the next
section, the concentration of enzyme in the test system is of the order of 10-g M,
the exact magnitude depending on the buffer system used and the amount of
enzyme added. Clearly, however, I 60cannot be lessthan half the enzyme concen-
tration. Two enzyme units in the barbital systemare 2 X 1W9 M, so it is probably
significant that several drugs yield 150’sin the range l-2 X lO+ M. In the case of
ethoxzolamide, the KI has been established by appropriate variations in enzyme
concentrations (330), but CL 13,580 yields values essentially of zero, suggesting
that the true affinity is of K1 < lo+ M. Further work on these exceedingly potent
inhibitors will be of interest and has been started (II, 6).
The relationship between the 150of these drugs and the concentrations needed
to approach full inhibition (190and 199)are both of theoretical interest and neces-
sary for the physiological treatment to follow. In the dilute assaysystem of Table
4, the 190may be estimated with some reliability. For 10 drugs, against enzyme
from dog and rat red cells, this value ranged from 3 to 18 times the 150(326, 454).
From the Michaelis equation, when I0 >> E, the theoretical relation of 19&o is
9. For the stronger inhibitors, where I, E E, , this ratio should approach 2; 3 of
the drugs (CL 13,580; CL 11,366; and ethoxzolamide) are in this category, and
the experimental ratios of 19&o ranged from 2 to 6. The ratio for acetazolamide,
whoseKr of about 10-a M puts it between the strong and the weak groups, was 5-8.
Additional experiments with acetazolamide, using both SE1 and EI order of addi-
tion (Table 4), show that the 195was 21 times and the 198was 70 times the 160.
Departures from this near-ideal relationship are seenin the caseof rat liver enzyme
and indicate that a mixture of enzymes is present with widely varying affinities for
drug (326). More precise evaluation of high degreesof inhibition (Igg+) is afforded
by the Jacobs-Stewart model (II, I and III, 54).
b) The fundamental relation between structure and activity in this serieshas
not been solved. In one sensethis is surprising, since, broadly, these drugs are all
related; in another sensethis is reasonable, since we have no knowledge of the
chemistry of the receptor site on the enzyme. Certain relations are briefly sug-
gested.
Miller et al. (356) saw that simple heterocyclic compounds were generally

more active than benzene derivatives. However, the later extensive screening of
Beasley et al. (20) revealed highly active diphenylsulfonamides, and, surprisingly,
esters of p-sulfamylbenzoic acid. Several of the latter compounds are among the
most active known. It will be noted that these were all tested in the EI system.
Miller et al. (356) also noted that within a group of closely related drugs, the
more acidic were the more active. This may be why acetylation of the -NH2
group (for example, N4 acetylsulfanilamide and acetazolamide) increasesactivity.
This is also consistent with the higher activity of benzenesulfonamidothiadiazoles
over benzamido- or acylamidothiadiazoles. However, pK, itself can clearly be
ruled out asa major determinant of activity, since,for example, CL 11,366 (pK, =
3.2) and ethoxzolamide (pK, = 8.1) have about the same activity at pH 7.4. It
must be noted that the pK, of CL 11,366 and many other drugs is essentially that
of a proton dissociatedfrom a group far from the R-SOZNHS moiety. The pK, of
ethoxzolamide, however, is that of one of the sulfonamide protons. Comparison
between these unlike situations could scarcely be fruitful. But even the comparison
based on pK, of the sulfonamide proton with activity (for example, CL 13,580,
methazolamide, ethoxzolamide, and sulfanilamide, in ascending order of pK,
from 6.6 to 10.4; data given below) does not yield a regular relationship. On the
basis of Coleman’s (88) work, discussedin section II, 3 above, it seemslikely that
the speciesR- SOzNH- reacts with enzyme at pH 6-8. It appears that a pK, of
this group within this general range is but one of several determinants of activity.
Becauseof the potency of heterocyclic compounds, and the enhancement of
activity by negatively charged groups, a hypothesis worth exploring would seem
to be one linking activity with electron density of the --SOJVHs group.
Among the thiazide drugs, it is evident that saturation of the 34 position re-
duces activity against carbonic anhydrase (chlorothiazide to hydrochlorothiazide)
and that change from 6 -Cl to 6 -CFa does the same. It is interesting that both
these maneuvers increase saluretic activity, a further evidence dissociating car-
bonic anhydrase inhibition from saluresisin this series(336).
c) Table 6 shows the affinity of six representative sulfonamides for the pure
human carbonic anhydrases. For the first three drugs, the & is a reasonable ap-
proximation of affinity (or Kr) since the concentration of either enzyme in solution
is much lessthan that of inhibitor. The surprising result is that for sulfanilamide
and acetazolamide about 12 times as much drug is needed for 50 % inhibition of B
asfor C, and for methazolamide the difference is about sevenfold. The criterion of
I60 is invoked becauseit doesnot involve any calculations of enzyme concentration,
and for inhibitors of this weak or intermediate range it can be effectively used. It
was mentioned in section II, 4B that acetazolamide was also a more powerful in-
hibitor of the esteraseactivity of HCA-C than of HCA-B.
For the other three (and more powerful) drugs, the ISOis about tenfold greater
for B than for C, but this now reflects almost entirely the greater molar concentra-
tion of B, in a situation where [loI E [EJ. Calculation of Kr showsno more than a
twofold difference in affinity among any of these drugs for B and for C.
Thus the six drugs are divided into two classes-those that discriminate be-
tween enzyme B and C (acetazolamide, methazolamide, and sulfanilamide) and

October 1967 CARBONIC ANI-IYDRASE 635
TABLE 6. Afinity of pure human red cell carbonic anhydrases to sulfonamides

I
Dw I B C lorresponding Ref.
-
Sulfanilamide
KI* 257 24
*
150 257 24
KD* 60 10
t112t 2 4
Acetazolamide
KI 2.7 0.2 (329 )
150 2.9 0.3 (329 )
LO (Co++) 5.1 (329 )
KD 6, 0.8 0.8 (527, 88)
KD (co++> 12, 1.5 (527, 88)
h/e 80 ^1500 (527)
Methazolamide
Kr 1.0 0.16
150 1.3 0.2
Ethoxzolamide
KI 0.02 0.02
150 0.40 0.04
CL 11,366
KI 0.08 0.04
I60 0.30 0.04
KD 0.004 0.01
h/2 >200 >200
CL 13,580
KI 0.01 0.004
150 0.47 0.04
h/2 400 400
* X 10’ M. KI from SEI system (of Table 4), equilibrated. 160 = KI + $@& . KD from
equilibrium dialysis. t Hr, escape time from EI in dialysis bag.
the three ‘UtrapowerfU’ inhibitors that do not. Independent meanswere taken

to confirm this finding. These are included in Table 6. Equilibrium dialysis showed
eightfold lessaffinity of acetazolamide to B. The half-time of escapeof acetazola-
mide bound to the enzymes in a dialysis bag at 0 C was 80 hr for B and 500 hr
for C. The tr/z of drug from human red cells in vivo also yielded two figures, 12 and
72 hr, which probably reflect decay from B and C, respectively; this is discussed
more fully in connection with the pharmacology of acetazolamide (section II, 7).
We have shown elsewherethat the ratio of decay rates should be as the square root
of the ratio of the dissociation constants (322). Thus the theoretical value would be
2/2.7/0.2 = 3.7. Corresponding studies for CL 11,366 showed no difference in
equilibrium dialysis or in vitro tl/2 betweenB and C. The t1j2in vitro for CL 13,580
was only approximated, since there was little decay after 2 weeks with either frac-
tion. This is consistent with the Kr of the order of lO+ M.
It will be noticed that the 1&s of these six drugs for fraction C are about the
sameas for crude cell hemolysate. This is in part due to the fact that in vitro the

636 T. H. MAREN vohne 47
“s1ow fraction B” contributes little to the rate (II, 4). Since the enzyme is virtually
saturated at the in vitro substrate concentration of 50-60 mu, the principal deter-
minant of I& is the turnover number, which is some36 times higher for C than B.
Thus, even though there is 5 times asmuch B, it is relatively “silent” in determining
enzymic rate measured in crude hemolysate. [This is borne out by the finding of
normal enzyme activity in a patient lacking B (435).] Only if B had a greater affinity
for drug than C and removed it from solution would crude hemolysates show a
very different 150or KI than enzyme C. This hasnever been observed. Model calcu-
lations of certain of thesesituations are given in references329 and 524. Somewhat
different affinities of methazolamide and acetazolamide for enzyme B could have
moderate physiological consequences,since in vivo, where [S] is very low, both
fractions do contribute to the overall rate (172, 174). “Complete” inhibition of red
cell activity should be more readily achieved with methazolamide.
C. Miscellaneous inhibitors, synthetic and natural
Few compounds other than the unsubstituted sulfonamideshave been reported

with activity against carbonic anhydrase. One is DDT (238), but this has not been
confirmed (P. Wistrand, personal communication). The reviewer does not know
of any unequivocal data showing activity (160< 10-2M) among organic compounds
other than the unsubstituted sulfonamides. In vivo activity of substituted sulfona-
mides, so far as they have been investigated (3 15, 336), is due to cleavage to the
free R-SOSNHZ compound.
A somewhat polemical literature arose some 25 years ago on the subject of
plasma inhibitors and activators of carbonic anhydrase; it has been reviewed by
Van Goor (502). Many of these claims have been disposedof by the finding that
they represented various artifacts of the experiments, including denaturation of
enzyme during violent shaking or protection of the enzyme from such effects (85).
These strictures apply more particularly to the activators, which are not discussed
here.
Several interesting papers have appeared on plasma inhibitors, and there
seemsno doubt that they occur in certain species.The inhibitor(s) originally
described by Booth (46) was found in sera and plasma of pig, rat, and sheepand to
a lesserdegree in horse and cat. Sera of rabbit and guinea pig had questionable
activity. No such factor was found in primate or bird sera. The inhibitor is not
dialyzable and is associatedwith a globulin fraction. Leiner and colleagues (284)
have revived interest in the sheep serum inhibitor, which they have found active
against enzyme of mammalian red cells, but not those of several speciesof bird
and reptile. Enzymes from somefish are inhibited, others not. Some isolation and
kinetic work was done; inhibition appears reversible. An inhibitor from fish plasma
against enzyme in fish red cells has been claimed (307). General interest in these
inhibitors has not developed, probably becauseof. their weak nature (about 0.050
0.01 ml undiluted serum in the reaction vesselis needed) and the fact that they are
not present in human serum.

6. Kinetics of Inhibition and Binding of Sulfonamides to Carbonic Anhydrase
A. Inhibition of the hydration reaction
The majority of the work has been done on the hydration reaction, using the
various methods outlined in section II, I above. The reaction of acetazolamide with
dog red cell carbonic anhydrase was shown to be reversible (328) by the criteria
of the Ackerman-Potter plot. We have recently run sulfanilamide, ethoxzolamide,
and CL 11,366 against different enzyme sources, including HCA-C. Equilibration
with enzyme was done with and without substrate present. Plots invariably showed
reversibility (Maren and Wiley, unpublished data). Binding experiments (see II,
SC), as well as characteristics of inhibition in vivo (II, 7), support the idea of a fully
reversible reaction.
Davis (115) showed that sulfanilamide (and also sodium sulfide) behaved like
a classic noncompetitive inhibitor in the hydration reaction; this is also the case for
acetazolamide (282). It is of interest that a guiding element in the successful search
for powerful inhibitors (356, 438) was the idea that the group R-SOzNHz was a
structural analogue and competitor for HC03 or CO2 ; this was taken more
soberly by others than its originator (Dr. Richard 0. Roblin, Jr.), and a diagram to
this effect can be found in several reviews. The realization that the sulfonamides
were noncompetitive inhibitors, or, more strictly, that they obey noncompetitive
kinetics (282)) is also of physiological importance, since it allows treatment of
inhibition in vivo in the absence of rigorous knowledge of CO2 concentration.
Bicarbonate ion in concentrations up to 140 mu did not alter the XI of ace-
tazolamide, chlorothiazide, CL 13,580, or sulfanilamide (Maren and Wiley, un-
published observations) for the hydration reaction. This is not used as an argument
for noncompetition, but to support the idea that HCO, and the sulfonamide may
share a site for dehydration (87, 283) and that CO2 and HCOa- sites may be sepa-
rate (II, 3).
Dobry-Duclaux (130) used Roussin’s salt, [Fe&(N0)7]K, as an inhibitor,
which she states to be a reagent against the nitrogenous ligands of the chelate for
Zn. A noncompetitive inhibition was described for hydration; 160 was about 3 X
10-T M . These and her dehydration experiments (see below) also lead to the idea
that CO2 and HCO, have separate sites.
Several reservations may be noted in connection with the general statement of
noncompetition. Benzthiazide (see Table 4) showed some deviation from this
behavior (282). There is also the suggestion that high concentrations of CO2 can
displace sulfonamides from their binding to pure enzyme fractions (527). Kernohan
(243) has criticized earlier kinetic work on the basis that dissociation rates between
drug and enzyme were neglected and that these rates are low enough so that the
system is not in equilibrium, but rather is frozen at [EI] during the assay and thus
appears independent of substrate concentration, i.e. appears noncompetitive.
Kernohan claimed competitive kinetics on the basis of inhibition of association
rate, but the full data are not yet available. Kernohan (244) shows a dissociation
rate constant for [EI] for benzenesulfonamide and acetazolamide of the order of

0.1-0.2 se@, whence the half-life is about 5 sec. This is a long time compared to
the rapid flow technique, but not for the ordinary estimates of hydration rates in
dilute enzyme, 20-60 set (282; Maren and Wiley, unpublished observations),
which yield noncompetitive plots. The final synthesisof the problem has not been
achieved.
What is essentially a titration of drug vs. enzyme has been explored for eight
different inhibitors (330) in the manner suggestedby Eassonand Stedman (138)
and Straus and Goldstein (477). Assuming reversibility and noncompetition, the
Michaelis-Menten equation is rearranged to form
[I 1
-=0
i 1 -il + CEO1 (9)

KI l -
[&J/i is plotted against l/( 1 - i), whence the slope yields the K1 and the ordinal
intercept yields [E,]. Accuracy is only obtainable when very powerful inhibitors
are used, so that measurable decreasesin rate are obtained when inhibitor (lo)
added is about the same concentration as enzyme (EO) in solution. Fortunately
such drugs (of KI about lo-” M) are at hand; ethoxzolamide was originally used
(330) and more recently CL 13,580 (326) and CL 11,366 (unpublished data). In
addition to obtaining accurate Kl’s, the finding of the molar value of [&I was a
considerable advance, which led directly to the knowledge of [&I in tissues(322).
This technique has been applied to the pure human fractions B and C to yield the
Kl’s reported in Table 6 and the [EJ/ e.u. values of Table 3. It is important to
note that [EJ values are, within experimental error, the same no matter which
drug is used. It will be recognized that this is a means of telling whether a given
enzyme or crude preparation is ccslow”or “fast.” Table 3 showsthat the samerela-
tion between human B and C was found by Gibbons (172) using rate data as by
this method of inhibition kinetics (329).
B. Inhibition of t/u dehydration reaction
Davenport (102) d id not do detailed kinetic studies,but on the basisof finding

no change in 160’sfor both thiophene-2-sulfonamide and sulfanilamide when sub-
strate (HCOs) was varied, it appeared that inhibition was noncompetitive.
However, Leibman and Greene (283) re-examined this question, since it seemed
important to know if the mechanism of inhibition was the samein two directions,
and this bears also on the problem of the active sitesfor CO2 and HCO~Y They
found that acetazolamide and sulfanilamide against bovine enzyme obeyed com-
petitive kinetics.
Dobry-Duclaux (130), in parallel experiments to those described above for
hydration, found that Roussin’ssalt (as anion) was competitive with HCOa. Her
conclusion is similar to that of section III, 3 above: the active site for dehydration
is a cation, which is able to fix Roussin’ssalt or HCOS-. We may add, presumably,
R-SOZNHZ or RSOZNH-.
Keller et al. (239) made titrations of drug vs. enzyme for the dehydration
reaction using the Straus-Goldstein plot, so that these results are comparable to

Octoba 1967 CARBONIC ANHYDRASE 639
those for hydration described above (330). Unfortunately the most powerful
inhibitors were not used, but experiments with acetazolamide yielded a reasonable
value for [&I.
In the dilute, cell-free systemsdescribed in section II, I, the observed catalytic
velocity (VoSt) is fully expressedand the enzyme concentration in the system is
proportional to catalytic rate. It is important to make the distinction between this
situation and those in which l& > VOba cat (expressedrate in an in vivo or intact
cell system) becauseof other limitations in the system. This was first described by
Davenport (102), who showed that 99.8 % inhibition of enzyme in intact red cells
was necessary to reduce the dehydration rate 50 %. I?& cat/V’no was 20, while
l?Oat/V’nois about 13,000 (III, 5A below). This is clearly the situation in the Jacobs-
Stewart red cell model (340), where l&B cat/Vuricis 90, and also applies to the
whole-cell method of reference 524. These casesall have in common a limitation
of the full expression or observation of Scat due to restrictions in diffusion or the
sensingof the end point. Such systemsare of value, however, in presaging the situa-
tion in vivo, where there are similar limitations to the full expressionof V’ (section
III). Recent attempts at rapid flow and thermal systemsfor high enzyme concentra-
tions and sensingsystemsin living red cells may make it possibleto measure Vg in
intact cells in vitro; this is discussedin connection with references 92, 246, 340 in
III, 5A be1ow.
C. Physical binding
The affinity of sulfonamidesfor carbonic anhydrase has also been measuredby

conventional equilibrium dialysis and by the useof red cells as their own dialyzing
membranes(333). Results agreed with those from inhibition studies.More recently
the binding to the pure human fractions (88, 527) has been measured. Some of
these data are included in Table 6. In general, the nature of these experiments is
such that data are lessprecise than those from inhibition studies.
Another estimate of physical binding is the decay rate of drug bound to en-
zyme in vitro or to tissues,particularly red cells, in vivo. Data from references329
and 527 are included in Table 6; these pertain to the decay of drug after its binding
to the two pure human red cell enzymes. These decay times have the expected
inverse relation to the dissociation constants among the several drugs and enzyme
fractions. The disappearance rate of drug bound to enzyme in vivo (EI) has been
studied for the dog (Fig. 1). The theoretical relation was developed that the decay
rates among different drugs, from red cells and other tissuescontaining carbonic
anhydrase, vary as the square root of their Kr , if other factors such as renal clear-
ance and metabolism are about the same. This was confirmed experimentally; for
example, the half-life of acetazolamide from canine red cells is about 50 hr and for
sulfanilamide 5 hr. Their Kl’s are about lOO-fold apart (322). The same relation
appearsto apply to the in vitro data of Table 6.
Several caseswere examined in which affinity to enzyme was high, but the
renal clearance or metabolism of free drug was very rapid. CL 11,366 (495) and
CL 5,342 (81) illustrate each of these two mechanisms.In both casestheir decay

from red cells of the dog in vivo is more rapid than that of acetazolamide (un-
published data) even though affinity for the enzyme is greater (Table 4). This is
quite predictable on the basis of equilibria shifting in favor of dissociation of [EI],
as Ifree is removed.
Additional properties of the reaction of sulfonamides with red cells were
studied (333). Of 18 drugs, only those with a free R-SO&H:! group were bound;
12 of these were investigated. The entry into red cells was not altered by metabolic
changes; there was no evidence for active transport. The diffusion rate constants
have been calculated; these are independent of inhibitory activity and vary as
much as 200-fold (220). Of great interest was the molar concentration of sulfona-
mides bound to the red cell. In man, for all the drugs studied (acetazolamide,
methazolamide, and ethoxzolamide in ref. 325 and CL 11,366 and CL 13,580 in
recent unpublished work) this was about 150 PM. This figure may be taken firmly
as the concentration of carbonic anhydrase, since it agrees roughly with the con-
centrations of the combined fractions B and C found by Lindskog (295); from
measurements of activity and inhibition kinetics (329) it is evident that the “fast
enzyme ” C has a concentration of about 20-25 PM, and from isolation work (Table
2) it is clear that fraction B is present in 5-7 times the concentration of C.
In the dog, the correspondence between binding and enzyme concentration
is less satisfactory. Among the 12 drugs studied in the dog, the total binding in red
cells varied between 32 and 92 PM (333), but [&I, at least from inhibition kinetics,
is reckoned as about 30 PM (330). Five of the drugs, including methazolamide and
ethoxzolamide (II, 7), do bind in the range 32-37 PM. The higher binding concen-
trations for the other drugs are provisionally interpreted as affinity to a red cell
protein akin to carbonic anhydrase but with little or no catalytic activity (60)-
qualitatively the same situation as the occurrence of enzymes B and C in man.
Among these drugs, the ease of washout from the bound component in red cells
could be correlated with the magnitude of the inhibition constant, Kr , modified
only slightly by the diffusional character of the drug (333). In bovine red cells,
there was good agreement between enzyme concentration (E,) and binding of
inhibitors (EI) in the range 40-70 PM (Maren and Wiley, unpublished observa-
tions).
7. Sulfonamide Inhibitors-General and Specijie Pharmacology
The absorption, distribution, and excretion of the sulfonamide inhibitors of

carbonic anhydrase differ, and they provide the investigator with an interesting
variety of ways and rates of delivering drugs to tissues. These are general properties
and are unrelated to the fact that they inhibit carbonic anhydrase, with the im-
portant exception of that small part of the usually administered dose that is specifi-
cally bound to the enzyme in tissues. To distinguish between these two facets, we
shall use for the first the term “general pharmacology” and for the second, ‘(Specific
pharmacology.” In this section we consider certain individual drugs in both of these
terms, and we show how drug dose and distribution are related to physiological

October 1967 CARBONIC ANEWDRASE 64’
inhibition of the enzyme. Only those drugs are included whose major physiological
effect is carbonic anhydrase inhibition. Thiazides and related diuretics belong to a
different area of physiology and pharmacology (41, 336).
A. Sulfanilamide
The general pharmacology of this drug was, of course, worked out thoroughly
before its inhibitory action was known (343). The chief property is its more or less
even distribution in tissues,according to their water content. This appears to be a
reflection of its low molecular weight (mol wt = 172), high pK, (10.4), low plasma
binding (about 10 %), and relatively high (0.15) ether partition coefficient (529).
We may add high water solubility of about 1 g/l00 ml and aqueous diffusion
coefficient of 0.85 X lo+ cm set- 1 (220). An unexplained anomaly in the earlier
time (343) was its high concentration in red cells, which we now recognize as
affinity for carbonic anhydrase (333).
Becausesulfanilamide is distributed in cell water, it is ideal for determining
the relation between fractional inhibition of enzyme and physiological effect. An
additional assetis its high Kr compared to [E,] in tissues,so that the precise value
of [&I need not be known; at total drug concentrations (lo) necessaryfor in vivo
or in vitro effects the amount bound to enzyme (E1) is small and may be neglected
in the relation [If*] = [loI - [EI]. Thus fractional inhibition (321) is approxi-
mated by the relation
[I 1
w
i = [IO] ; KI
in place of the formally correct

~~fmi31
i = r~f*eel + KI
[&I may be determined in the tissue or estimated from the plasma (321). It
may be shown that after 100 mg/kg sulfanilamide intravenously, [lo] is about 700
PM; using the KI at 37 C of 5.7 PM, i is 0.992. No physiological effect is seen on
kidney (321), eye (529), pancreas (498), or stomach (104). When the doseis raised
to 1000 mg/kg orally, and [ZJ = 2300 PM, renal HCOS- excretion is elicited; the
calculated i = 0.9976 (321). About the samevalues for [I,] and i would follow 300
mg/kg intravenously, which. does produce a fall in intraocular pressure(321).
Sulfanilamide is too weak an inhibitor to allow unequivocal recognition of
binding to carbonic anhydrase containing tissuesin vivo, particularly since its
decay from plasma is relatively slow. The half-lives from red cells and plasma of
dog are each of the order of 5-7 hr, and only a careful study, involving washing of
red cells, shows that this red cell decay pattern is indeed a reflection of specific
releasefrom a binding site rather than lossfrom cell water (333). The relation of
this decay time to the KI is discussedin section II, 6 above. The comparison with
sulfadiazine is illuminating, since here a single washing removes all drug from red
cells; this is never observed even with very weak carbonic anhydrase inhibitors.

The same is true in vitro (333). Because of its weak affinity, the specific pharmacol-
ogy of sulfanilamide was not explored further and was reserved for the stronger
inhibitors.
B. Acetazolamide (Diamox)
The physical and chemical properties of this drug are important and are
summarized largely from references 220, 328, and 529. Activity, structure, and
chemical name are given in Table 5; mol wt is 222, and solubility in water is about
50 mg/lOO ml. A solution of the sodium salt may be achieved by addition of 1.6
moles NaOH per mole of drug; this is used in parenteral administration. Ace-
tazolamide is weakly acidic with pK,, = 7.4 and pK,, = 9.1 (3 15). Partition
coefficient for CHCla is 0.001 and for ether 0.14. The aqueous diffusion coefficient
is D = 0.38 X lo+ cm2 see-l (220). Plasma binding varies markedly with species
(Table 7).
Becauseso much of the knowledge of the physiology of carbonic anhydrase is
based on experiments with acetazolamide, the general toxicity and distribution for
the immediate period after drug administration are reviewed. This is followed by
an account of its specific reaction with the enzyme in tissues,as studied in a period
when drug has essentially disappeared from the general circulation.
TABLE 7. Binding of carbonic anhydrase inhibitors to plasma
Percent Binding
Drug -
MaXI Dog Rat Rabbit Cat Mouse
I . .- ..
Acetazolamide 2-3 60 69 63 16
lo-20 95 45 67 85 51 5
25-50 90 33 50 75
70-100 36
260-350 24
Ref. (332) (328, PF) (4% (529) (W
Methazolamide Z-50 55 55 55 66 68
Ref. (W PF) (4% (529) ow
CL 11,366 1 97 94 90 82
5 95 92 75 62
10 94 89 58
24-108 96 59
Ref. uw (495) (494 > (529) (W (4% >
Ethoxzolamide 1 97
4 96 95
10 96 95 94 95
100 95
Ref. (PF) (W (PF) (529)

The toxicity of acetazolamide was extensively studied at a time when the

general effect.sof inhibiting carbonic anhydrase were not known (328) and had in
fact been predicted to be dire (200). The exceedingly low acute and chronic toxic-
ity for many specieswas then surprising: single intravenous dosesof 2000 mg/kg to
dogs were not fatal, and the murine ~~60was in the range 3000-6000 “g/kg, about
equivalent to sodium chloride. Growing rats survived 900 mg/kg per day in the diet
for 6 months, with some retardation in weight and length. At 100 mg/kg per day
all toxicity data, including histological, were identical with those of control rats.
Dogs survived singledaily oral dosesof 100mg/kg for as long as 16 months. Growth
and development of dogsw ho received acetazolamide from the day of birth (in their
mothers’ milk) were normal; this included careful attention to skeletal and en-
docrine organs. In all these animals there was a marked metabolic acidosis,usually
with a respiratory component (seeIII, 5A and B), and these experiments constitute
the first demonstration of the fact that this chemical alteration is remarkably
benign to the host. It was also clear that Na+ and IS+ loss,observed after the initial
dose, was not continuous. The animals had a steady-state deficit of about 10 %
body K+ and Na+ while on drug. These chemical changesreverted to normal when
drug was withdrawn. Calcium and phosphorus concentrations of plasma were
unchanged (328). Rabbits exhibited a special toxicity to acetazolamide, probably
based on renal damagi (52). This matter has not beenadequately explored.
Pregnant rats, guinea pigs, rabbits, and dogs were given acetazolamide in
various stagesof term. Parturition was normal and no abnormalities were observed
in the offspring (328). Recent work in which rats received the drug throughout
pregnancy revealed a special lesion, discussedin section III, XJ.
Because of the pathological connection between acidosis and decalcification
and renal stones, particular attention has been given to this phase of the problem
(328). In these initial studies, only one rat on the highest dose showed renal casts
suggestive of calcium, but later and under somewhat different conditions, renal
stoneshave followed acetazolamide administration in this species(200). In man,
there is some evidence, but not a compelling reason, to link acetazolamide treat-
ment with renal stonesin a moderate number of cases(467). This could be based
on the depression of citrate output and renal citrate in metabolic acidosis (96).
Further discussionof this matter is given in reference 323.
The lack of toxicity at the very high dosesof acetazolamide is now explicable
on the grounds that carbonic anhydrase inhibition is essentially complete at 5-20
mg/kg in all organs (321). Higher doseshave no effect on this systemand the drug
has no clearly known action elsewhere. The enzyme itself is not essential to life,
or usually even to smooth physiological function, becausesuch factors as the rate of
the uncatalyzed reaction and processeslike renal HCOa- reabsorption or blood
carbaminohemoglobin may accomplish the sameoverall purpose for the organism.
These are described in section III, 5.
The distribution of the drug in body fluids and tissueshas been studied almost
entirely by measuring the activity of homogenatesagainst carbonic anhydrase in
a standardized and reproducible system (324). This activity represents and is
expressedas a concentration of acetazolamide, based on the following evidence.

644 T. H. MAREN v01u??le47
In man (328) and rabbit (521) over 90 % of orally administered drug is recovered
in the urine by this procedure. The corresponding average in the dog is 70%,
with somevalues as high as 82 %. In this species,the authentic drug has been re-
covered and isolated from the urine; in one example 53 % of the dose was crystal-
lized from urine and an additional 21% accounted for in the filtrates. There is,
therefore, clear agreement between the enzymic method and direct chemical
analyses (328). The differences between thesefigures and 100% may be account-
able to biliary secretion (326). In the rat, urinary recovery by the enzymic method
averages 30 % (335). In this species,however, biliary secretion is prominent (326).
No evidence of metabolism was found in this speciesusing +labeled drug and
chromatographic analysis of urinary and plasma fractions (V. Nair, personal com-
munication). It is therefore a fortunate circumstance that this drug is stable in
vivo, both for validation of the analytical method and the attempts to determine
accurately the fractional inhibition during physiological experiments.
In a few casesother methods for acetazolamide have been used, including a
polarigraphic procedure (510) and 3SS-labeleddrug (442).
Absorption, blood concentrations, and urinary excretion were studied in the
dog (Fig. 2 and ref. 328) and in man (328, 332). The main difference between
these two speciesis in plasma binding, as indicated in Table 7. The drug is quan-
titatively absorbed from the intestinal tract, and in both these specieshas a plasma
half-life of about 100 min. About 80 % of the drug is excreted by tubular secretion
in man (332). In the dog, clearance of unbound acetazolamide is equal to or slightly
below creatinine (328), but Weiner et al. have shown that probenecid decreases
this clearance (510). As for many weak acids (pK 7.4), the excretion of acetazola-
mide is dependent on both passive tubular reabsorption as the unionized species
and secretion of the anionic species.The short half-life in the rabbit and the com-
plete recovery of drug in the urine also suggesttubular secretion (510).
Penetration into red cells follows a small lag, both in vivo and in vitro, but
within a few moments after 5 mg/kg iv there is enough drug present to saturate the
enzyme (333) and produce physiological effects (494). The statement that this red
cell binding is unrelated to carbonic anhydrase is incorrect (332), although a large
component in the dog is bound also to a noncarbonic anhydrase receptor (333)
and in man to the “slow” enzyme B (II, 6). The kinetics of entry have been quanti-
fied in terms of a first-order rate constant, from saline or unbound moiety in plasma
to human or canine red cells; the value is approximately 25 hr+ (220). The exit of
free drug from red cell water not in equilibrium with carbonic anhydrase follows
the plasma decay (0.4 hr-1 in dog or man) , which is much slower than the actual
inward rate constant, obtained for the appropriate speciesby multiplying-25 hr.1
by the fraction unbound.
It is evident that the pharmacology of acetazolamide is such that inhibition
of red cell enzyme may be achieved; this is discussedfurther in III, 54. A model of
the general distribution of acetazolamide in man 1 hr after a moderate intravenous
dosehas been given (332).
The distribution into special fluids has been of considerable interest. In the
dog the ratio aqueous humor/plasma and CSF/plasma is about 0.05 (335, 526)

Octobm 1967 CARBONIC ANHYDRASE 645
under conditions approaching equilibrium. In the cat the corresponding value is

0.1 [Sisson and Maren (470; and unpublished data)], which agreeswith data on
CSF and brain in this speciesusing 36S-labeleddrug (442). Single doses in the
rabbit give the same result for aqueous humor (14, 335). Results of continuous
infusions in the rabbit for 120 min are given in Table 8, adapted from reference
529. It is evident that transfer of drug into the aqueous is a relatively slow process;
the first-order rate constant is about 0.2 hr-l, or 1% that for red cells (220). In
these and other studies the aqueous concentration is always substantially lower
than that of unbound drug in plasma. Clearly, removal by bulk flow or possibly
by secretion (24) is more rapid than entry, which is almost certainly by diffusion.
The ratio of CSF/plasma, for acetazolamide, in man is 0.01 (44, 332) after pro-
longed treatment and continuous concentrations in plasma. The ratio of CSF/
plasma unbound is then 0.1-0.2, still a low figure for a drug half undissociatedand
relatively soluble in ether (529). The low CHCl3 partition coefficient (0.001) may
be significant (220).
Table 9 showsthe plasma half-life in the various speciesfor the first few hours
after administration of dosesin the range 5-20 mg/kg. These yield a first-order
decay curve; when extrapolated to zero time the apparent volumes of distribution
are about 20 % in man (332), 40 o/o in dog (335), and 100% in dogfish (319). These
differences reflect, in part, great differences in plasma binding, which in these three
speciesare respectively 90 %, 50 %, and 0.
Table 10 showsthe distribution in tissuesof the rat 0.5 and 2 hr after adminis-
tration of 20 mg/kg orally. It is evident that acetazolamide reaches several tissues
in concentrations higher than plasma; the notable exception is the brain. The up-
take in red cells is due to affinity for carbonic anhydrase. Concentration in liver
and kidney is due to secretion by these organs (326, 510). A three- to fourfold
TABLE 8. Entrance of carbonic anhydrase inhibitors into aqueous

humor of rabbit (.529), comparison with red cell entry
Acetazolamide Methazolamide CL 11,366
pg/ml at 2 hr
Plasma 101 88 92
P1 unbound 35 30 4
Posterior aqueous 14 24 0.4
Anterior aqueous 25 31 0.6
Ciliary process 41 41 24
Ki* 9 posterior aqueous

O-20 min 0.14 1.5 0.11
20-120 min 0.22 * 0.04t
Kin * red cells (dog) (220) 21 181 12
20 mg/kg iv prime, followed by 1 mg/kg per min. I& calculated from unbound plasma
concentration, accumulation at the times noted. * Equilibrium was achieved in this
period. t This may reflect some secretion out (see ref. 48).

TABLE 9. Plasma half-life of various sulfonamides*
Man Dog Rat Rabbit Mouse Cat Dog&h Alligator Sea Gull
w-P--Y-P-
Acetazolamide 95 100 65 27 w 120 1.5d -2d

Methazolamide --w 150 53 66$ 180 Id 300
CL 11,366 dot 20 15$ 35 1st 200
Reference8 given in text. Data based on intravenous doses except where noted. * In
minutes, except where d is used, denoting days. t Based on oraf dosage. $ ip or im.
TABLE 10. Tissue distribution of sulfonamides in the rat, 0.5 and 2 hr after drug
[concentration in pmoles/kg (494) and (PF)]*
Plasma Kidney
I
rime, Hr RBC Muscle Liver Brain
-
Lung
Total Un- Zortex
lound
.. .- m .
Acetazolamide, 20 mg/ 0.5 101 33 112 56 290 4.8 246 370 ~ 28

kg per 08 2.0 32 11 101 42 115 3.5 111 197 24
Methazolamide, 20 mg/ 0.5 103 46 110 35 21 21 86 49
kg per OS 2.0 76 34 120 45 18 20 62 30
CL 11,366,32 mg/kg im 0.5 182 18 96 19 96 3 865 836 106
2.0 10 1 70 2 8 co.2 56 65 11
Ethoxzolamide, t 26 mg/ 0.5 122 6 66 7 17 11 39 23
kg im 2.0 14 1 111 2 10
I 3 12 8
* At least 6 animals used for each point. All values corrected for blood in tissue. t At
0.5 hr and 2.0 hr concentration in fat was 26 and 9 C(M, respectively.
gradient from plasma to renal cortex has also been shown for the dog; the excessin
kidney above the amount bound to carbonic anhydrase (10 PM) decays at the same
rate as drug from plasma (321). The values in muscle may reflect an initial en-
trance when plasma concentration is high, with slower egress.
Additional studies were done to find the concentration of acetazolamide in
special secretory sites.Two hours after injection of 10 mg/kg in dog, plasma con-
centration was 60 PM (13 pg/ml), half of which was unbound (Table 7). Choroid
plexus concentration was 50 PM, but CSF concentration was 3 PM (unpublished
data). Table 8 showsthe samesituation for ciliary process;in the immediate period
after dose these tissuesclosely reflect plasma concentration of acetazolamide, but
aqueous humor, CSF, and brain tissue have much lower concentrations. The
physiological implications of these findings are discussedin section III. In another
seriesof experiments cats received 50 mg/kg iv; 3 hr later plasma and inner ear
tissuescontained an average concentration of 200 PM (148). The distribution of
%-labeled acetazolamide in specific areas of brain has been studied after injec-
tion of 150 mg/kg iv in the cat (442). Although the published radioautographs
show more intensive staining in the ventricles than in brain, the actual counts
show about the same concentration in CSF as in most brain areas; at 1 hr these
concentrations were 0.02-0.14 those of plasma [see recalculation of data and dis-

cussionby Rall and Zubrod (419)], which agree with the grossfigure of Table 10.
The differences among the tissues are conceivably of interest: cerebral white
matter is initially low, but caudate nucleus is high. It was proposed that drug
reaches the brain via CSF, aswell as from the blood stream. If acetazolamide does
reach brain tissuefrom CSF (441), it may be due to binding to glial cells that con-
tain carbonic anhydrase.
The low concentration of acetazolamide in brain (186) is due to slow trans-
port in, not to lack of spacewithin, tissue. In similar ventricular perfusion experi-
ments to those showing an inulin space of about 12 % (418), acetazolamide dis-
tributed in about 40 % of brain volume (Oppelt and Rall, unpublished data).
Doses used for the usual physiological effect yield initial plasma and tissue
levels (&*i) considerably higher than those of [EJ, the molar concentration of
enzyme (14,321). [EJ is obtained from enzyme activity; in the carbonate buffer
system e.u./g X 0.017 = pmoles/kg or PM. On this basisrat kidneys contain 5 PM,
dog kidney cortex 10 PM, and rat ciliary processabout 0.5 PM (from Table 16).
Since [&J >> [EJ the resulting inhibition is over 99 %. The drug concentration
in this phase is then essentially [&J; the data of Table 11 show that this compo-
nent leaves tissuesin a fashion roughly parallel to plasma. This is the basisfor the
pragmatic finding that when the first-order plasma decay curve declines below
about 2 pg/ml (9 PM) in dog, the renal effect wanes (335). It is now clear that this
is about the minimum concentration that would provide free drug to renal tissue
in reasonable excessof that bound to enzyme (321).
The relations among the concentration of acetazolamide in tissuesand fluids,
the fractional inhibition, and the physiological effect have been described in detail
and are not repeated here (321). For the purpose of general exposition, the data on
methazolamide are simpler in showing quite uniform distribution and inhibition
and are documented below (Table 12). For acetazolamide, it is evident from the
preceding discussionand Tables 8 and 10 that the drug reaches different tissuesat
somewhat different rates and equilibrium concentrations, so that fractional inhibi-
tion may not be precisely the samethroughout the body. To a much greater extent,
this is shown to be the casewith CL 11,366, and this drug is taken as the example
of the opposite in type from methazolamide (seebelow); acetazolamide occupies
an intermediate position.
The remaining discussion on acetazolamide concerns the drug bound to
enzyme, after [.&J has essentially disappeared from plasma and tissues.More
precisely, we mean the disappearance of that portion of [Itree] that was not origi-
nally bound as [Ea. The following showshow, after the usual dose, acetazolamide
is partitioned between drug unbound to enzyme and that portion bound to car-
bonic anhydrase: supposethat 5 mg (22 pmoles) is injected into a l-kg dog. The
weights of major carbonic anhydrase containing organs and their molar concentra-
tions are:
red cells, 0.04 kg X 24 pmole/kg = - 1 pmole

kidneys, 0.01 kg X 10 pmole/kg = 0.1 pmole
stomach, 0.005 kg X 8 pmole/kg = 0.04 pmole

Contributions of other tissues are negligible, either because of low weight or con-
centration.
It is evident that at this dose 95 % of drug is not bound to enzyme; this frac-
tion, as mentioned above, follows simple first-order kinetics (decay constant 0.4
hr-1) in its disappearance from plasma and body fluids, so it is presumably not
bound to any other receptor, except the loose binding to plasma protein. As indi-
cated, during the first part of this decay curve (about 4 hr) [&I is great enough so
that i is > 0.995 and physiological effect is maximal. By 8 hr [lfre] is small and
the physiological effect is finished; of the 22 pmoles injected, < 1 pmole remains
free and essentially all of the remaining drug is bound to enzyme. It is this phase
that is termed the specific pharmacology and is discussed in the following para-
graphs. Figure 1 and Table 11 provide the data (322).
The molar concentration of enzyme (E,) in the tissues of untreated dogs agrees
well with the total molar concentration of inhibitor (lo) found in the tissues 24-48
hr after injection of acetazolamide (Fig. 1). At this time, [EJ /v [&I z [lo]; drug
is a measure of enzyme. In the expression [lo) = [EI] + [&J, the last term is
small, and almost all of the inhibitor is specifically bound to enzyme (Table 11).
[&-I is small because the excess drug, as shown above, has been eliminated by this
time and because the dissociation constant of [EI] is small. The following gives
Ace tozolamide
Junol/Lm 0
F’LASMA
0.09 - ‘I
I
o*45 I I
f I 2 3 4 5 6
5mg/kg i.v. DAYS
FIG. 1. Diagram of the decay of acetazolamide from enzyme sites in vivo. On left of ordinute
are shown enzyme concentrations (I?,,) in untreated dogs, in red cells, renal cortex, and choroid
plexus. On right of mdinate are total concentrations (I) of acetazolamide found in these organs at
a time when there is virtually no free drug present, as judged by plasma concen trations. Values
of (I) shown are thus equivalent to @I). Note the striking difference in the plasma decay curves,
the first reelecting loss from the mobile fluid compartment of the body and the second, from the
enzyme sites. From Maren (322).]

TABLE I I. Distribution of acetazolamide in tissues of the dog

(pmoles/kg) after 5 mg/kg iva
Red Cell Kidney Cortex ghoroid Plexus

Eo = 24 - 30~~ Eo = 8 - 11~ 0=6-8pM
Time, Hr
IO Ibound If ree 10 Ibound Ifme IO Ibound Ifree
.-
0.25 40 54 4!jb 9 75 loo 65
1 32 103 97b 6 58 10” 48
2 50 7c 43
4 13 97 91b 6
24 0.9 31 30d 1.4d 11 10d 0.8d 9 8d 0.7d
48 0.30 27 26 1.3 9 8 0.7 7 6 0.6
72 20 19 1.1
96 0.18 16 15 1.0
144 0.09 12 11 0.8 6 5 0.6 3 3 0.2
6 Table shows decay of drug fractions free and bound to carbonic anhydrase (321,322,
PF). b &, from total concn (lo) minus that washed out of red cells (Z’f) according to pro-
cedure in ref. 333. Note that only in the case of red cells, &, > E. . See text and ref. 333.
a 1b from E. = EI, on basis that drug is bound only to enzyme. d These and later entries
calculated from the expression I’ = 4-1, using I . E EI. (See eq. 12 in text.) & is always
directly measured. [In the case of red cells, 1f was also sought by washing the red cells at
24-144 hr, but no drug was detected. The calculated 1’ values are actually less than could
be detected (333).] Then Ib = I, - If . * These and later entries calculated from urine
concentration.
the concentration of [&J in equilibrium with [EI], when all the enzyme has
reacted with all the inhibitor and there is no excessof either. Then,
[IfI l [&I = KI l [EKI but [If] = [Et], then [IfI = 1/~- ua>
[HI is approximated from [E,] or [lo]. In red cells

[If] =l/S x 10-a l 24 x 1V
= 1.2/&M
A model of this situation for the various tissueshas been published (322).
Since over 90 % of drug in the body is now bound to enzyme, it is possiblethat
radioautography of tritium-labeled acetazolamide in tissueswill reveal intimate
sites of acidification in kidney and stomach and perhaps carbonic-anhydrase-
dependent secretion elsewhere.
The decay of free drug, as discharged from enzyme to plasma, was studied by
urinary excretion, since the amounts in plasma were far too small to determine
directly (322). Figure 1 shows that curves from 2 to 7 days for the various tissues
and plasma (assuming a constant U/P ratio) are roughly parallel, with a first-
order decay constant of 0.3 days-l, about 30-fold slower than the initial (l-4 hr)
rate. Table 11 showscomposite data from published (322, 333) and unpublished
experiments, showing concentrations of drug in tissuesduring both the general and
specific phasesof distribution. Up to 24 hr there is free drug in tissues;thereafter
the drug concentration can be accounted for by binding to enzyme. At 24-48 hr

650 T. H. MAREN volume 47
the drug values [lo] correspond closely to [EJ values in untreated dogs; [loI -
[EO] - [El. Thereafter drug decline from [Eg is apparent. It is shown below that
enzyme activity is fully restored as [EI] declines. The binding to red cells in the
excess of the value for [.E,J during the first 24 hr corresponds with in vitro experi-
ments (333), and may reflect attachment in this species to an inactive form of the
enzyme with properties akin to carbonic anhydrase (section II, 6).
The decay rate from enzyme-containing tissues and plasma in this “specific”
phase reflects the Kr and the rate of renal excretion that shifts the equilibrium
[Efl + [Ifl * [Efl to the left by removing free drug (322). We will return to this
point connection with this phase for other drugs.
C. Methasolamz’de (Neptazane)
This was developed with the idea that eliminating one dissociable proton from
acetazolamide would yield a more diffusible drug (470, 539). The basic data on
this substance have not been published in detail, as pharmacological editors
deemed it too much like those of acetazolamide for general acceptability. Certain
important similarities and differences are of physiological and medical interest and
are reviewed here.
Table 4 shows that methazolamide activity against crude red cell carbonic
anhydrase is identical to that of acetazolamide. In the usual in vitro test (SEI),
methazolamide, like sulfanilamide, requires no measurable equilibration time,
which is unlike most of the other drugs. Methazolamide, like acetazolamide,
discriminates in its inhibitory activity between the two human red cell fractions,
although its activity against B is greater than that of acetazolamide (Table 6);
this may have important consequences in terms of its physiological effect in man,
since inhibition in vivo will be relatively greater than that of acetazolamide.
This is discussed below; an additional factor is greater diffusivity into body fluids
than acetazolarnide, exemplified by the relative rate constants of Table 8. Other
physical and chemical data of interest are: mol wt = 236, solubility in water 150
mg/lOO ml. A solution of sodium salt is prepared by addition of 1 mole NaOH per
mole drug; pK, is 7.2. Partition coefficient for CHC13 is 0.06 and for ether is 0.62
(529). Plasma binding is quite uniform among the species at about SO%, except
for the mouse (Table 7). Diffusion coefficient in water is 0.44 X lo+ cm2 set-l
(220).
Methazolamide is well tolerated in animals (470) but the detailed experiments
have not been published. In acute studies, dogs survived 250 mg/kg iv but died at
500 mg/kg with convulsions and extensor rigidity. Mouse LD 50 was between 1000
and 2000 mg/kg. Dogs were given 33 mg/kg per day orally for a year, with no
abnormalities except a slight decrease in urinary concentrating ability, which
reverted to normal when the drug was stopped. The usual mixed metabolic and
respiratory acidosis ensued; other plasma electrolytes were normal. Dogs did not
tolerate 100 mg/kg per day by the oral route. It thus appears that methazolamide
is more toxic than acetazolamidc, perhaps by threefold, but in both cases these

manifestations occur at such greater doses than required for inhibition that it is
not of serious import (G. M. Sisson and Maren, unpublished data).
The t1/2 in plasma of dog is 150 min, of cat 180 min, and mouse 66 min. In
man, after an oral dose, t1/2 may be as long as 10 hr (Table 9). In the dog the
renal clearance is 8-16 ml/min uncorrected for plasma binding; probenecid does
not alter this figure. This is about one-fourth the rate for acetazolamide; since the
t1/2 values are roughly the same, a considerable fraction of methazolamide is
eliminated by nonrenal routes. In the dog 16 % of an administered intravenous
dose (5 mg/kg) was recovered in the 3-hr urine and 30 % in 24 hr, as a carbonic
anhydrase inhibitor with the activity of methazolamide. After the very large dose
of 30 g iv, 12 % was isolated and crystallized as the original compound from the
5-hr urine. There is no evidence of hepatic accumulation (Table 10) and little
hepatic secretion (326), so it is presumed that the remaining 70 % is metabolized
to an unknown compound, not a carbonic anhydrase inhibitor. In man and in the
cat, the total urinary recovery of inhibitor is also 20-30 %.
Table 10 shows the even distribution of methazolamide in tissues, in marked
contrast to acetazolamide, which accumulates in secretory organs and is excluded
from brain. Sisson and Maren (470; and unpublished observations) showed that
in the-cat, aqueous humor and CSF approached within about 80 % of plasma
concentrations 3-6 hr after methazolamide; the corresponding figure for acetazola-
mide was about 25 %. In man, where CSF/plasma ratio for acetazolamide is 0.01
(332), it is 0.15 for methazolamide. Table 8 shows the very rapid penetration into
aqueous humor compared with acetazolamide. Gray et al. (186) showed the same
relation for brain and Holder and Hayes for red cells (220). It will be recognized
that in terms of rate of entry into cells and general tissue distribution, methazola-
mide resembles sulfanilamide. Since it is a much stronger inhibitor, however, it
can be used for physiological analysis in a manner that could not be achieved for
sulfanilamide. Differential centrifugation in sucrose showed methazolamide to be
about 90 % in the supernatant fraction of brain cells, after intravenous injections
in mice (187). Comparisons with other inhibitors have not been made.
The functional reflection of differences in distribution between methazolamide
and acetazolamide has been studied in certain of the physiological systems. Reduc-
tion in intraocular pressure (22, 270, 529) and cerebrospinal fluid flow (386) by
methazolamide are achieved with about one-third the dose of acetazolamide. This
does not necessarily reflect the concentration in the whole tissue such as ciliary
process, which is the same for these two drugs as well as the less diffusible and less
effective CL 11,366 (529). Rather, it appears that the concentration of drug that
reaches the posterior aqueous may represent drug in equilibrium with enzyme in
ciliary process water. Their quantitative activity in reducing intraocular pressure
is in the order of their concentrations in aqueous humor: methazolamide, ace-
tazolamide, CL 11,366 (529). Further consideration of this wi.th respect to aqueous
humor dynamics is deferred to section III, 5C. The point emphasized here is that
tissue or special fluid concentrations of methazolamide are roughly that of un-
bound drug in plasma (529; Table 10 in this paper), with no evidence of exclusion
or accumulation apart from binding to enzyme. [If] in tissues approximates that

of plasma unbound, so that i may be calculated from [lo] in tissues,which yields

I31 = [lo1 - PO19or directly from unbound concentration in plasma, yielding
[If] directly. As an example, 3 mg/kg of methazolamide injected into four dogs
gave an average plasma concentration 15 min later of 25 JUM, or 11 PM unbound.
At the same time the renal cortex concentration [lo] was 25 JUM, of which 10 PM
[(the value of [&,I) (322)] is bound to enzyme and 15 PM remains free. In the renal
l
medulla [IO] was 12 pM, of which [&] E [Efl was 1 PM (322), leaving [Ifl = 11 PM.
The same general equality was observed between free concentration in plasma and
ciliary processand aqueoushumor (Table 3 in ref. 529).
The diffusion velocity from saline or unbound drug in plasma to red cells is
very rapid, both in vivo (526) and in vitro (220). The difference in rate constant
from acetazolamide is about sixfold. The functional result of this difference is that
dogs are more sensitive to methazolamide in eliciting CO2 retention, as measured
by acute rise in intracranial tension (526). No quantitative comparison between
methazolamide and other drugs has yet been done on the apparent CO2 gradient
between blood and alveolar air (1x1, 5A). However, an interesting clinical observa-
tion is that children treated for hydrocephalus showed no respiratory symptoms on
20 mg/kg acetazolamide by mouth, but on switching to the samedoseof methazola-
mide, pronounced hyperventilation occurred (Dr. Richard Schain, personal com-
munication). It should be noted that in man, the factor of relatively low plasma
binding of methazolamide will add to its diffusibility into cells.
Although this drug has immediate accessto all tissues,it may discriminate in
its functional effect on another basis,namely the different concentrations of enzyme
in the tissuesin relation to what is needed for physiological effect. The drug yields
similar fractional inhibition (zJ in all tissues,since free drug in tissues[1/1 is about
that in plasma, and equation II showsthat i is a function only of [Ifl and K1 (Table
12). The concentration of [&,I does not affect i when considered in these terms,
since the plasma concentration of inhibitor provides an infinite source of [Ifl.
However, there is some evidence to suggestthat at the same degree of inhibition
(N 0.99), ciliary processsecretion is partially reduced whereas renal H+ output
is unchanged (Table 12). The concentration of carbonic anhydrase in renal cortex
is 10 PM, in ciliary process0.5 JIM. It has been shown that total function is possible
for kidney when 0.1 JUM is present (320), and it is only necessaryto assumethat the
eye requires about 0.02 PM to satisfy the findings of Table 12. This line of experi-
mental and theoretical reasoning had been anticipated by the clinical finding that
control of intraocular pressure (at the dose of l-2 mg/kg by mouth) was possible
without evidence of the renal effect of methazolamide (23, 270).
The specific pharmacology of methazolamide has not been explored in the
fashion described above for acetazolamide. Observations have only been made on
the red cell, but these have clarified several critical questions. The canine red cell
binding in vivo agreed with the in vitro concentration (333; seesection II, 6 above),
both of which reflect the carbonic anhydrase content of red cells. In an experiment
analogous to that of Table 11, [lbounJ in red cells at 15 min-4 hr was about 35
PM. There was neither the diffusional lag nor the initial binding to other receptors
that is characteristic of acetazolamide. From 24 to 144 hr the decay of methazola-

TABLE 12. Relation between enzyme inhibition and physiological efect

for a rapidly dzyusible drug, methasolamide
mg/kg iv
1 2 3 4 5 10
--
Renal effect in dog

HCOa-output, peq/min, at 30 3 26 67 92 122
min
y. Max effect 0 21 55 76 100
Ifree in renal cortex, /~rn+f 5 10 15 25 50
i (PF) .988 294 996 998 .999
Intraocular pressure in rabbit

Pressure, mm Hg 21.5 19.5 18.6
y0 Decrease in outflow pressure 11 25 31
y. Max effect 31 _ 71 89
Ciliary process Ifree , PM 4 7 14
i (529) .985 ,992 ,996
Rise in CSF pressure in dog

y. Max effect 33 100
Red cell Ifree , PM 4 40
i (526) .988 299
Lowering CSF production in dogs

70 Decrease 2 35 34
y. Max effect 6 100 100
Ifree (from plasma unbound), PM 4 18 36
i (386) .985 997 399
-
Certain of the values for If fee were interpolated or calculated from distribution data in
other experiments. i (fractional inhibition) calculated from the usual expression 1,/(2, + Kf),
using the values for 1f ree given in the table for the particular organ studied.
mide was identical to that of acetazolamide, with rate constant of 0.3 days-? This
suggeststhat the removal of drug bound to enzyme in tissue is not limited or in-
fluenced by its diffusional characteristics. The similarity of discharge rate from
enzyme between these two drugs is due to their similar Kl’s and decay rate of free
drug from the body (unpublished observations).
D. CL II, 366
This new drug (structure given in Table 4) was developed, like methazola-
mide, becauseit was felt that changes in physical and chemical properties among
these sulfonamides would lead to special physiological and perhaps medical uses
(495). CL 11,366 may be regarded as antipodal in character to methazolamide,
asthe following discussionshows.

Activity against carbonic anhydrase is extremely high (Table 4); in the test
system used, enzyme and inhibitor are present in about the sameconcentration so
that a special type of kinetics is applicable (329, 330). Kl’s against the two human
red cell enzymes, as well as physically measured dissociation constants, are about
the same. All four of these constants are in the range 3-8 X 10-g M (Table 6).
CL 11,366 has strikingly lessdiffusivity into aqueous humor than methazolamide,
as shown by the relative rate constants of Table 8; a similar relation holds for red
cells (220) and for distribution into brain (Ta.ble 10). Chemical and physical data
are: mol wt = 320; solubility in water 45 mg/lOO ml; soluble sodium salt prepared
by addition of about 1.2 molesNaOH per mole drug; pK, is 3.2; CHC& partition
coefficient is lo-4 and ether ‘lo- 3. Plasma binding is over 90 % for all speciesexcept
for the cat and the mouse (Table 7). The diffusion coefficient in free solution is in
the same range as given for acetazolamide and methazolamide, 0.7 1 X lo-” cm2
set-l (220).
The intravenous ~~60 in mice is about 1300 mg/kg. Rabbits tolerated single
dosesof 300 mg/kg but not 500 mg/kg. Chronic studiesshowedthat dogs tolerated
daily dosesof 20 mg/kg very well for 4 months and rats survived 260 mg/kg per
day in the diet for a month. These and other toxicity data are reported in reference
494.
The plasma half-life in the dog is about 20 min; essentially all the drug is
excreted into the urine (495). The renal clearance approaches that of p-aminohip-
purate (PAH), whose pharmacology it resemblesexcept for the high plasma bind-
ing. Taking this property into account, it is clear that virtually all of CL 11,366
is eliminated by tubular secretion in the dog. Probenecid reduces renal clearance
about 90 % (494). The rate of decay from plasma suggeststhat the samemechanism
applies to man.
Table 10 showsthe anatomical correlate of tubular secretion, a gradient from
unbound drug in plasma to renal cortex and medulla of some 50-fold. This has
been shown also in the dog (495), along with the physiological data indicating
that this drug secreted acrossthe tubule is available for inhibition of carbonic an-
hydrase. Thus the drug in kidney [&j minus the concentration bound to enzyme
[Ea is taken as [Ifl in the calculation of fractional inhibition. Accordingly, renal i
of a very high order (0.999) and physiological responseare achieved in the low
dose range of 0.3-3 mg/kg, at which i in other tissues,particularly red cells, is too
low to be physiologically effective (495). The lack of accessto other tissuesis due to
high plasma binding and ionization, factors associatedwith uptake by kidney and,
to a lesserdegree, liver. It is of interest that this drug also showsuptake into the
kidney of the dogfish, S. acanthias,along with a U/P ratio indicative of secretion
(327). As noted below (III, 2), this kidney contains no functional carbonic anhy-
drase.
CL 11,366 is effective at 3 mg/kg in elevating biliary Cl- concentration, the
typical effect of hepatic carbonic anhydrase inhibition (326; section III, .5Fin this
paper). As with most anionic drugs, the hepatic and renal concentrating and ex-
creting mechanismsare similar and there is a moderate concentration of drug in

October 1967 CARBQNlC ANHYDRASE 655
bile (326) and liver (Table 10). The pancreas, on the other hand, does not ac-
cumulate the drug, but its concentration in the tissue and juice is roughly that of
unbound drug in plasma. At 3 mg/kg, CL 11,366 produces the typical effect of
reducing pancreatic HCOZ- output; 1 mg/kg is inactive (unpublished observa-
tions).
In organs other than kidney we have found that the gross tissue concentration
may greatly overestimate effective drug level at the secretory site; the concentra-
tion in the fluid formed, i.e. aqueous humor or CSF, appears more of an index of
drug in equilibrium with enzyme. Thus Table 8 shows for CL 11,366 a 60-fold
ratio of drug in ciliary process/posterior aqueous. The aqueous/plasma unbound
or CSF/plasma unbound ratios do not exceed 0.1. The tissue concentration, were
it in equilibrium with enzyme, would certainly be enough for reduction of secre-
tion; the fluid concentration would not. At this dose (10 mg/kg) no reduction of
flow is observed (529). A similar situation exists for choroid plexus, where 5 “g/kg
causes no reduction in CSF flow in the dog, but the tissue concentration is high and
CSF concentration is low (495 and unpublished results). The probable explanation
lies in high plasma binding, which permits delivery of a large fraction of drug to
vascular spaces of tissue, but it is not available to enzyme.
Thus, CL 11,366 may be regarded as a selective renal carbonic anhydrase
inhibitor when used at 1 “g/kg. At 3 mg/kg a pancreatic and biliary effect is
observed. When the drug is given in single (495) or daily (494) doses of less than
5 “g/kg, a pure metabolic acidosis ensues, secondary to renal HCOS- loss (III, 5B).
There is no component of respiratory acidosis, as seen with acetazolamide or
methazolamide. The initial renal effect is sustained as long as renal [If] remains
above 5 PM, and it has been shown that this fraction declines in proportion to
plasma concentration (495).
When plasma concentration drops to the 0.5~PM level, the remaining drug in
the body is bound to carbonic anhydrase in the way described above for the “speci-
fit” pharmacology of acetazolamide and methazolamide. Two hundred minutes
after 1 “g/kg, renal [IO] drops from the initial value of 30 PM to 9 PM, the latter
reflecting drug all bound to renal cortex enzyme with no excess [If] (321). The
decline of renal bound drug has not been followed. After a high (20 mg/kg) dose
to saturate the red cells, the decline of drug has been studied for 6 days in dog
erythrocytes. There are two phases of decline for bound drug (495), as for ace-
tazolamide (333), the initial phase (&JU 1-2) reflecting the “additional binding”
site described above for some of these drugs in canine red cells. The second phase
(2-6 days) has a half-life of 3 days (k = 0.23 days-l). The similarity of this rate
constant to that for acetazolamide and methazolamide is regarded as due to the
fortuitous balancing of the two factors contributing to this number: the smaller
& but greater free drug decay of CL 11,366. When the 2- to 6-day first-order decay
curve for CL 11,366 in red cells is extrapolated back to 0 time, it also yields a
concentration of about 30 ,UM, about the value for [I?,] in dog red cells (495). This
phase of the decay curve, then, certainly reflects dissociation of drug from carbonic
anhydrase.

E. Ethoxzolamide (Cardrase)
This drug (structure and activity in Table 4) was derived from the parent
benzothiazole-Z-sulfonamide (CL 5,342), which Roblin et al. (356, 438) found
highly active in vitro in the original description of new heterocyclic sulfonamidesas
carbonic anhydrase inhibitors, from the Chemotherapy Division of the American
Cyanamid Company. Subsequent investigation (81), however, showed that CL
5,342 was so rapidly and completely metabolized (half-life about 5-10 min) that
it had little in vivo activity in the dog. The metabolite was of particular interest,
being the first example of a glucuronide of a mercapto compound. In a later and
most elegant study (go), the pathways of this and several accompanying reactions
were worked out and found to depend largely on an initial cleavage of the C-S
bond in CL 5,342, with subsequentcoupling of the carbon atom to glutathione.
Meanwhile ethoxzolamide, the 6-ethoxy derivative of CL 5,342, was found,
by the Upjohn Laboratories, to have considerably greater in vivo stability while
retaining the high activity against the enzyme. Their data have not been published,
but it will be shown that the 6-ethoxy substitution does yield a drug of reasonably
long half-life and high in vivo activity. It is not known whether the metabolic
pathway describedfor the parent CL 5,342 (81, 90) may be traversed by ethoxzola-
mide, perhaps at a slower rate.
Becauseof its very high activity, ethoxzolamide was usedin a pharmacological
(529) and kinetic (330) analysis of carbonic anhydrase inhibition. The following
data are derived from these two studies, unlessotherwise noted.
Chemical and physical properties are: mol wt = 258; solubility in water 4
mg/lOO ml; pK, = 8.1; ether partition coefficient is 140 and CHC13 25; aqueous
diffusion coefficient is 0.73 X 10e5cm2/sec (220). Plasma protein binding in man,
rabbit, dog, and rat is all over 95 % (Table 7).
The toxicity of this drug was studied at the Upjohn Laboratories under the
direction of Dr. Boyd E. Graham. The following summary is taken from their
unpublished data. Drug diet experiments in the rat, similar in design to those of
reference 328 but for only 3 weeks, were done at the dose range 100~00 mg/kg.
At thesehigh doses,occasionalgastric petechiae and hydronephrosis were observed.
There was no mortality attributable to the drug. Dogs received 10, 30, and 100
mg/kg per day by mouth, divided into 3 doses/day. At the highest dose, the dogs
died or had to be sacrificed within a month: there was someevidence of K+ deple-
tion. At 30 mg/kg per day the dogs did well for at least 4 months. A direct com-
parison was made with the 2-isobutyryl homologue of acetazolamide, which
showed, like acetazolamide itself (328), no toxicity or progressiveelectrolyte deple-
tion at 100 mg/kg per day. Occasional plasma determinations of ethoxzolamide
suggested moderately good absorption and duration of drug level. These data
suggesta somewhat greater toxicity than for acetazolamide. The clinical toxicity
of ethoxzolamide-numbness, tingling of extremities, sleepiness,anorexia-ap-
pears identical to that of acetazolamide, but occurs at a lower dose (375 mg/day).
However, clinical reduction of intraocular pressure (134) is also maximally ob-
tainable at a lower single dose (125 mg) than for acetazolamide.

Pharmacodynamic effects were investigated in a standard preparation using

a barbitalized dog. At 10 mg/kg iv, which would induce complete carbonic an-
hydrase inhibition (compare ref. 321) if the drug were well distributed (see below),
there was no evidence of cholinergic, anticholinergic, anticholinesterase, adrenergic
blocking, histaminic, or ganglionic blocking activity. There was transient anti-
histaminic activity. It thus appears that ethoxzolamide, like acetazolamide, is a
very specific agent and has no known properties apart from carbonic anhydrase
inhibition.
A general outline of the distribution pattern of this important drug is available
from our own unpublished experiments. Table 10 shows that unbound drug in
plasma diffuses freely into muscle and brain. There is a small uptake in liver and
kidney and inexplicably in lung (compare CL 11,366). After intravenous injection
into the dog, there is rapid drug entry into red cells, as might be predicted from
the lipid solubility and in vitro entry kinetics (220); the same is true for entry into
aqueous and CSF. The actual concentrations of I free in these tissues, however, are
relatively small because of the high plasma binding. It is likely that free drug in
tissues comes into diffusional equilibrium with that in plasma within a few minutes,
i.e. [If] = plasma unbound. Table 10 shows that by 2 hr If is so low that drug
remains only at enzymic sites or in fat.
Renal secretion elicits a cortex/plasma unbound ratio of about 6 in the rat
(30-min value in Table 10). In the dog this ratio is about 3, and probenecid reduces
clearance two- to threefold. Renal UV/P, uncorrected for plasma binding, is
normally about 20 ml/min. Since plasma binding is 95 %, a large portion (some
70-90 %) of excreted drug reaches the urine by secretion, an interesting point in
view of the high lipid solubility and pK, . About 40 % of drug appears in urine so
that the relatively rapid disappearance from plasma reflects both renal secretion
and metabolism. The apparent volume of distribution is 55 % and tl/2 is for dog 50
min, rabbit 25 min, and man (oral) .300 min. After a single dose in dog, the drug
is deposited and stored in fat for several months. Table 10 shows rapid uptake into
fat, with some turnover. The renal activity has been documented for the dog (323)
and for man (364); the minimal dose for maximal effect is 1 mg/kg. The limitation
imposed by plasma binding on the diffusion into the kidney is not compensated
for by high active uptake (as with CL 11,366), so it is not possible to take fullest
physiological advantage of the very high activity against the enzyme.
The activity in reducing intraocular pressure in the rabbit is the highest of
nine carbonic anhydrase inhibitors studied, whether based on dose or on plasma
concentrations (529). It seems certain that this is due to the combination of very
high activity against the enzyme and ready diffusibility into tissue. The quantitative
relations among these nine drugs, in terms of their in vitro and in vivo activity,
are considered at the end of this section.
The binding of ethoxzolamide to red cells and other tissues is of considerable
importance as a label for enzyme. In vitro, human cells bind 140 PM, dog cells 32
PM (333), and bovine cells about 40 PM (unpublished data). This corresponds
almost precisely to the values for red cell carbonic anhydrase. In vivo, the same
value was obtained for the dog, either following a low dose (1 mg/kg) yielding

small unbound plasma concentrations (0.1 PM) or after a large dose (20 mg/kg)
and washing of excessdrug in the red cells. The decay of drug from canine red
cells in five experiments gave quite variable results; the half-life cannot be defined
with precision within lo-60 days. A complicating phenomenon is the prolonged
sojourn of drug in the fat, so that there is interplay between the enzymic and lipid
site. This remains to be clarified. In kidney cortex and medulla 1 mg/kg also yields
the concentration of drug equal to that of enzyme, 10 and 2 PM respectively. Decay
from such siteshas not yet been studied.
F. Miscellaneous other inhibitors
Several other compounds, for various reasons,have thrown light on this system,
either in vivo or in vitro.
I) Benzothiasole-2-sulfonamide (CL 5,342). Reference has already been made to
the very rapid disappearance of this drug from plasma (81) and its unique meta-
bolic fate (81, 90). This offered the opportunity to retest the idea that such sub-
stances would be removed from binding to red cells relatively rapidly, despite a
very low dissociation constant (333). A 20-mg/kg dosewas injected intravenously
into a dog; plasma concentrations at 30,60, and 120 min were 6, 1, and 0.2 pg/ml.
Red cell concentration (unwashed) declined from 7.4 pg/ml to 0.7 pg/ml in the
period 0.548 hr, yielding a t1/2 of 15 hr. The red cell decay curve extrapolated
back to 35 JIM; the original carbonic anhydrase concentration of red cells was 27
PM. Thus, despite a greater affinity for carbonic anhydrase than acetazolamide
(Table 4), decay from red cells is some3-5 times more rapid becauseplasma and
free drug removal is about 10 times asfast.
The concentration of CL 5,342 bound to canine red cells and its activity
against the enzyme are similar to those for its closerelative ethoxzolamide (333).
In both cases,comparatively little drug is bound to the “silent” receptor of dog red
cells. Both also require some lo-15 min of equilibration in the enzyme activity
test (Table 4).
There is some suggestion,from the lossof drug from red cells on washing and
incubation, that the metabolizing systemfor CL 5,342 is present in erythrocytes.
As might be predicted from the foregoing, CL 5,342 has only weak physiologi-
cal effects on carbonic anhydrase. Urine is slightly and transiently made alkaline
by 5 mg/kg iv in the dog (81). Effects on other organs have not been studied.
2) 2-o-Chlorphenylthiadiazole-5-sulfonamide (CL 13,580). This belongsin the class
of the most active inhibitors (Table 4). It is structurally of interest because it is
the only thiadiazolesulfonamide studied without a 2-amino substituent. Chemical
properties follow :‘p& , 6.6; ether partition coefficient, 79 and CHC13, 10; 95 %
bound to plasma protein in the rabbit (529) and 97 % in the dog (unpublished).
It is a most useful drug for kinetic work becauseit is the only one of the “high-
activity” group that does not require measurable equilibration time (Table 4).
No systematic toxicity work has been done, but we have found that this sub-
stance, unlike others discussedhere, causesrespiratory and cardiovascular symp-
toms at relatively low doses(lo-20 mg/kg) in the rabbit and dog. This may be a

thiadiazole characteristic, since some toxicity is reported for this ring; such pqop-
erties appear to be modified in the other drugs by the amino substituent.
The plasma half-life in rabbits is about 45 min, and its activity in reducing
intraocular pressureis about that of acetazolamide (529). In dog the half-life is 60
min; there is no renal activity at 1 mg/kg but full responseat 10 mg/kg. It is thus
somewhat lessactive than expected on the basisof in vitro results, and this may be
related to its very low volume of distribution (11%) and high plasma binding. Free
drug diffuses very rapidly into red cells, as expected from the high ether partition
coefficient (unpublished observations). Rate is comparable to that for methazola-
mide in (220).
3) 20p-AminobenzenesuIfonamido-1,3, Mhiadiazole-5sulfonamide (CL 13,475). This
drug. is structurally of some interest as a derivative of sulfanilamide, but its chief
significance is as a pharmacological twin of its benzene analogue, CL 11,366. The
data show clearly that CL 13,475, despite its very high in vitro activity, is also
unable to lower intraocular pressurein the rabbit at 10 mg/kg. Like CL 11,366,
it highly ionizes at pH 7.4 and is plasma bound and lipid insoluble. The data re-
inforce the idea that such drugs may lack accessto an intracellular enzyme because-
there are limitations to diffusion within tissues(529).
4) 2+Chkwben<enesulfonamido-1, 3t 4-thiadiazole-5sulfonamide (“siccamid ’).
This drug is chemically, and almost certainly pharmacologically, akin to its close
structural homologues CL 11,366 and CL 13,475. These and other 2-sulfonamido
and 2-acylamido thiadiazole-5-sulfonamides were synthesized by Vaughn et al.
(505) and tested for in vitro and in vivo activity. Many of thesewere more active
in vitro than acetazolamide (Table 4), particularly those that had a benzene ring
as part of the 2-substituent.
The renal effect of 2-p-chlorbenzenesulfonamido- 1,3,4-thiadiazole-5-sulfon-
amide was studied by Kuhn et al. (265) in the rat. On a weight basisit is more
active than acetazolamide, but the pattern of renal excretion was identical. There
is no reason to believe that the properties of this drug are different than those of
CL 11,366, so it may be assumedthat the renal effect observed at 2 mg/kg (265)
occurred without other evidences of carbonic anhydrase inhibition.
5) Diphenylmethane-P4’-disulfonamide. As Table 4 shows, drugs of this general
type are surprisingly active in vitro (20,5 18). Diphenylmethane-4-4’.disulfonamide
was tested in the rat and showed maximum diuretic effect at 50 mg/kg (518). This
is lessactive than would be expected on the basisof in vitro activity; perhaps the
compound is rapidly metabolized, or poorly absorbed. The samelack of correlation
was reported for other dicyclic sulfonamides and estersof p-sulfamylbenzoic acid
(20) .
6) Dichlorphenamide (Daranide) and 0th 1,3-disulfamylben~enes. Dichlorphena-
mide occupies a curious position, since it is both a potent carbonic anhydrase
inhibitor and a renal chloruretic of the thiazide type (41). Elsewhere we have
developed the position that these two actions are entirely separate (336) and this
evidence is not given here. This discussionis confined to the carbonic anhydrase
inhibiting properties of dichlorphenamide.
Dichlorphenamide is insoluble in water, but like the other sulfonamides will

660 T. H. MAREN ‘volu?ne 47
form a soluble.sodium salt; pK, is 8.3, ether partition coefficient is 11 (529), and
CHC13 partition is 0.017. The activity against canine carbonic anhydrase,is about
one-third that of acetazolamide both at 37 C and at 0 C (Table 4), but against the
bovine enzyme their activity appears to be about the same (41). In rabbit plasma,
83 % is bound (529).
Toxicity studieshave not been published, but reports from the Merck, Sharp &
Dohme Laboratories cite the oral LD 60in rats as 2.6 g/kg and in mice as 1.7 g/kg.
Chronic administration to dogsand rats has been carried out for 6 months with no
stated evidence of toxicity. It is of interest that, despite the profound difference in
structure between dichlorphenamide and acetazolamide, the chief manifestations
of toxicity in man are the same: numbnessand tingling of extremities and.anorexia.
The pattern appears stereotyped and specific for carbonic anhydrase inhibition in
the human. An additional symptom with dichlorphenamide is headache.
Little has been done on the distribution in tissue. From its p&and lipid
solubility, ready diffusion into cellswould be predicted; limited data on canine red
cells show a large diffusible component over the amount bound (333). In general
the disposition of dichlorphenamide is similar to sulfanilamide, which it resembles
structurally. Both drugs have a relatively long half-life in the rabbit (about 80
min), suggesting no element of secretion, and a volume of distribution equivalent
to that of body water.
The comparative quantitative effect of dichlorphenamide in vivo has only
been measured on intraocular pressure in the rabbit where its activity is roughly
equivalent to that of acetazolamide, either on the basisof doseor plasmaconcentra-
tion (529). In man a doseof dichlorphenamide (100 mg) lower than acetazolamide
(250-500 mg) is clinically effective (22,179), and it is reasonable to invoke possible
differences in plasma protein binding (not available in man for dichlorphenamide)
and in diffusibility between the two drugs in this species.Acetazolamide is at a
relative disadvantage in man compared to the experimental animals on the basis
of plasma binding; this in part is reflected in the human CSF/plasma ratio of 0.01
(332) compared to that of 0.05 in rabbit and dog (328). Dichlorphenamide is
widely used in the treatment of glaucoma (179), although this can be criticized on
the basis of the additional and unwanted action as a “thiazide saluretic” with
danger of K+ depletion.
As would be expected for a drug of this K1 against carbonic anhydrase, some
renal lossof HCOa occurs at 2.5 mg/kg iv in dog (41) and orally in this range in
man (78, 133, 372, 439). Complete dose-responsecurves for Cl- and .HC03
output have not been published, and it is not clear whether the chloruretic and the
carbonic anhydrase effect can be dissociated on the basisof dose.
Although little basic renal or respiratory pharmacology has been published on
dichlorphenamide, a number of papers have appeared dealing with the use of the
compound in respiratory insufficiency (78, 133, 372). The basis for the specific
attention to this drug is not entirely clear; in part it seemsdue to the mistaken
notions that dichlorphenamide was 30 times as active aninhibitor as acetazolamide
and that the chloruresis would limit the development of metabolic acidosis (372).
In reality, a powerful inhibitor of red cell carbonic anhydrase in vivo is contraindi-

cated in respiratory disease (133) and it seems clear that dichlorphenamide does
freely enter the red cell (333). In any event, dichlorphenamide clearly produces a
metabolic acidosis secondary to the renal loss of HCO,; but this is imperfectly
compensated, particularly in patients with the most advanced disease. In a clear
and thoughtful paper, Dorris et al. (133) show why a drug of this type (i.e. a general
carbonic anhydrase inhibitor, which would presumably include acetazolamide and
methazolamide) has no rational place in the treatment of respiratory acidosis. The
complex effect of these drugs on respiratory function is considered in detail in
section In, 5A.
A second drug of this type is 5-chloro-2,4-disulfamyltoluene (disulfamide). Its
renal effect (108) and pharmacology (45), as well as the inhibitory activity of many
of its congeners (45), has been well studied. The following are chemical properties:
solubility in water at 25 C, 60 mg/ 100 ml; solubility in chloroform, 1 mg/lOO ml.
The drug is readily soluble in dilute aqueous alkaline solution. Activity against
carbonic anhydrase was 0.4 that of acetazolamide, which on our scale is an I50 of
approximately 5 X 10e8 M against dog or human red cell enzyme. As with many
drugs of this class, incubation without substrate at 0 C markedly enhances activity
(Table 4). Ultraviolet absorption spectra in various solvents are given (108).
Rats withstood oral daily (except on weekends) doses of 300 mg/kg for 10
weeks; there was no behavioral or pathological change. No symptoms apart from
slight lethargy were seen after single oral doses of 9000 mg/kg ( 108). After an oral
dose of 10 mg/kg, 22-48 % was recovered in the urine as authentic 5-chloro-2,4-
disulfamyltoluene (45). The pharmacological actions were like those of acetazola-
mide, except that the typical renal effect of chloruresis due to the 1,3-disulfamyl
group was also present. The acetazolamide-like actions included anticonvulsant
activity and an excess of Na+ and K+ over Cl- in the urine ( 108). In summary,
this drug is virtually a twin of its close chemical relative, dichlorphenamide.
A structurally important compound of this type is 5-chloro-2,4-disulfamylani-
line (Salamide); it is both a chemical precursor and metabolite of chlorothiazide
(41). However, the* aniline group appears to lower carbonic anhydrase inhibition
markedly; this drug is only about l/40 as active against the enzyme as either
dichlorphenamide or 5-chloro-2,4-disulfamyltoluene. Only a moderate alkaliniza-
tion of the urine is observed at the dose range of 10 mg/kg (40) but 20 mg/kg
appeared to give full renal effect for both HCOS- and Cl- excretion (336).. Sixty
percent of the drug is recovered unchanged in the urine 3 hr after injection into the
dog (344) and no metabolites were found (5 19). Nonrenal effects have not been
studied but are presumed to be minimal except at high doses. The metabolism of
its N-methylsulfamyl derivatives has been studied (336,5 19) and found to undergo
N-demethylation.
G. Thiaside diuretics
This subject is only mentioned to indicate that as sulfonamides (II, 5), they all
have activity against carbonic anhydrase in some degree in vitro (Table 4) and in
vivo. None are used physiologically or medically in this context, however, since all

are substantially weaker inhibitors than the major drugs that have been discussed.
For the thiazide saluretics that are moderately active against carbonic anhydrase,
there is much to commend the principle that drugs with dual action be avoided, if
possible. Thus chlorothiazide (Table 4 gives structure and activity) in dosesof
over 1 mg/kg produces dual renal effects (41), which are certainly unnecessarily
complex for the physiologist. The low dose required for carbonic anhydrase in-
hibition in viva is due to the high concentration of drug in the kidney (321). How-
ever, at high doses,chlorothiazide can also reduce intraocular pressure (529).
Hydrochlorothiazide (Table 4) is a lessactive inhibitor in vitro, and the renal
chloruretic effect can readily be achieved at doses(< 5 mg/kg) where renal and
other carbonic anhydrase inhibition is not apparent. However, typical HCOa-
excretion occurs at 50 mg/kg (336). Again, this is due to high uptake of drug in
the kidney; effects on other organs cannot be demonstrated at this or considerably
higher doses.
Benzthiazide, another powerful sulfonamide chloruretic, requires special
mention becauseunder different conditions of in vitro testsother than the standard
(SW) procedure of Table 4, it is a highly potent carbonic anhydrase inhibitor.
This occurs when drug and enzyme are incubated (E) in the absenceof substrate
(282, 330). At 37 C, there appears to be no difference between incubation with
and without substrate (320), but activity now equals that of acetazolamide (Table
4). The relation of this phenomenon to the type of inhibition has been discussed;
paradoxically, the kinetics are probably those of noncompetitive inhibition, as for
sulfanilamide and acetazolamide (282). The matter hasnot been resolved, but does
explain why benzthiazide alkalinizes the urine at low doses(390).
A comprehensive and very useful review on these and other “newer” diuretic
agents is available (41). The general excellence of that monograph is marred, in
this author’s opinion (336), by the continued implication that the chloruretic action
of the thiazides is in somefashion related to their afEnity for carbonic anhydrase.
A second and more fundamental difference between that review and this one is
their doubt and our belief that a closeand meaningful relation can be established
between in vivo events and enzyme inhibition in vitro. It is inevitable that a reason-
able number of factors concerning drug, enzyme, and host must be considered, but
the preceding treatment suggeststhat it is quite possible to accomplish this. The
final part of this section summarizesdata of this type from representative situations
to emphasize the reality of these correlations in the carbonic anhydrase system.
H. Pharmacology of three representative inhibitors correlated with

their enqme and in vivo efects
Three sulfonamides of different structural type are used to correlate the phar-
macological disposition of the drugs, their enzyme effect, and the physiological
events that follow. Sulfanilamide, methazolamide, and ethoxzolamide are selected
becausethey all diffuse freely into tissue; evidence has been presented above that
the unbound concentration in plasma is a reasonable approximation of [Ifl in
tissue.

Table 13 shows that for these three drugs the & for the enzyme varies almost
6000-fold. (Equation II showsthat Kl is equivalent to the concentration of I, that
will reduce enzyme activity 50 %.) It is implicit that among different drugs with
the same mechanism, the concentrations of If for any set degree of inhibition will
bear the samerelation as their Kl and that theseconcentrations are independent of
enzyme concentrations. Thus the use of the unbound concentration of drug in
plasma as [Ifl furnishes a means of testing whether in vitro and in vivo inhibition
may be correlated.
Table 13 showsthe concentration of the free drugs necessaryto achieve 50 %
of the maximum reduction of outflow pressurefrom the eye after intravenous in-
jection in the rabbit. The relations among these concentrations, expressedas rela-
tive activity, are remarkably like the relative in vitro activity. The 50 % physiologi-
TABLE 13. Summary of relations between pharmacological disposition, inhibitory activity,

and physiological response in 3 d#usible drugs of widely d&rent &
In Vitro In Vivo
k$Eit reFt:iv
y$, Maximal AI
effect physi;iical O i at 500Jo effect
KI, X Act+ity Dose iv,
l$ $ rela:ix to Species al =Q,w
mg/lrg
Kidney
Eye tR) (D) Eye Kidney Eye Kidney
Ethoxsolamide
0.01 5700 0.5 R, D 0.07’ 37 20
co 7 0.1 50 501 8700 6700 0.99 0.997
1’ R,D 0.15 71 65
2 D 0.3 100
4 R 0.6 100
Methasolamide
0.65 88 1 R, D 4 32 0
Cl 5 R 6 501 145 0.99
2’ R 8 71
3 D 11 50 182 0.995
4 R 16 loo
10 D 36 100
20 D 72
Suljan amide (SAA)
5050
I I
5.7 1 200 R 870 0.992
1000 D 1 0.997
R= rabbit
(oral)
and refers to experiments on lowering

I I
of intraocular pressure (529). D =
dog and refers to renal HCOa- excretion (323). Data in brackets were interpolated to yield
50% effect. The column PI, = 1’ represents unbound plasma concentration taken from dose-
response curves. For the 3 drugs selected, this has been shown equal to 11 , free drug in tis-
sues, with the following small exception. * In the case of ethoxzolamide, I/ in kidney is
3 X Pl, . For 50% effect, 1’ is taken as 0.3 C(M.

cal effect occurs when i = 0.99. Full physiological effect occurs when i = 0.996;
this could not be achieved with sulfanilamide. For the other two drugs, the 53-fold
difference in plasma [Ifl at this level of inhibition agrees well with the 64-fold
difference in & .
The samesituation holds for the kidney: at either 50 % of the maximum renal
effect (where i = 0.995-0.997) or at the full renal effect (where i = 0.999) the
relation among [Ifl values, or the activity in vivo, agreesclosely with the relative
in vitro activity. It is worth noting that for similar magnitude of effect, greater
inhibition is required for the kidney than for ciliary process,which may reflect the
higher [&I in kidney.
These data support the idea that carbonic anhydrase inhibition is solely re-
sponsible for the observed physiological effects and show that, under quantitative
conditions of comparison, in vivo inhibition faithfully reflects in vitro kinetics.
Although the three drugs selected achieve similar concentrations in tissue
water to that free in plasma, the correlation of in vitro with in vivo activity is not
restricted to such drugs but merely simplified. In other casesin the carbonic an-
hydrase system, complicating factors involving the disposition of the drug may
make the analysis from plasma concentration impossible, but it is felt that the
fundamental relation involving [Ifl in tissue, when known, holds rigorously. It is
recognized that this measurement may not always be directly made from gross
tissueanalysis, asfound for CL 11,366 in ciliary process(529), and discussedabove.
The analysis from Table 13 is facilitated by a number of other simplifying
factors: location of the enzyme in cell water; noncompetitive and reversible inhibi-
tion; lack of toxicity of the inhibitors; simplicity of the catalytic reaction being
inhibited. It is suggestedthat becauseof these fortuitous factors, the carbonic an-
hydrase systemmay be regarded as a model for correlation between enzyme kinetics
and physiological response.
III. DISTRIBUTION AND PHYSIOLOGY
1. Distribution and General Signzjcance in Plants and Invertehtes
Table 14 showsthe distribution of enzyme in plants and invertebrates. Certain

functional aspectsare also indicated.
The plant enzyme is intriguing, since it does not contain Zn and is not in-
hibited by sulfonamides (156). Nothing is known directly of its function although
its connection with photosynthesis is challenging (56); perhaps work has been
hampered by lack of a specific and potent inhibitor. Cyanide ion is a poor inhibitor
(little or no activity at lo+ M) ; azide is somewhat more active at 100~M (509).
In arthropods and worms there is no carbonic anhydrase in blood, but it is
present in secretory organs, several soft tissues,cuticle, and digestive and genital
tracts (73, 84). It is of interest that the adaptation to life in larval Gastro@ilus,
without blood carbonic anhydrase, appears to include blood pCO2 of 300-500 mm
Hg (286). The short-circuit current due to active secretion of K+ by the midgut of
Cecropia in vitro is reduced by high concentrations of ethoxzolamide (but not by

TABLE 14. Distribution of carbonic anhpdrase, and.probabZe roles,

in tissues of plants and invertebrates
Species Organ Reference Role and Comment
A. Plant
Wide distribution. See ref. Leaf cytoplasm (47, 124; Possibly involved in pho-
502 tosynthesis (56). Enzyme
lacks Zn and not inhib-
ited by sulfonamides
(156).
B. Bacteria (506) In certain strains of Neis-
seria only.
C. Invertebrates
General treatment and re- Usually none in blood, Role in gill not known.
view by Van Goor (502) High in gill and
and Polya and Wirtz some in muscle
(412).
Mollusca
Oysters, various species Mantle and related (165, 516) Catalyzes COa’ formation
tissues in shell. Also in internal
organs.
Snail, several species Mantle and related (84, 164) Also in internal organs.
tissues
Arthrojoda
Balanus imfirovisus (bar- Body and mantle (94) Also in internal organs.
nacle)
Coelenterata
Hexacorall ia subclass Body and mantle 080) All of 24 species contain
(coral) enzyme. COa’ forma-
tion.
Anne1 ida
Arenicola Stomach, calciferous 03%84) Role not clearly known.
glands, esophagus
Lumbticus In arenicola, in (502)
plasma
chlorthalidone), and carbonic anhydrase was thought to be involved (202); how-

ever, we could find no enzyme in the midgut of Cynthia, a closely related speciesof
caterpillar.
The enzyme is present in the gills of many speciesbut its function is not known.
There appears to be a definite and widespread role for the enzyme in shell forma-
tion, since CO2 is a substrate for synthesisof CaCOa (84,350). Table 14 summarizes
recent physiological work; older distribution studies are tabulated by Van Goor
(502). It appearsthat carbonic anhydrase inhibition could have more seriousimpli-
cations for marine invertebrates than for mammals; if the seahad 10-CM acetazola-
mide, oysters would be unprotected by their shell (165) and coral reefs might in
time disappear from the oceans(180). Yachtsmen, however, would no longer fight
barnacles (94), and if their eggscame from marine birds, there would be no shells
to break (30). Details are given in section III, 5R below.
Enough has been done on enzyme inhibition in the invertebrates to suggest

that drugs such as acetazolamide have rough&y the same potency as for the mam-
malian enzyme. For example, acetazolamide is reported to “completely inhibit”
enzyme in homogenate of oyster (c~assostrea virginica) mantle at 4 X 10-a M, while
benzothiazole-Z-sulfonamide was 10 times as active (516). It was also possible to
achieve nearly “complete inhibition” with these two drugs at about 100~M against
enzyme from the barnacle Balanus im~ovisus (94).
Certain bacteria of Neisseria and Streptococcus genus contain carbonic anhy-
drase; one strain of A%sicca had about the sameconcentration as partially purified
horse red cells (506). The physiological implications of this finding are discussedin
section III, 5V.
2. Distribution and SigniJicance ,in V’tebrates Other than Mammals
Table 15 shows the distribution of carbonic anhydrase in fish, amphibians,

reptiles, and birds. The reader is also directed to references 104 and 502. In this
section, the general phylogeny of the enzyme is considered; a fuller treatment is
given in reference 322a.
A. Fish
All fish, and indeed all known vertebrates, have carbonic anhydrase in red
cells. Marine fish lack functional renal carbonic anhydrase; the presence of en-
zyme, in small quantities in the elasmobranch and rather more in the teleost, is
almost certainly due to hematopoetic tissue (in, 5B below and ref. 339). The
physiological correlate of this is a fixed urinq pH of about 5.8. Fresh-water
teleosts,on the other hand, have functional renal enzyme, respond to the inhibitors,
and have a flexible urinary pH (2 13, 3 17). It is here that renal carbonic anhydrase
appears in the phylogenetic line, and all “higher” animals thus far examined have
renal enzyme (see, however, the comment below on the toad).
All fish appear to lack enzyme in the lens (308, 3 19) although it is present in
the secretory tissue of the choroid plexus and ciliary body (319). Gill invariably
has enzyme. The stomach, pancreas, and liver of the elasmobranch have small
amounts of enzyme (339) Table 15 showsthat the succeedingclassesof vertebrates
always have enzyme in these organs.
The special secretory organs of fish-rectal gland, alkaline gland, and swim
bladder-all contain enzyme (Table 15) and are discussedbelow. It is of interest
that certain speciesmay lack the enzyme (asin the alkaline gland of R. stabulofwis)
but carry out the usual function at a lesserrate via the uncatalyzeh reaction. (331).
B. Amphibians
Renal carbonic anhydrase in this classhas an important historical role, for

it wpasin the frog that Hijber first showed alkalinization of urine after administra-

TABLE 15. Distribution and role of carbonic anhydrase in vertebrates other than mammals
Concen-
Organ tration ,* Role and Comment
cu./g
PiSCCS
Elasmobranchii (319, 339) Gill 37 Physiological aspects of inhibi-
Squalus acanthias Red cells 40 tion studied in refs. 213, 319.
(dogfish) Stomach 40 Lacking in kidney (see text),
Ciliary process 21 pancreas, and lens. See ref.
Choroid plexus 23 420 for rectal gland inhibi-
Brain 24 tion.
Rectal gland 92
Retina 29
R. ocellata and R. erinacea Alkaline gland 200 See ref. 331 for physiological
(skates) (331) Red cells 32 effects. Enzyme lacking in
Gill 31 kidney as above, no enzyme
in alkaline gland of R. sta-
buloforis.
Teleostei (see also ref. 502)
Ameiurus nebulosis Gill 55 Physiological effects in ref. 213.
(catfish) (319) Red cells 300 Freshwater teleosts typically
Kidney 47 respond to renal inhibition,
Swim bladder 42 as do mammals.
Pseudojdeuronectes ameri- Red cells 200 Renal enzyme for hemopoiesis.
canus Kidney 42 See text (III, 5B). Inhibitor
(flounder) (317) has no renal effect (421) in
marine teleosts.
Lophius piscatorius Red cells 300 As above (317).
(goosefish) (317) Kidney 63
Sphyacna sp. Kidney 80 As above.
(barracuda) (PF) Pancreas 6
Perca fruv ia t il is and Serra- Red cells 2400 Data of ref. 308 have been con-
nus cabrilla Pseudobranch 3200 verted to units of ref. 319.
(perch) (308) Gill 1200 Data of the two species noted
See also teleost data cited Swim bladder (152 are not notably different and
in ref. 502 Retina 1800 are averaged. No enzyme in
Choroid layer 700 lens.
Choroidal gland
Kidney 70
Amflh ib ia
R. climatans (317, 502, PF) Pancreas 27 Renal role and effect of inhibi-
and R. catesbiana (538 Distal tubule 33 tors as in mammal (212,538).
Proximal tubule 27
Stomach 28
Red cells 190
Lens 23
Ciliary body 15
Skin 0
Bufo marinus (PF) Bladder @I

668 T, H. MAREN Volume 47
TABLE 15. (Continued)
Concen-
Species Organ tration, * Role and Comment
e.u./g
Reptilia
Chelonia
Terrepene Carolina and Pseu. Red cells ps Renal acidification in Chelonia
demys scr$ta troosti (7) Stomach P has not been studied.
Bladder (PF) 0 Bladder HCOS- transport un-
affected by acetazolamide
(455) *
Chrysemys, Chelydra,Sterno- Red cells 950
therus (PI?) Kidney loo
Pancreas 0
Crocod il ia
Alligator m ississij ienses Kidney 30 None in lens or pancreas.
and Caiman laitrostris Red cells 100 Renal role and effect of in-
WQ) Gastric mucosa 10 hibitors different from other
Choroid plexus 8 classes (95).
Ciliary process 10
Aves
L. argentatus (317, 376) Red cells 600 Role similar to that in mam-
(gull) mals.
Salt gland 30 Role in NaCl secretion (376).
Chicken (PF, 91) Red cells 800 Inhibition elicits more CO2
retention than in mammal
ow l
(PF) Kidney 225 Similar to mammal (533).

(91) Oviduct 80 Role in egg-shell formation.
Inhibition leads to soft-shell
eggs (30) l
(91) Uterine epithelium 200

(91) Pancreas 75
(91) Proventriculus 600
(91) Small intestine 35
(502) Pecten P
* All units in terms of the bicarbonate buffer system used in this laboratory. See Table
16 for conversion to other units. t It should be noted that in a few cases no enzyme is
found in the bicarbonate system, and a low concentration is found in the barbital assay [cf.
pancreas of fish (339) and toad bladder (PF)]. $ P = present. Quantification not possible
from data given.
tion of a sulfonamide (2 12). The frog was also used to show the presenceof enzyme
in both the proximal and distal tubule (3 17). Curiously, the African clawed toad
doesnot respond to acetazolamide; but renal enzyme is present, probably subserv-
ing a hemapoietic function (339). Other distribution of enzyme in the frog is like
that of the mammal. Becauseof the importance of frog skin in physiological work
it is important to note that enzyme is absent.

C. Rep tiles
There appears to be renal enzyme in the several orders studied (530 for
Crocodilia;unpublished data for Chelonia). In Crocodilia the urinary composition,
and effect of inhibition, is entirely different from that in the mammal and is ex-
tremely instructive (95; and section III, 5B). Nothing is known about urinary acid-
base excretion or the role of the renal enzyme in the other orders. There is no
carbonic anhydrase in the bladder or pericardial membrane of the turtle. As in
fish, enzyme is absent from lens and equivocal in pancreas of the caiman; other
aspectsof distribution in this speciesare like that of the mammal (530).
D. Birds
This classhas a distribution of carbonic anhydrase similar to that of the mam-

mal. In addition the special organs contain enzyme asfollows: salt gland, oviductal
system, proventriculus, and pecten. Functional aspectsof the first two are discussed
in III, 5. Bird red cells show a different relation between concentration and catalytic
effect in the in vitro system; each increment of cells produces a larger effect than in
the mammal (3 17). This might be related to more rapid respiratory exchange;
inhibition produces greater elevation of pCOz (376) and greater toxicity than in
mammals.
In general, there are certain common features of vertebrate distribution of
carbonic anhydrase. All speciesexamined have enzyme in red cells, choroid plexus,
ciliary process,stomach, and gill. The pancreas has not been systematically studied
but its presencein frog probably denotesgeneral vertebrate distribution. Functional
renal carbonic anhydrase is associatedwith the appearance of vertebrate life in
fresh water. Special secretory organs that have arisen usually contain the enzyme,
which is involved in the elaboration of acidic (154, 376), alkaline (33l), or neutral
(54) fluids or gas (152).
Something is known of the composition and kinetics of red cell and other
carbonic anhydrasesin nonmammals from the work of Leiner (284; seesection II, 3)
and Maren and Wiley (338, 339). This work reveals quantitative differences of
someimportance in structural make-up and reactivity. So far as has been studied,
the carbonic anhydrases mentioned in this section are all inhibited in vitro by
acetazolamide and several other inhibitors, although there are some differences
(not exceeding sevenfold) in the Ibo)sof a given drug against the various enzymes
(339).
3. Distribution in Mammals
A. Normal: molar equivalent of activity
Table 16 setsout the distribution of enzyme activity in dog, rat, rabbit, and
cat. Speciesdifferences are not common. Interestingly, these are confined largely

to the reproductive system. Some of these variations are discussedin connection

with the physiological findings in section III, 5.
The concentration of enzyme in red cells of 15 mammals, ranging in size from
the 6-g bat to the 52l-kg horse, has been studied by Larimer and Schmidt-Nielsen
TABLE 16. Distribution of carbonic anhydrase in tissues of mammals*
Organ Dog Rat (PF) Rabbit Cat Notes
RBC MO (322) !4Qo 1100 (14) 2oooW) See ref. 281 for
data on possi-
ble enzyme in
carotid body.
Kidney
Whole 400 (325) 245 275 (44) Finding no en-
180 (102) 260 (107) zyme in me-
546 (322) dulla (107)
Medulla 64 (322) 0 (107) appears to de-
0 (107) note only low
sensitivity of
method.
Eye
Ciliary process 23 (14, 520)
Lens 500 (12) 50 195 (14) 650 (12)
309 (319)
Retina (12) 450 50
Whole stomach 434

Parietal cell (100) ‘200
Pancreas 20 (422) 10
Prostate (285, 345) 0 8 Marked ana-

Posterior 1400 (285) tomical and
Ventral 28 (285) species differ-
ences. No
functional
data availa-
ble. See III,
5J.
Salivary glands (502,

537)
Parotid
Submax
Sublingual 52
Brain 60 77 (W In glial celL,

Choroid plexus 386 (322) 262 (44) not neuron8
(171)
Liver (326) 20 20

TABLE 16. (Continued)

I
Organ Rat (PF) Rabbit Cat Notes
-- ~~
Uterus (301) 0 40 0 See section xxx, 4
Pregnant uterus 130 200 for values of
W) sheep, cow,
and pig.
Lung 30
Inner ear 2500 (148) Average of vari-

ous sites in
saccus.
Intestine See also ref. 267

Colon 345 for additional
Duodenum 77 data on dog
Jejunum 26 and agree-
Ileum 19 ment with
pattern cited
for rat. “Ap-
pendix” of
both species
(cecum) has
C.A. concn
like colon.
Breast (50) 116 39 In rat, Iarge in-

crease due to
pregnancy,
parturition,
and nursing.
* The figures are units/g of wet tissue, as defined in ref. 324. Figures from work done
elsewhere are converted, as best could be done, into these units. The following relation ap-
plies roughly: units of Table 16 (bicarbonate buffer system), 1000; barbital buffer system
used in the same way, 3000; Roughton unit, 3000; Philpot unit, 500. Conversion of units
to molar terms is given in the text; for the bicarbonate buffer system, 1 e.u./g tissue = 0.017
PM. Where the literature notation is given with the organ, it applies to all the data in that
row. PF (unpublished data from this department) applies to all the rat data unless other-
wise indicated.
(273). In a general sort of way, the smallestanimals have the highest concentration,
although there are several exceptions: The overall range is 1l-fold, with dog and
man about at the median. In view of the variability and the great excessof enzyme
(at least in dog) for physiological needs,it is doubtful if these variations are signifi-
cant for CO2 transport. Table 16 also showsenzyme in lung and possibly in carotid
body, but its role in respiration has not been elucidated (III, 5A).
The different species of Table 16 show a reasonably consistent pattern in
enzyme concentration among the different organs: pancreas and ciliary processare
low, kidney is moderate, and salivary gland-and inner ear are very high. Both in
this respect and in physiological responses(III, 5) there is little difference among

the mammals studied. The matter of concentration in whole organs, as against that
in single secretory cells, must be discussed; it is considered in the following para-
graphs in terms of the kinetics of the secretory processes, the calculation of the
fractional inhibition, and the light it may throw on the anatomy of secretion. The
problem is simplified by the fact that the enzyme is largely in cell water rather than
in particulate matter (99, 187, 233, 326). These studies do show as much as 20 %
of enzyme in particulate fractions of submaxillary gland, kidney, liver, and cerebral
cortex, but there is reason to suppose that physiological effects and inhibition are
based on enzyme in solution in the cell (326). In the red cell, all enzyme is in cell
water; ghosts have none (unpublished data, Pharmacology Dept., Univ. of Flor-
ida).
To place the data of Table 16 in a kinetic context, it is first necessary to convert
the enzyme units per gram into molar concentrations. The rationale behind this
important step is given in section II, 4. The constant used is derived from dog red
cell data: e.u./g X 0.017 = pmoles/kg. This arises from the fact that in the test
system used, 1 unit in 7 cc of solution is equivalent to 2.4 X 10Bg M, as judged by
titration of the enzyme with ethoxzolamide (330). It is shown (II, 4) that this con-
stant can reasonably apply to crude enzyme in various tissues when values of [&]I
e.u. agree. Although this has not been tested for all tissues, work to date does suggest
that, except for the “slow enzyme” B in human red cells, the enzyme in tissues is
kinetically like human red cell C, which like dog blood has the equivalence of
about 2 X lo-” M/e.u. (329).
When the enzyme content of tissues is entered into equation 21 to obtain the total
secretory rate of an organ, E, has the dimension of weight, specifically of m2les in
the whole organ (320). Thus the intimate localization or concentration of the
enzyme is not an issue in this context. The term E. is used in III, 5 for several of the
tissues.
The uncatalyzed reaction rate, however, depends on the volume of solution
(or cells) involved: rJuncat = kl [CO,], where [CO,] is in moles/liter. Thus it is
necessary to estimate the relation of the cell volume to the total volume of the organ.
This may be done by using the enzyme concentration itself as an index. In the
stomach the secretory cells contain about 20 times as much enzyme- as the whole
tissue (calculated from ref. 100 by T. F. Muther; see also ref. 369); presumably the
inverse of this gives the fraction of secretory cells. This is not a surprising figure in
view of the extensive muscularis and serosal layers and other cell populations. In
the choroid plexus the enzyme concentration in secretory cells is about 40% that
of red cells (17 1); the whole organ has about 27 % the concentration of the secretory
cells (Table 16) so that the secretory volumk in this case approaches one-third of
the whole organ. Again this is not at odds with the histology. Unfortunately, data
for other organs do not exist at present but guesses have been made as to secretory
volume in kidney and pancreas (III, 5).
Intimate localization of the enzyme, while it may not be essential for kinetic
calculation, is an interesting issue in itself that has not been solved in a satisfactory
manner. It is possible to show that both distal and proximal tubule cells of the
frog (3 17) and rat (232) contain enzy*me, and functional studies (reviewed in 323)

support this. On a cellular level, radioautography using labeled inhibitors has not
yet been successful,due probably to lossof soluble enzyme-drug complex during the
procedure. Histochemical methods have been alluded to elsewherein this review
(II, 1); they do not appear specific for carbonic anhydrase and probably measure
binding sitesin tissuefor divalent transition metals (367).
Certain tissuesof Table 16 either have no known physiological role for enzyme
or have’ not been adequately studied in this regard. These include lens, retina,
breast, uterus, and prostate. It is likely that future research will clarify this, for
carbonic anhydrase isnot found indiscriminately in tissues;it is absent from many
tissues,as shown in part C below.
B. Abnormal. states: 2% concentration and dejciency
Several studieshave been made on the carbonic anhydrase content of tissuein

abnormal physiological states or in disease.Only rarely have the findings shown
significant variations from the normal. Data are briefly discussedhere.
Severe metabolic (314) or respiratory (70) acidosis did not change renal
carbonic anhydrase activity. A variety of electrolyte and* dietary manipulations,
including metabolic acidosis and alkalosis and K+ administration and depletion,
changed renal carbonic anhydrase only slightly, in view of the large (+25 %)
standard error of the control group (366). The 5Q% drop after NH&l does not
seemsignificant, particularly in the light of evidence showing a very large excessof
enzyme in this and other organs (320, 321).
No variation from normal red cell carbonic anhydrase activity was found in
14 patients with chronic respiratory disease,ages27-79 (469). In pernicious ane-
mia, enzyme activity per red cell doubled, so that whole blood activity was,about
normal. In other anemias the enzyme activity per red cell did not change (501).
In a dog with severe renal disease,analogous to chronic glomerulonephritis, renal
carbonic anhydrase (and glutaminase) wasnormal, even though the animal showed
no responseto acetazolamide and could not make urinary ammonia. It was con-
cluded that defects in filtration and transport, rather than in these enzymes, were
responsiblefor the pathophysiological findings (3 16). There has been much interest
in the possibleconnection between renal tubular acidosisand carbonic anhydrase
deficiency or inhibition; however, a critical difference is the strict limitation on
sodium loss when the enzyme is inhibited (328). Decisively, patients with this
syndrome do respond to acetazolamide with typical alkalinization of the urine
(235). An ancillary finding of interest is that such patients show normal gastric
acidification (472). It is clear that this is not a carbonic anhydrase deficiency-state.
An early attempt to produce Zn deficiency in growing rats did not result in
any change in blood carbonic anhydrase. Bone zinc was reduced about one-third
(123). In a variety of normal and diseasedindividuals, the correlation between zinc
concentration and carbonic anhydrase activity in red cells appears quite doubtful
(501). The concentration of zinc in human red cells, 200 JAM (501), exceedsthat of
both human carbonic anhydrase fractions (II, 3), and since activity dependslargely
on fraction C (25 k~) a correlation of this type is not expected. Almost certainly

there are Zn-containing diphosphopyridine nucleotide (NAD) dehydrogenasesin

red cells.
Rats and mice bearing tumors of several types did not show a significant varia-
tion from normal carbonic anhydrase concentration in blood, liver, kidney, or
stomach. Hepatomas in mice had roughly the same concentration of enzyme as
normal livers (466). Rat mammary tumors of several types were notably lower in
enzyme than normal tissue (Table IS), probably due to proliferating fibrous tissue.
Even such tumors, however, do increase enzyme content with lactation (50).
Absence of HCA-B from red cells of a Negro woman has been reported (435).
Also of interest is the deficiency of both HCA-B and HCA-C in a patient with per-
sistenceof fetal hemoglobin. Overall carbonic anhydrase activity of the red cells was
also reduced appropriately. The interesting question is raised of the relationship
between the fetal pattern for hemoglobin and carbonic anhydrase, although the
synthesisof these two proteins is under separate genetic control (146).
The most recent work in this field correlates absenceor marked reduction of
HCA-B with hyperthyroidism. Overall enzyme activity of the red cells was slightly
but significantly reduced. An increased intensity of the B band was observed in
hypothyroidism. The only other consistent alteration was the increase in intensity
of the B band and in total activity in patients with untreated megaloblastic anemia.
In all thesesituations, treatment elicited return of the normal pattern. The intimate
connection, if any, between HCA-B and human diseaseremains to be discovered;
in the present context this work emphasizesthe relative stability of HCA-C and
that even total activity varies over only a twofold range in these diseases(509a).
C. Tissues and fluids lacking carbonic anhydrase
This has in part been discussedby Davenport (104). and only a few emenda-
tions are necessary.Enzyme is present in lung and in retina (Table 16), but absent
from saliva. There is no doubt in this reviewer’s mind of its absencefrom the fol-
lowing organs (104 and personal observations) : muscle, blood vessels,peripheral
nerve, skin (including frog skin), tongue, toad bladder, cornea, and thyroid. A
low concentration has been reported in thyroid (233), but the presence of blood
was not*excluded. In our hands appropriate correction for red cells reducesthyroid
carbonic anhydrase to zero. The same is true of adrenals (104) and pituitary, al-
though no critical study has been published on the carbonic anhydrase content of
this or other endocrine organs. There would seem to be no reason for carbonic
anhydrase in these various tissues;indeed in such sitesit would impede the evolu-
tion of metabolic CO2 and be “more an enemy than a friend to the organism”
(444).
Absence of enzyme in turtle bladder (unpublished observation and lack of
acetazolamide effect in ref. 455) is of particular significance in that the tissuetrans-
ports HCOa- ion as such, rather than by its formation from CO2 (455). Analogy
to a mechanism in the proximal tubule will be recognized (III, 5.).
Fluids such as plasma, urine, milk, CSF, and aqueous humor are absolutely
free of enzyme. The significance of a trace (0.1% that in red cells) in human serum

is not clear (397). Curiously, its presencein allantoic fluid (347) and sperm (58)
has been reported. We cannot confirm this latter finding (for bull sperm, using
method of Table 16) and Meldrum and Roughton had reported but a trace in the
rabbit (350). Saliva was also reported to contain a high concentration of enzyme
(479); again we cannot confirm this. We believe that for both fluids there are
artifacts hidden in these published data; for saliva the use of undiluted fluid (479)
adds to the HCOa- concentration and hence the rate, and for sperm the problems
may be due to the manometric assayat 37 C (58). The matter should be resolved,
particularly in view of conflicting theories about calculus formation involving
carbonic anhydrase (11, 348) and the effect of sulfonamideson glycolysis in sperm
(453).
“Brain carbonic anhydrase” has been the subject of considerable controversy.
Certainly the enzyme is present in homogenates(104); the issueis that of localiza-
tion and function. This is discussedin III, 5D below. Here it may be stated that the
reviewer and his colleagues believe that brain enzyme is in glia, not in neurons
(17 1). In neurons, as in muscle, it would truly be an enemy to the organism.
4. Development in Birds and Mammals
All organs that contain carbonic anhydrase show an increase in their activity
from prenatal and neonatal life to the adult. These are considered here according
to organ systems and with some comparison of developmental rates in enzyme
among the tissuesof birds and mammals. Emphasis is given to work since Van
Goor’s review (502).
Carbonic anhydrase first appears in the rat embryo at day 13, at which time
there is an enzyme concentration (scaleof Table 16) of about 3 units/g. Thereafter
it roughly triples on successivedays. Only very careful methods can show activity
before day 15 (517). In the amnion of this species,enzyme was found at day 12. An
intriguing claim by Tgutu and Voiculet (483) is that enzyme for hydration ap-
peared on day 7 in the chick encephalon, whereas that for dehydration appeared
at day II. If this is confirmed, it has important implications vis-h-vis enzyme struc-
ture, but it is possible that the difference found has its basisin the different sensi-
tivity of the two methods.
A. Red cells
A careful study by Clark (83) showsthat enzyme appears in murine red cells
at day 15 of development, when 1% of the adult activity is detected. At birth (day
21) about 10% of the adult level is found. Enzyme concentration plateaus at about
7 weeksafter birth. This is roughly the sequencefound in man (3 1) and in monkey
(159) when appropriate corrections are made for gestation periods and life span.
The discovery of the human red cell enzyme forms B and C has naturally
raised the question of whether these may be interrelated in terms of development.
Neonatal enzyme activity has been analyzed kinetically in parallel fashion to

676 T. H. MAREN Vohmze 47
experiments of Table 3 and 6. In our experiments neonatal blood activity averages

about one-sixth that of the adult. The kinetic and inhibition values exactly parallel
those from adult blood, which are, as discussed above,. the characteristics of the
fast enzyme C. Binding and inhibition data taken together suggest that there are
40 PM carbonic anhydrases in neonatal blood, of which 4 PM is C and the remainder
B. In the adult the total is 150 pM with 25 PM C and the remainder B (329). In the
newborn, whole blood shows an immunological reaction only with human B,
which is tentatively thought due to the very small component of C present (528).
Acetazolamide was slightly more active against neonatal than adult human blood
(524).
Red cells of premature infants occasionally contain no detectable carbonic
anhydrase activity (329, 475). No clinical symptoms were associated with this
deficiency. In our case (329) the observation was made on cord blood in a 2400-g
male nonidentical twin; by the 2nd day of life carbonic anhydrase appeared in the
blood. From the onset, there was a component of red’cells that bound acetazola-
mide; presumably this is a protein related to the ,active enzymes, perhaps a pre-
cursor. In a group of five premature infants, binding and inhibition data taken
together suggest that HCA-C is present in about 1 PM concentration (4 % of t’he
adult level) and that HCA-B or an even less active related protein is present at
about 40 JUM (329).
In the chick embryo, carbonic anhydrase appears in the blood at day 12, 8
days after the onset of hemopoiesis. Enzyme increases rapidly until the adult level
is reached at day 18. In this valuable study (83) it was shown that enzyme is absent
from primitive red cells, i.e. blood islands, blastoderm, and endothelium. It is also
likely that red cells from spleen and yolk sac lack the enzyme; carbonic anhydrase
is confined to red cells arising from bone marrow.
B. Eye
Van Goor (502) has reviewed his own and other work prior to 1948. Recent
work has confirmed the very early appearance of carbonic anhydrase in the retina
[day 6 in chick embryo (83)]. In the rabbit embryo the whole eye, without lens,
shows considerable activity at day 18. The lens develops carbonic anhydrase in the
chick embryo much later, &zy 12 (83), than retina. In the rabbit, at day 15, lens
enzyme has the highest concentration of any organ in the body (311). It is clear
from these studies that the enzyme develops at different rates in the various organs.
As noted elsewhere, there is no obvious role for carbonic anhydrase in lens or retina,
despite its prominent early appearance and high concentration. Some speculations
on the matter are of interest (503). No studies have been reported on the develop-
ment of carbonic anhydrase in the ciliary process.
C. Kidney
Chick embryo develops renal carbonic anhydrase by day I5+omewhat later

than red cells (83). In human fetuses, renal enzyme appears as early as 2 months

October d967 CARBONIC ANHYDRASE 677
after gestation, at which time and throughout uterine development it is in higher
concentration than in blood (32). A comparison among the carbonic anhydrase
concentrations in the renal cortices of premature and mature infants and adults
revealed minimal differences in activity (125). It is of interest that mammals can
produce a strongly acid urine during fetal life. In a few preliminary experiments
we demonstrated the renal effect of acetazolamide in the newborn rabbit. After
birth, in the rat, the concentration of renal enzyme increasesrather slowly, but at
2 months has increased sevenfold to the adult level. A similar sequencefrom fetal
to adult life has been reported for the rhesus monkey (159). It is evident that car-
bonic anhydrase is continuously synthesized in this vital organ, from early fetal to
postnatal life. Rates vary with the species.Of compelling interest is the fact that
the pig mesonephros contains high concentration of enzyme (347). There still
remains to be done, however, a complete study of renal acidification and HCO,-
reabsorption in the newborn.
D. Stomach
This organ shows moderate concentration of enzyme at day 12 in the chick

embryo; this increasessixfold by day 21 (83). Human fetusesconsistently show en-
zyme by the 4th month; the concentration rises moderately with development
(32), as in the monkey (159) and rabbit (311).
E. Pancreas
This organ also showsa progressionin carbonic anhydrase concentration from

fetal life to the adult. In both humans (32) and monkeys (159), there is about a
fivefold increase.
F. Lung
This is of particular interest since the adult lung contains little enzyme rela-
tive to blood and there has been somequestion whether that found in tissuecould
be due to erythrocytic contamination. In the 75-day fetal monkey, however, lung
and red cells have about the same carbonic anhydrase content. Unlike the other
organs studied, however, lung concentration only increasestwofold from fetal to
adult life (159). A few determinations done on human fetuses, 6-8 weeks before
term, also show carbonic anhydrase in lung, at a time when enzyme in red cell was
roughly at the sameconcentration (32).
G. Liver
Embryonic liver in both rabbit (3 11) and man (32) contains the highest car-
bonic anhydrase concentration of any organ, including blood and lens. The sig-
nificance of this is certainly not clear, although the role of the enzyme in hepatic
physiology is beginning to be understood (III, 5G).

H. Allantoic membranes
These contained considerable amounts of carbonic anhydrase in the pig. The

allantoic fluid had a pH of 5.8-6.1 with moderate concentration of titratable acid,
NH& and CO2 A surprising finding was that of carbonic anhydrase in allantoic
fluid itself; -if this can be confirmed it is unique (347).
5. Function of Carbonic Anhydrase in Organ Systems and E$ects of Inhibition
A. Respiration and red cells
It is convenient to divide this section into three parts: I> a consideration of the
physiological events connected with CO%* H&Oa equilibria in red cells; 2) the
chemical kinetics underlying CO2 evolution or uptake in red cells, including a
consideration of inhibition kinetics; and 3) a short part on aspectsof the system in
fish and birds. An attempt is made to link the physiological findings with the rates
of the catalyzed and uncatalyzed reactions.
I) Physiology in the mammal. The role of carbonic anhydrase in CO2 carriage
by red cells has been exhaustively studied since the pioneering work of Roughton
(444, 445). When it is appreciated that in the first of these reviews, 30 years ago
in this journal, 25 pageswere devoted to this problem, the task to the present writer
is evident. The advent of acetazolamide 15 years ago made it possible to study
respiration in the virtual absenceof the enzyme. This section attempts to synthe-
size the best data into a model that will describethe various forms of CO2 in plasma,
both at equilibrium and disequilibrium, induced by inhibition of enzyme.
The nature of the overall uninhibited processis clear: metabolic CO* diffuses
from tissuesinto the red cell, where it is in part converted into HCOI;-. The equi-
libria of equation 7 are achieved, essentially instantly. In the lungs some CO2
diffuses into the alveolar air, whence HCOa- generatesmore CO2 to restore equi-
libria in the blood. Questions involving Cl- and H+ shifts between red cells and
plasma are not considered here.
Almost at once after his discovery of carbonic anhydrase, Roughton attacked
the problem of the relation between the time for enzymic and for nonenzymic
unloading of CO2 from HCOa- in the lung. The following equation relates the
time required (t) for the uncatalyzed dehydration reaction to liberate CO2 in
the lungs from blood HCOS- asa function of the percent (X) of HCOa- reacted:
100
In - = 5 . f HCQ- . &J+ . t WI
100-x K,,
This equation was published in reference 444 without the term t because it was
initially used to solve for X when t = 1 sec. The constants used and the implica-
tions of this equation are discussedin some detail becauseit has become widely
used (446) and occasionally misunderstood. I am indebted to ProfessorsF. J. W.
Roughton and A. B. Otis for discussionsthat clarified its original meaning and the

physiological application. f HCOa is the activity coefficient, usually taken as 0.6,

and aH+ is the activity of H+ taken directly from the pH. Roughton’s numerical
value of the first term on the right was
70 set-1
- = 3.5 x 1w
2x10-%
From the data of section II, 2 above, the modern value (extrapolating k-1 to 37 C)
would be
30 set-l
=l.l x 106
2.7 x lo-%f
whence the new value of t will be about 3 times greater than originally given.
The equation is derived from the kinetics of opposing first-order reactions and
takes into account the dehydration of HCOa- from blood and the “back reaction,”
that is, the hydration of alveolar CO2 . Roughton’s derivation, which has not yet
been published, depends on the setting of 100 as an equilibrium situation (not an
initial concentration as in a first-order decay equation). 100 is proportional to the
CO2 liberated from HCOa- under conditions of equilibrium between blood HCOa-
and alveolar CO* . X follows the same relation, except in the absenceof catalyst,
with the approach to equilibrium now being a function of the other elementsstated
or implied in equation 13, specifically h-1 , &, pH, time, and pCO2 . The first two
of these are constants discussedabove. pH was assumednot to change during
inhibition, although it is clear that if [H+] increased t would drop proportionately.
The significant variables discussedbelow are t and pCO0.
Roughton solved for X using t = 1 set, the time of blood transport through
the lungs. X, the calculated quanta of CO2 evolved without enzyme, compared
with the normal or equilibrium state of 100%, was only l-2 %. Conversely, in
the more frequently cited model, the equation as written above was solved for t,
setting X = 90, to calculate how long it would take for arterial HCOa to reach
90 % of equilibrium with the samealveolar pCO2 in the absenceof enzyme. Put in
another way: in this formulation 100 is the CO2 liberated by the normal A-V
HCOa- difference, and 90 is the CO2 liberated by the A-V HCOa difference fol-
lowing inhibition, which in turn is 90 % of the normal A-V difference. This carries
with it the implication that HCOs in both situations equilibrates or tends to
equilibrate with the same alveolar pCO2. Under these conditions, t is the only
variable of X; when X = 90 (for Roughton’s constants) and at the pH of serum,
t = 227 set for a subject at work (444).
This calculation led to the prediction that total inhibition of carbonic anhy-
drase would lead to “speedy death” (110). The matter was unresolved until Cain
and Otis (65) furnished experimental data for the value of X of 5.9 %. Using Rough-
ton’s (444) or Constantine’s (92) k-1, t = 5.4 set; if k-1 is extrapolated to 37 C
from data of section II, 2, t is about 12 sec. If, as suggestedby Cain and Otis, the
pH of the red cell is 7.0, these times would be lower by almost half. In any case,
there is a profound quantitative difference between the numbers in Roughton’s
(444) model and Cain and Otis’ (65) experiments, and this is essentialto analyze.

Roughton (444) tacitly assumeda simple system with no respiratory adjust-

ments or alterations in the uncatalyzed state, other than failure of unloading of
HCOa- itself. On the other hand, the experimental findings after 50 mg/kg iv of
acetazolamide were of a greatly lowered alveolar pCO2 (15 mm Hg), a large
venous-alveolar pCO% gradient (64 mm Hg), and increase of participation of
carbamino CO2 from 26 to 38 % (65). Becauseof these changes,the role of HCO,
in the total unloading of blood CO2 decreasedfrom 55 % to 17%. The A-V HCOa
difference was 4 mM, whence the HCOa- reacted was 0.7 mM. This divided by the
normal uninhibited A-V difference (X 100) for the same /CO2 will yield X. Cain
and Otis (65) calculated that for the venous alveolar pCOz gradient of 64 mm Hg,
the HCOS- A-V difference (i.e. HCO, available to react) was 12 mM. Thus
0.7
x = 12 x loo = 5.9%
Alternatively, the left side of equation 13 could read

12
in -
12 - 0.7
thus using the actual values rather than percentages. It should be clear that equa-
tion 13 showsthe relation between CO2 that could be unloaded catalytically from
EICOa within the circulation time through the lung against a given pCOz , with
CO2 that is unloaded noncatalytically, at a rate set by constants of the equation,
against the samepCO2 .
It should be emphasized that Roughton’s original formulation (eq. 13) did
provide the key to the problem. The wide discrepancy between his value of t (or X)
and the physiological consequencesof inhibition, which have long been puzzling,
is clearly resolved by the experimental findings cited above. Such ‘Ginvivo adjust-
ments” were in fact foreseen by Roughton et al. (450) in early work on sulfanil-
amide in man. It is also evident that the solution to the physiological problem is
not necessarily realized in terms of approach to equilibrium, but in terms of the
quanta of CO2 released. Cain and Otis’ data (65) show a new steady state after
inhibition, with a large increase in CO2 transport due to nonbicarbonate sources
and with the HCOa- contribution reduced to a rate in the range of the uncatalyzed
reaction. In the second part of this section, these matters are considered anew in
terrns of reaction rates.
Table 17 summarizes the literature on the respiratory effects of carbonic
anhydrase inhibition in both the unanesthetized and anesthetized animal, using a
variety of techniques and species.The changes from the normal are: elevated
venous and capillary pCOz , disequilibrium between CO2 and H&Oa,, lowered
alveolar pC0~ , net CO2 retention, and increased ventilation. The chemical
changesare given in idealized form in Table 18. This model is taken from experi-
ments in which 5-100 mg/kg acetazolamide were given intravenously to the dog.
At the low sideof this range fractional inhibition of erythrocyte carbonic anhydrase
is 0;998, and effects after intravenous injections are maximal for at least 45 min
(495). It is of interest that oral dosage of lo-25 mg/kg produces much lesseffect

TABLE 17. Efects of carbonic anhydrase inhibition on CO2 exchange in blood, tissues, and alveolus
Author and Ref. Species AorN* PACO*p”co, pvCOt PA02 irE vcoz Comment
Roughton et al. (450) Man N 35 .oral SAA 1 During exercise.

Becker et al. (28) Man N 50 oral Failure of effect due to long sam- \i
pl ing periods.
Maren et al. (335) Dog, N 5-100 oral, iv t Measured a - VA for CO2 + pH
rat not altered by inhibition.
Tomashefski et al. (492) Dog A 100 iv 1 t 1 First demonstration of a - A gra-
dientt
Carter and Clark (68) Dog N 100 iv 1 t t Samples of 30 min; no CO2 reten-
tion noted.
Mithoefer (360) Dog A 20 iv 1 t 1 Ventilation controlled in some ex- 9
periments.
Mithoefer and Davis (361), Rat N 50 ip Tissue pouch pCO2. 2z
tt> 0
Cassin et al. (72) %
Pocidalo et al. (404), Amiel Dog A 50 iv 1 t 1 Vent. constant; Vco2 effect lasts 8
et al. (4) 2 hr, elevated a - A gradient
6 hr.$ %
51
Lissac et al. (296) Dog A 50 iv 1 1 Spont. vent. ; ho2 drop lasts 30
min, other effects 2 hr. 3
Pocidalo et al. (403 1 Man N 15 iv 12 oral 1 t Vent. constant. Lower doses inef- E
fectiv& iii
Collier (89) Dog A dose response 1 t 15 mg/kg gave max. response. RBC
concn 90 PM.
Burton et al. (57) Man N 10-20 oral 25- 1 t No effect at oral dose. iv effect
50 iv when RBC concn 200 PM.
Cain and Otis (63, 64) Dog A 50 iv (T‘1 9~02 down only 1 hr in. spont.

breathing. If VIZ is controlled
ho2 .stays down.
Dog A 50 iv 1 t Quantification of CO2 transport.
Travis et al. (494, 495) Dog N dose response 1 t Gradient# at 5 iv acetazolamide
and 10 iv CL 11,366.
-
- Means no change; ( t ) Denotes true PVC-, obtained from tissue pouch (361) or rebreathingbag (64) *‘A = anesthetized, N = not
_.
anesthetized. t Dose is of acetazolamide, unless otherwise noted. $ By gradient is meant apparent alveolar-blood difference of PC@.

in dog (492) and in man (57, 403), even though the drug is well absorbed (492).
This is probably because slower onset of effect permits adjustments (see below)
that are accomplished before critical measurements are made. There is a sugges-
tion that a larger intravenous dose and red cell concentration of acetazolamide are
required in man than in dog for the same respiratory effect (57), and this can
reasonably be related to the higher concentration of enzyme in man (section II, 4;
Table 3).
The basis for and sequence of the model of Table 18 follow: normal capil-
lary = venous plasma values are derived according to equilibria demanded by
p&, of 3.57 and p&l1 of 6.17, when total [COJ is 21.2 m.M (see section n, 2).
Arterial-end pulmonary capillary plasma is shown in the sameway for total [COJ
of 19 mu. A large body of data is available to show that tissue pCO2 tension (40
mm Hg) is normally nearly equal to venous pCO%, and alveolar pCO2 tension
(33 mm Hg) is equal to arterial pCO2 . In the normal there is always a ratio of
400: 1 between [CO21 and [H&08]; this equilibrium (K) demanded by the rate
constants kl and k-1 (II, 2) is achieved instantly in the face of any shift in tissue or
lung, due to the very large concentration of red cell carbonic anhydrase.
Table 18 showsthat after inhibition there is failure of metabolic CO* to reach
equilibrium with H&08 , due to lack of carbonic anhydrase in peripheral capil-
laries. Tissue and capillary CO% are elevated. As blood flows to the heart, CO*
slowly becomes hydrated through the uncatalyzed reaction, but equilibrium is
not reached. When a sample is drawn, equilibrium is gradually achieved in the
syringe, but [CO 2] and [H&08] are still above normal, since no buffers are added
to the “frozen” system. When blood enters the lungs CO2 is releasedto the alveolar
air, but true gaseous[CO21 in arterial blood and alveolus is low becauseof failure
of the dehydration reaction to reach equilibrium and becauseof hyperventilation
due to high tissuepCO2 or [HSCOJ (493). These two factors were dissectedin the
early and important work of Mithoefer (360). As blood flows through the lungs,
the disequilibria increases,since the uncatalyzed rate cannot keep pace with CO2
diffusion. Nevertheless, hyperventilation, the increased gradient of CO2 from
mixed venous blood to alveolus, and other adjustments [increase in participation of
carbamino-CO2 (65)] ensure adequate release of CO2. True arterial blood thus
contains almost the same total [COS] as normal, but it is slightly acidotic due to
excess[H&O*], and equilibrium is not yet restored. As collected in a syringe, meas-
ured arterial blood, like measured venous blood, necessarily shows equilibrium
between gaseous[CO21 and [H&O& with both slightly elevated. Neither arterial
measurement can be regarded as a quantitative reflection of events in lung or tissue.
Alveolar pCO2 is the best and most accurate measurement of red cell carbonic
anhydrase activity or inhibition, since it reflects pulmonary end capillary pCO2 .
On the venous side, there is no comparable sample in the normal. The artifice of
gas tissuepouch (361) or rebreathing bag (64) must be used. Increased pulmonary
ventilation is assumedto be an indirect measureof increased CO%tension in tissues.
Actual description of the immediate environment of respiratory receptors during
inhibition is lacking.
The overall pattern of carbonic anhydrase inhibition is simply one of greatly

TABLE 18. CO2 equilibria in plasma throughout the circulatory system

after carbonic anhydrase inhibition in the dog
HCOa-, HzCOs, coa H+, IM

CtH HaCOa PH
Normal
Capillary and venous 20 1.2 40 3.0 7.39 41
End pulmonary capillary and arte- 18 1.0 33 2.5 7.43 37
rial
Inhibited
Capillary 19 2.2 73 3.2 630 7.34 46
Venous 19.3 1.9 63 3.3 540 7.34 46
Mixed venous 19.6 1.6 53 3.4 7.33 47
Measured venous, in syringe at [19.8 1.4 46 3.5 7.32 481
equilibrium
Beginning pulmonary capillary 19.6 0.8 27 3.4 235 7.33 47
End pulmonary capillary 18.5 0.5 17 3.2 156 7.33 47
Large artery 18.2 0.8 27 3.0 266 7.35 45
Measured arterial, in syringe at [17.9 1.1 37 2.8 7.38 421
equilibrium
The model is derived mainly from work with the anesthetized animal, which somewhat
exaggerates CO2 retention on the venous side. Data and references given in Table 17.
increased gradients of CO2 from tissue to alveolus, the former being abnormally
high and the latter abnormally low. It is probable that all the sequelaeof red cell
carbonic anhydrase inhibition can be derived from this pattern. For example, the
elevation of CSF pressure after intravenous acetazolamide results from CO2 ac-
cumulation in brain tissue (reviewed in ref. 332), whereas the increase in exercise
tolerance at altitude (72) can reasonably be explained by the increase in alveolar
~02 that follows the drop in alveolar pCOz (68; and other data of Table 17).
Several additional problems may be briefly discussed.It is not entirely clear
whether the situation of Table 18 persistsas long asred cell carbonic anhydrase is
inhibited or whether it only represents an initial new steady state. Reduction in
pulmonary CO2 output, when it does occur, lasts but 15-30 min after 50-100
mg/kg iv acetazolamide in spontaneously breathing animals (63, 474), since
hyperventilation ensuesand elicits normal CO2 output even at very low alveolar
pCOz (63, 68). In the free-breathing anesthetized dog, true mixed venous pCO2
continues to be elevated 120 min after drug administration (64). Carbonic anhy-
drase inhibition, after large dosesof acetazolamide in the normal dog for 8 hr,
elicits a continued rise in ventilation and in measured venous pCO2 and a drop in
alveolar pCO8 (494). Similar inhibition for months produces a small rise in venous
pCOz in the dog (335) but not in normal children (44); it is, however, implicit in
Table 18 that a normal measured venous pCO2 reveals little about the situation in
capillaries or tissue after carbonic anhydrase inhibition. In general, then, the data
of Table 18 seem to apply to continued carbonic anhydrase inhibition, but one
important reservation must be made. This is at the tissuelevel, where gas pouch
tensionsof CO* appear to decline 60 min after 50-100 mg/kg ip of acetazolamide

684 T. H. MAREN Vollmle 47
in the normal rat (72, 361). Further documentation of this effect, coupled with
measurementsof inhibition in the red cell, should be made. A second effect, which
may directly reflect tissue pCOz , is also transitory; this is the elevation of CSF
pressure after intravenous acetazolamide or methazolamide in man (93, 332),
monkey (lo), and dog (526); inexplicably it does not occur in the rabbit (496) or
cat (254). The effect lasts but 30-60 min, even after as much as 80 mg/kg. Either
brain pC0~ becomeslowered in this time in the face of maximal red cell inhibition,
or the vesselsthat initially dilate in responseto CO2 become refractory to the gas or
other volume adjustments take place. This alsorequires further study. In summary,
the unsolved issuehere is whether the initial CO2 retention (shown in Table 18 as
capillary and hence tissuepCO* twice normal) persistsor is qualitatively or quanti-
tatively modified with time.
Although changes in acid-base balance may mimic or oppose those of car-
bonic anhydrase inhibition in other tissues(seekidney and pancreas), such varia-
tions do not seemto alter greatly the effects of acetazolamide on red cells in widen-
ing the apparent Pa - PA gradient for CO2 in the anesthetized dog (402). This
supports the idea that when the effect of acetazolamide (or other inhibitors) is
reduced in vivo, it is not due to any change in drug enzyme interactions, but to
change in local secretory events that reduce the participation of carbonic anhydrase
in the overall secretory scheme.
A number of studieshave been concerned with the contributions of the several
forms of CO2 to the pulmonary releaseof CO2 gas, transit time of HC03, and the
possiblerole of enzyme in the lung. Widely differing techniques have been em-
ployed. Chinard et al. (76) used isotopically labeled CO2 and HCOao and deter-
mined their blood transit time through the lung to CO2 in the normal dog and
after carbonic anhydrase inhibition. It is clear that in the latter situation equilibra-
tion is not complete during transit. A corollary of theseexperiments is that HCO,
as such does not leave the confines of the vascular system in the lung.
Using plethysmographic criteria for CO2 evolution in ventilated anesthetized
dogs, Soni et al. (473) compared rates after injection into the pulmonary artery of
NaHCOs , lactic acid, and tris buffer. In other experiments carbonic anhydrase
and acetazolamide (5 mg/kg) were added. Comparison was made with the transit
time of an inert substance (ether) through the pulmonary tree. It was concluded
that the reactions involved in the movement of CO2 acrossthe alveolar-capillary
membrane normally reach completion within the time HCO,- spendsin the tissues
of gasexchange. In further studies (155) this time was found to be 2.2 set, consid-
erably longer than ether transit time, presumably becauseof the larger volume of
distribution for HCOa- and CO2 within the lung. When acetazolamide is given in
submaximal doses(2 mg/kg), the rate of CO2 evolution from HCOS- is decreased,
but is neverthelesscomplete by 2.2 sec. At 4-10 mg/kg evolution is not complete;
this dose range yields essentially complete inhibition of red cell enzyme in this
species(495). Thus, it is demonstrated in still another fashion that acetazolamide
causesimmediate CO2 retention in the absence of hyperventilation. Expansion of
the total CO2 spacein the lungs, as observed by Feisal et al. (155), must occur by
catalytic dehydration of HCOa” in the red cells and catalytic hydration of CO2

in the lung tissue. As a result of this extracapillary route, injected HCO,- ions
spend 2-3 set in contact with gas exchange surface, about 3 times longer than
erythrocyte transit through the capillaries. Presumably, this extracapillary route
would not be available in the absenceof red cell and lung enzyme, since HCOa
as such is confined to the vascular bed; Chinard et al. (76) have shown that transit
time of HCOa (from pulmonary to carotid artery) is decreased after acetazol-
amide. Chinard et al. (77) went on to show directly that HC03 injected into the
trachea was rapidly converted to expired CO2 , and this was slowed by acetazol-
amide. Clearly lung carbonic anhydrase exists, but its role is not defined. Perhaps
expansion of the HCO a- spaceis functionally desirable; perhaps enzyme has some
role in the secretion of certain ions (including H+) by the tracheobronchial mucosa.
These, together with the unexplained fact of accumulation by the lung of the
anionic inhibitor CL 11,366 (494), are matters for further speculation and study.
Chinard et al. (76) also made the significant finding that acetazolamide (20
mg/kg iv) reduced the incorporation of l*O in water into expired CO2 , but only
by lo-fold. The authors interpreted this to mean that, in the normal state, the
enzyme is not rate limiting and that under normal conditions there is a margin of
loo-fold or more over minimum requirements. The normal rate is thus much slower
than the catalytic potential; this is unquestionably the case also for kidney and
pancreas (320). We will return to this important point later on in connection with
enzymic rates and inhibition kinetics.
The means of adjustment to CO2 retention is suggestedby Cain and Otis
(65) in their study discussedabove, which gives the normal contribution of dissolved
COa (19 %), carbamino-CO2 (26 %), and HCOa (55 %) to pulmonary CO2
output. After acetazolamide (50 mg/kg iv) in the anesthetized dog, vco, dropped
sharply but returned to normal in 30-60 min. At that time, the partition of pul-
monary CO* changed from the above values to: dissolved CO2 (45 %), carbamino
(38 %), and bicarbonate (17 %). The new, high levels of pCOz (80 mm Hg) and
carbamino-CO2 insure a net gradient of gas from tissue to alveolus, while the
decreasein the HCOa component now places its contribution within the range of
the uncatalyzed reaction. Returning to our earlier discussion,the time to unload
the requisite amount of HC03 via the uncatalyzed reaction was calculated as
5-12 set or perhaps less (65), while 1 set is transit time through the capillaries
(473). The CO2 partition values of Cain and Otis (65) must be regarded as tenta-
tive, for they are essentially calculated values. Their figure for dissolved CO2 is
based on direct measurement, carbamino-COP by calculation from CO2 pressures,
and HCOa- contribution by difference. In the isotope study of Chinard et al. (76),
the increase in contribution from dissolved CO2 after carbonic anhydrase inhibi-
tion was confirmed, but not the decreasein HCOa- fraction. Despite these uncer-
tainties, the work of Cain and Otis (65) yields a reasonablepattern for CO2 trans-
port after carbonic anhydrase inhibition, a pattern that may be subjected to
further inspection.
The effect of carbonic anhydrase inhibition on exercise tolerance has received
relatively little attention. Roughton (450) found that sulfanilamide (0.2 mu in
the blood) decreasedCO2 output during strenuous exercise. Unpleasant subjective

effects were also observed. No ventilatory or subjective signswere seenat rest or

moderate exercise. He calculated the fractional inhibition of blood enzyme to be
0.99. The implication of thesestudieswas that carbonic anhydrase inhibition would
be a considerable handicap in exercise. In rats trained to run on a treadmill, 100
mg/kg ip of acetazolamide had no effect on performance at sealevel, but increased
their running ability at simulated high altitude (72). The adjustments to the greatly
increased load of CO2 , when the enzyme is inhibited, are not presently understood.
Altitude tolerance is clearly increased by carbonic anhydrase inhibition. Law-
son (278) showed that rabbits treated with sulfanilamide (blood level about 1
mM) could tolerate an additional 3000 ft when decompressedat a rate equivalent
to 1000 ft/min. These data are rather difficult to interpret in the light of present
knowledge. There were no certain changes in blood oxygen; ‘and sulfapyridine,
which is not a carbonic anhydrase inhibitor, caused changessimilar to those after
sulfanilamide. An interesting light is cast on the radical implications of the appar-
ent alveolar-arterial pCO2 gradient [first described by Tomashefski et al. 10 years
later (492)] by Lawson’scomment that if such an expanded gradient was observed,
the whole experiment was discarded. Carter and Clark (69) and Tomashefski et al.
(492) found that arterial OS at low ambient oxygen was increased about 7 mm Hg
after 100 mg/kg acetazolamide in dogs. 9~ was consistently increased. This follows
the pattern of Table 17 for experiments done at sealevel. The main basisfor the
acetazolamide effect appears to be an increase in body CO* storage, which effec-
tively counters the usual elimination of CO%during hypoxia. Increased ventilation
plus low alveolar pCO2 conspire to raise ~02. More recently, Cain and Dunn
(61) decompressedunanesthetized dogs to 335 mm Hg (equivalent to 21,000 ft).
Treated animals received 10 mg/kg iv of acetazolamide 27, 15, and 3 hr before
decompression.At those times arterial ~02 was about 15 mm Hg above normal;
during 8 hr of decompressionarterial ~02 was 9 mm above normal; equivalent to
a gain of 5000 ft. Another improvement was the absenceof excesslactate that was
observed in controls. Similar experiments in man yielded results in the samegen-
eral direction (62). Since the dosage schedule used yielded a frank metabolic
acidosisat the time of the test (323), it is impossibleto say to what extent the in-
creasein ~02, secondary to increased alveolar ventilation, is due to the respiratory
component of the acidosis. Unquestionably this is a large factor, since there is also
increased ~02 in the acute experiments of Table 17 where there is inadequate time
for baseloss.The role of metabolic acidosiscould readily be evaluated by repeating
the protocol of Cain using CL 11,366.
Hypoxia was studied in another context by Weinstein and colleagues (3 12,
511). They showed that 5-8 % 02 increased the latent period in a learned motor
responsein mice; the responsewas avoidance of 6 % CO2 . When CL 11,366 (2
mg/kg ip) was given 2 hr before the test, the latency period was reduced at all
oxygen levels. It was then demonstrated in rats that the drug prevents the develop-
ment of respiratory alkalosis, which usually attends hypoxia. At 2 mg/kg this is
entirely due to its renal effect. These experiments, together with those of Cain (61)
and Cassinet al. (72), suggeststrongly that both the respiratory and the metabolic
acidosis of carbonic anhydrase inhibition may provide a useful pathway to in-

creased oxygenation during hypoxia. Factors responsible may include increased

ventilation, increased cerebral blood flow, and increased dissociation of 02 from
oxyhemoglobin in the tissues.
Several new, and to this writer tentative, observations on the red cell or respira-
tory function of carbonic anhydrase are worth mentioning. Enns (147) studied
transport of CO2 in millipore filter disks and showed that carbonic anhydrase
facilitated its diffusion-not only by its effect on equilibria with HCOa- (299)
but, at pH 5.2, by acting as a CO2 carrier. The effect was not large; its significance
must await further data. Another unexpected finding was that of carbonic anhy-
drase in the carotid body (281). Glomus cells were estimated to have about the
same enzyme concentration as renal tubules. If this is real and not due to red cell
contamination (the concentration reported was about s that of red cells), it opens
up an aspect of the problem susceptibleto critical experimentation. In preliminary
experiments, Travis (493) has made the important observation that the carotid
body responseto CO* is in reality a responseto H&OS. CO2 may be itself inert,
but whether hydration occurs in red cells or carotid has not yet been determined.
2) CL~mistry: rates and ewyme inhibition. This part deals with a) the relation
between physiological rates of HCOa- dehydration and those of the catalyzed and
uncatalyzed reactions, and b) the relation between enzyme inhibition and physio-
logical response.
a) The amount of enzyme in bovine red cells will increase the uncatalyzed
rate 13,000 fold, i.e. Vcst/V”nc = 13,000 (245). Since [E,]/e.u. for whole bovine
cells is about the same as for dog and man (Table 3), we need not be concerned
with speciesdifferences or problems of differing chemistry of isoenzymes, at least
for these fist approximations. What is the relation between I&, I&, and usual
physiological requirements, which we may call V&, 3 VUncis calculated as follows,
first for the contribution in the normal state, when there is enzyme present, and
then during inhibition.
V UUO = k-1. [H&OS] - kl. [CO21 WI
k-1 is 13.7 set-1 at 25 C (173) and we may take it as 30 set-l at 37 C. kr has been
determined directly at 37 C at 0.045 sec.l, but in the present context it is essential
to preserve the ratio k-&i = 400, so kl is used again by extrapolation from 25 C
(173), yielding 0.075 set-1. [H&OJ is taken from the first line and [COJ from the
second line of Table 18. Then
V uno = ((30 sec.l.3 pnt) - (0.075 sec-1400 PM)) 60 sec/min
= 900 pba/min (15)
For the actual value we must know the volume of reacting fluid. Pulmonary capil-
lary volume in man is about 80 ml, and we will use 20 ml for the dog. Of this 80 %
is in solution, yielding 16 ml. Effective exchange volume in the lung is 3 times
capillary volume (155), so it is estimated at 50 ml. VUncis then 45 pmoles/min in
the normal state.
Vuncduring inhibition, however, must take into account the seriousperturba-
tions recorded in Table 17 and put into the numerical model of Table 18. We use

the same constants as equation 14, but [H&03] is now 3.4 PM and [CO,] is 500 JUM,
whence VUno= 195 E.cmoles/min,without carbonic anhydrase.
VR, the potential enzymic rate, is simply the rate obtained from equation 15
times 13,000, 12 M/min or 600,000 I.cmoles/minfor the 50.ml volume reacting.
How do these calculated rates compare with observed rates in vivo? The rate
of CO2 release (Voba)in the resting dog is about 50 ml or 2200 pmoles/min. Some
55 % of this (1200 pmoles/min) is normally derived from HCOSu (65), whence
Vobs cat should be 1200 - 45 = 1155pmoles/min. Enzyme, thus, is found to be in
520-fold excessof actual needsand can never be a limiting property. In the normal
state, the contribution of l&c is negligible-49<2oo or 3.7 %.
During inhibition the situation is quantitatively altered. Due to the low alveo-
lar pCO2 the net rate of dehydration is increased fourfold, but is still only 195
pmoles/min. If CO2 releaseis unchanged, this is 9 % of total output of 2200 pmoles/
min. Cain and Otis found 17%, but this may be regarded asreasonably closesince
their value was arrived at by difference after partitioning of CO2 among the vari-
ous forms. Thus, if the carbamino contribution, which involved someassumptions,
was increased after inhibition from 26 % (in the normal) to 46 % (rather than 38 %,
as calculated) their value for the HCOa- contribution would be, like ours, 9 %.
Finally, these figures should make it clear that with the profound drop from an
almost unlimited rate of HCOa dehydration to 195 pmoles/min, failure of rapid
adjustments should inevitably lead to CO2 retention.
In an interesting and neglected experiment by Anderson and Thompson
almost 20 years ago (5), drops of whole human blood were allowed to fall through
tubes 8-30 ft long and the decrease in total CO%measured. Since the time of
transit was accurately measured, the rate of evolution of CO2 can be calculated,
and is equivalent to vobs = 36,000 pM/min. This figure is very much lessthan
VE , the catalytic potential of the system, calculated above as 12 M/min, even with
the back reaction taken into account. But it is remarkably close to the observed
unloading of HCO, in the lungs, 2200 pmoles/min in the dog with a 50-ml reac-
tion volume, or 44,000 PM/mm. (It will be recalled from section II that tempera-
ture has little effect on the catalyzed rate, so we would not expect much influence
f’rom the presumed 18 C conditions of the in vitro experiment.) This suggestsagain
that enzyme is not rate limiting, and perhaps the geometry and membranes of the
red cell impose a limitation on CO2 release over the physiological limitations im-
posed by lung transit time. In the Anderson-Thompson model,
V obs = 36,000 pM/min = vOb8 cst -f- Vunc + other processes WI
Vobscat may be obtained by finding Vobsbefore and after inhibition; fortunately

the authors (5) used a final blood concentration of 3 mM sulfanilamide, which
inhibits 99.9 % enzyme. The rate after inhibition, Vinh , was 6000 PM/mm, yield-
ing vobs cat = 30,000 pM/min. Vinh may be further inspected:
V- mh = 6000 pnd/min = Vunc + other processes (17)
We can test whether “other processes”are operative, since Vunc = k-1 [H&03],
now unopposed by the back reaction. At 18 C, k-1 is approximately 8 see-1 (ex-

trapolating from the 25 C value used in II, 2 and the Qlo of 2 for V”,,) whence for
[H&OS] of3 PM, Knc = 1400&min. We can now insert the numbers in equations
16 and 17: in pM/min, VObscat = 30,000; yUnc= 1400; other processes,presumably
carbamino and dissolved CO%, 4600. These relationships are roughly analogous to
those obtained in vivo; the correspondence for r/7unc/l?&,at about 4% is particu-
larly close.
The most sophisticated attempts to measure CO2 exchanges in human red
cells directly are those of Constantine et al. (92). They used a Hartridge-Roughton
continuous-flow, rapid-reaction apparatus, in which induced changes of pCO2 in
red cells at 37 C were measured by a glasselectrode sensedby a vibrating reed
electrometer. Their important findings are summarized. Depending on conditions,
the apparent hydration constantsfor catalysis were 236 set-1 and 772 se@. Taking
from literature the extrapolated kl value of 0.11 set+ for 37 C, they computed the
relationship between Vcat/V’uncas 2000 - 7000. If they had used Maren’s meas-
ured value (320) of Q.043 se& for k 1, they would have neatly encompassedKerno-
han’s (245) value of 13,000 (obtained from ox red cells) for Vcat/Vunc in hemoly-
sates.Thus the apparent greater activity of hemolysatesover red cells, to which con-
siderable discussionis given, is probably not real (see also ref. 246). The reviewer,
therefore, believes that the catalytic potential of the enzyme can be measured in
this system. Constantine et al. (92) reported k-1 at 37 C as 83 se@, which is at
somevariance with recent 25 C data (173, 2 11); if this is the true value, it means
that the solution for t in equation 13, using the data of Cain and Otis (65), yields
about 5 set, which is only twice transit time. Added under proper conditions of
incubation, 1 mM acetazolamide, which should inhibit 99.995 % enzyme present,
only reduced the apparent rate constant 90 %. This can only mean that other sub-
stanceswithin the cell are capable of carrying CO 2 . This result and this conclusion
are pleasingly closeto those given above for the Anderson-Thompson experiment.
The question of whether carbonic anhydrase in red cells has activity directly
proportional to that in dilute hemolysateswas first raised by Roughton (444) and
now has been settled (246) by Kernohan and Roughton 31 years later. Using the
method of rapid thermal reaction for intact cells, it was possibleto show that ob-
served enzymic activity in the red cells agreesto within 10 % of that calculated from
the activity of cells after hemolysis, dilutiop, and assayby a chemical method. V’ ,
in the present usage,was thus equivalent to Scat .
Enzyme kinetics in the intact red cell in vitro have also been studied by the
hemolysis method of Jacobs and Stewart (227) described in u, 1. The data (340)
provide a useful model in showing again that the true I& is extremely rapid-
approaching that of ionic reactions- and can never be rate limiting in the physio-
logical milieu. Since the actual VE is never achieved (in this model or in vivo), it
is clear why enzyme inhibition to 90 or 99 % may not be recognized. As an example
(340), the theoretical time for the catalyzed reaction is 0.1 set; observed time is 20
sec.Only a small fraction of enzyme is invoked in this case,sinceother factors limit
the observed rate. When enzyme is completely inhibited, however, the rate of
CO2 hydration doesbecome limiting and the observed time is greatly prolonged, to
1800 sec. When enzyme inhibition is studied from 20 to 1800 set, the calculated

values of i are from 0.995 to 0.9999+; this provides an unusual opportunity to

make accurate observations of inhibition far from the usual 50 % point.
b) The relation between enzyme inhibition in red cells and physiological
response, according to the criteria of Table 18, has been critically evaluated by
Travis et al. (495). Fractional inhibition is calculated as discussedin section II,
i = [1J/([l,] + &). [ZJ istaken from the unbound plasma concentration when it is
known to be readily diffusible into red cells, or from that concentration of drug
in red cells that is readily washed out or exchangeable. It may be shown for any
carbonic anhydrase inhibitor that the respiratory effect (widening of the blood
alveolar pC0~ gradient) does not occur unless i = 0.998 or more. For acetazol-
amide this threshold correspondsto 5 mg/kg iv in the dog. These data yield another
approach to the question of “excess” enzyme discussedabove. There it was found,
from rate data, that the excess( V,/K,aa cat) was 520; now from inhibition data it
also appears to be about XX&fold since 1 - 0.998 = 0.002 parts of enzyme present,
which is enough to maintain normal function. It is shown in succeeding sections
that the same approximate relation holds for other organs as well.
3) Birds and&/z. In birds there is also an elevation of venous pCOz after car-
bonic anhydrase inhibition (376). The rise is perhaps greater than in the dog,
measured values without anesthesiareaching about 60 mm Hg.
In fish the elevation of peripheral blood pC0~ after inhibition is very great.
Normal value is about 4 mm Hg; this is the gradient acrossthe gill, since pCO*
in the ocean is negligible, and corresponds to the A-V difference in mammals.
Carbonic anhydrase inhibition raisesthis to about 15 mm Hg in teleost and elasmo-
branch (2 13,3 19). The situation is not fundamentally different from the model of
Table 18; there is the significant variation that fish probably do not hyperventilate,
thus they retain CO2 during inhibition. A single doseof acetazolamide (30 mg/kg
iv) lastsfor a week in S. acantllias,during which time the fish appear well and active
despite the fourfold elevation in pC0 2 , .which, by day 2-3, results in compensatory
increase in total CO2 (3 19). By the use of 1 mg/kg of CL 11,366, which inhibits
the small concentration of red cell enzyme in S. U&&S but lacks accessto gill
tissue, it was possibleto show that the elevation of pCO2 and total CO2 is entirely
due to the red cell effect (327).
It should be mentioned, as a matte; for further exploration, that the carbonic
anhydrase activity of fish blood is about 2 % that of mammals. In the absenceof
definitive data relating this to function, it would be rash to say certainly, as we
have done for carbonic anhydrase in other sites and species,that enzyme is in
excess, It is conceivable, although not likely, that the yet unexplained gradient
between pCO2 of fish blood and ocean is maintained in part by a limitation on rate
of equilibrium achievement between CO2 and HCOa-.
B. Kidney
A major proportion of work on this enzyme has centered on the kidney; events
in this field have been of considerable theoretical and practical importance. Ber-
liner and Orloff have critically and extensively reviewed the literature through

1955 (37); the present author has reviewed renal work on the mammal, with
particular emphasis on pharmacological aspects and the effects of carbonic an-
hydrase inhibition on the excretory patterns of drugs (323). Here we follow the
scheme used in the respiratory. part above : 1) physiology in the mammal with a
note on phylogeny leading to this pattern; 2) chemical kinetics and fractional
inhibition in the mammal in vivo; 3) comparative aspects, emphasizing the fact
that renal carbonic anhydrase inhibition can produce an alkaline, acid, or unal-
tered urinary pattern, depending on the species.
1) Physiology in the mammal: whole-animal studies. There appears to be a direct
phylogenetic line in which renal acidification is partially dependent upon carbonic
anhydrase. In the following animals, acetazolamide produces an, alkaline urine:
fresh-water teleosts, exemplified by Ameirus nebulosus (2 13); frogs, exemplified by
the pioneering study of Hiiber in Rana pz$Gens (2 12) and the later quantitative work
of Yoshimura et al. (538) in the bullfrog, R. catesbiana; birds, as indicated by Wol-
bath’s (533) work in the chicken; and all mammals thus far studied (323). These
speciesare characterized also by flexibility of urinary pH in responseto buffer or
salt loading. Thus, all of the vertebrate classesare represented for this function
except ReptiZia; alligators have a different role for renal carbonic anhydrase (see
below), but it is possiblethat snakesand turtles have the acidification mechanism.
This remains to be investigated. A phylogenetic tree for this function hasjust been
published (322a).
In the speciesjust discussed,acetazolamide produces the general pattern of
renal excretion of HCOa-, Na+, K+, and osmotically obligated Hz0 (Fig. 2, Table
19). Nechay and Sanner (377) made a study of Na+/K+ ratios in the chicken, in-
cluding the effect of carbonic anhydrase inhibition in the reserpinized animal. This
latter procedure had the effect of increasing the ratio; the effect is unexplained but
illustrates the typical situation in which (Naf + K-t) and HCOS- output are quite
stereotyped, but the individual cations may change in responseto other stimuli. A
major effect of acetazolamide in these speciesis to lower urinary H+ and NH,+; an
exception is the lack of NH,+ responsein the frog (538). Other ions are generally
unaffected by carbonic anhydrase inhibition (Tables 19, 20).
It cannot be stated too strongly that renal carbonic anhydrase inhibition in
the mammal and bird is immediately recognizable by the pattern of urinary re-
sponse,the main criterion being increase in HCO,- output. All known inhibitors,
if given in the proper dose, produce this effect. The inhibitors, ethoxzolamide,
CL 11,366, methazolamide, and acetazolamide have been compared quantita-
tively; all produce the.same HCOa- output at a certain maximal dose,which differs
according to activity and pharmacological disposition of each drug (323). It is
emphasized that, despite the widely differing chemical and physical properties
of these drugs (II, 7) and their different means of accessto tubule cells (diffusion,
secretion, reabsorption), their renal effects are indistinguishable. Unless sulfon-
amideshave someancillary function [thiazides and congeners, and 1,3-disulfon-
amides (41)] they do not produce an increase in renal Cl- output, no matter what
physical or chemical property they may possess. No monosulfonamide of any type
or potency (II, 5 and II, s> has ever been shown to produce a chloruretic effect.

BLOOD
DRUG DAY ONLY
.
PLASMA HCOf AND pH _ 1.40
‘” v~” DAMOX IN BLOOD AND URINE Ly
11 \ L 1 /rCb I A IIAm *
wC~I~JL UAT5 (X-X) AND LWUG PAY (-1
HOURS
FIG. 2. Pharmacologic and renal effects of a single dose of acetazolamide in the dog (5
mg/kg orally at arrow). For urine X-----X is average of 2 control days, O--O is drug day.
Dog was fed at 0 time, 9 AM. @?rom Maren et al. (335).]
The renal action of acetazolamide in the dog, with respect to the time sequence
of changes, is shown in Figure 2; as in all other physiological systems,the effects
are reversible. In the caseof acetazolamide, they last while plasma concentrations
of drug exceed 2 pg/ml (335), which was later shown to yield sufficient [If] in the
kidney to sustain full effects of inhibition (321). When the plasma and renal con-
centration falls below these levels, reacidification of the urine occurs and the base
deficit is made up. In the dog, it takes about 3 times aslong to restore a basedeficit

TABLE 19. Efect of carbonic anhydrase inhibition on renal electrolytes

in the normal dog and in acute metabolic acidosis (314)
Plasma Urine, peq/min Cre;rGne

Time, min
pCO2, HCOs-, Cl-, pH Na+ K+ H+ NHI+ HCO3- Cl- POI- mlzi:
PH mmHg mu mu
-P-----P---
Normal
-20 to 0 7.34 40 21 96 6.9 8 18 1 3 6 8 5 38
5 mg/kg iv acetazolamide at 0 time
O-20 17.36 1 38 1 21 1 96 1 7.8 1 70 1 100 1 0 1 0 116 1 18 1 5 1 35
1.2 meq/kg oral NH&l every 20 min X 7, from 40 to 160 min
180-200 17.12 ( 36 1 11 1 116 1 5.2 1 28 1 70 ( 12 1 10 0 11201 3 1 37
5 mg/kg iv acetazolamide at 200 min
200-220 17.15 / 31 1 11 / 117 j 7.5 1 40 / 140 1 0 1 0 42 j 125 / 2 j 38
Trained female beagle, wt 11.5 kg. Fasted on day of test. Urine flow approx. 1 ml/min.
The dose of acetazolamide, if not interrupted by NH&I, would produce an alkaline urine
for about 5 hr. (See Fig. 2.)
TABLE 20. Effect of carbonic anhydrase inhibition during maximum bu$er loading in
the normal dog and during metabolic acidosis (337)
Plasma Urine, peq/min Cre;rGne

Urine
Time, min
pH
1Hco3-, m 1 po4'
m, ml/An
F1ow PH. 1 Na+ 1 K+ / H+ /HCO3-1 PO,- 1 Cl- rng;
At -180 to - 120 min: 20 mg/kg iv thiopental. Inject 18 ml of 5% creatinine iv. Infuse

5% dextrose with 0.15% creatinine at 10 ml/min. Wt 18 kg.
-100 to -80 1 7.21 1 23 1 1.7 1 5.4 1 5.8 1 8 1 4 1 6 1 1 10 1 18 1 45
-80 Add phosphate to infusion: 38 mM NaH2P04 + 92 mM Na2HPOJ

-20 to 0 1 7.24 1 24 1 10.4 1 10 1 6.3 1 578 1 100 1 306 1 18 498 1 18 1 44
0 20 mg/kg acetazolamide iv
O-20 17.231 23 112 ) 13 1 7.1 11181 ) 169 1 52 1 340 1 545 / 17 ) 42
NH&l by mouth started on previous day. 10 meq/kg given from -6 to -4 hr. From
- 160 to - 100 min: thiopental and creatinine as above. Wt 17.6 kg.
-90 to -60 I 6.96 1 10 1 1.4 / 9 I 4.9 l 77 58 1 42 j 0 1 15 1 173 1 41
-60 Add phosphate to infusion as above

-20 to 0 16.99 1 11 1 11 1 9 5.7 1 527 75 1 341 1 14 1 544 1 50 1 42
0 20 mg/kg acetazolamide iv
O-20 1 7.00 1 12 1 13 1 10 1 6.8 I1008 1551 40) 65152211151 38

as to produce it by carbonic anhydrase inhibition. Specifically, if the renal effect

of acetazolamide lasts 6 hr (as after 5-10 mg/kg), an acidosis will ensue that will
be corrected in the next 18 hr. It is becauseof theserelations that 5 mg/kg per day
of drug in dog does not elicit chronic acidosis; but higher and more frequent doses
lead to around-the-clock lowering of plasma [HCO,-1 and pH (335). Temporal
relations within this cycle are different for man becauseof slower activation of the
NH&secreting mechanism. As long as 3-7 days may be required for recovery
after a single dose of acetazolamide (150).
The fundamental carbonic-anhydrase-dependent step in the normal mammal,
bird, amphibian, and fresh-water teleost is the urinary production of acid from
water and CO2 within the cells of both proximal and distal tubule. Enzyme is
present in both segments(409). Renal cells may be regarded as specialized for the
separation of H+ and OH- ions; this will in fact be shown to be a general property
of many secretory organs. Figure 3 showsthat OH- is buffered by CO%and passes
into the blood, while H+ is secreted at the luminal border. In the proximal tubule
this subservesthe reabsorption of about 30 % of filtered HCOs’-, after its luminal
conversion to H&O 8 + CO2 . This latter equilibrium is considered below in con-
nection with micropuncture and other work that attempts to localize further the
H+ secretory function in the kidney. From 10 to 20 % of filtered HCO,- may escape
into the distal tubule. The remaining large fraction (about 60 %) appears to be
reabsorbed proximally by a mechanism apart from the primary production of
H+-in this writer’s view, active reabsorption of HCOs ion. Of the filtered HCO,-
that is proximally reabsorbed by reason of H+ secretion dependent on CO%hydra-
tion, about s may be assignedto the uncatalyzed and s to the catalyzed reaction
(seenext section on chemical kinetics). It is this latter portion, about 20 % of fil-
tered HCOa-, that appears in the urine of the normal dog after maximal carbonic
anhydrase inhibition (Table 2 1, row A). Under special conditions of increasedflow
or buffer load, this figure may approach 40 % (Table 21, row B). Na+ is the chief
cation accompanying HCOa- as it leaves the proximal segment during inhibition.
In the distal tubule, H+ secretion from the same source, CO2 + Hz0 e
H&O8 , chiefly subservesthe output of acid and NHA+ (Fig. 4). Enzyme inhibition
reducesthese to virtually zero. In this region, H+ and K+ secretion share a common
mechanism, so that when one is reduced, the other is generally increased (36).
Thus, KS- output is increasedin the alkaline urine of carbonic anhydrase inhibition.
The anatomical separation of HCOa- reabsorption and H+ output is not absolute,
since the former function also occurs in the distal tubule and the latter (including
NHd+ formation) takes place in the proximal segment. The matter is discussed
more fully in reference 323. The functional distinction between proximal and distal
tubule in the present context (i.e. between HCO,- reabsorption and H+ secretion)
is important and is shown by the evidence to follow.
Insight into the renal role of carbonic anhydrase is afforded by studies of the
effect of acetazolamide in acidosis. Regardlessof whether acidosiswas caused by
administration of acid (Table 19), or CO2 (49), or continuous useof acetazolamide
(Fig. 5), or the intracellular acidosisof K+ depletion (334), the further administra-
tion of acetazolamide causeslittle increase of HCOS- in the urine. These various

Ocfobm 3967 CARBONIC ANHYDRASE 695
Proximal Tubule
Lumen Cell Plasma

+ 52mV 0 mV ‘2 mV
NO+
(800
fib.
HCOi (465)
HCO; (235)
co2
(100) HCO; to distal tubule

FIG. 3. A conception of the CO, + H+ system in the proximal tubule. Numbers in paren-
theses are rates in ~oks/min. Circular arrows show active processes. Dotted lines show lesser
degrees of certainty. The model is taken fkom dog data: 800 pmoles/min of HCOa- are filtered
and are disposed of in the various ways indicated. V oat in me is observed catalytic rate, denoted
VObr oat in text. Processes that tiy also occur in this region such as H+ and NH*+ output are
neglected for the sake of exposition of the fate of HCOI-. (See ref. 323 for data and discussion.)
situations are illustrated in Table 2 1, rows C-G. The explanation advanced is

that in acidosisthe reabsorption of ionic HCOa- per se is activated, lesseningthe
role of proximal carbonic anhydrase, or indeed of CO2 hydration. Becauseof this
phenomenon, there is no progressive loss of HCOa- when inhibition is continued
(335). The initial acidosisof HCOa- lossis sustained, but not increased. This very
important point is frequently misunderstood. It is for this reason that continuous
carbonic anhydrase inhibition (as in the treatment of glaucoma) is not toxic, and
this certainly setsit apart from situations of progressive baseloss,asin renal disease
or renal tubular acidosis.
On the other hand, the distal carbonic anhydrase function, H+ and NH4+
output, is repressed-by acetazolamide equally in normal acid-base balance and in
acidosis.This is illustrated by Table 20, which showsexperiments carried out under
conditions of maximal H+ secretion due to buffer loading. As under the normal
(non-buffer-loaded) conditions of Table 19, acidosisvirtually abolished the HCO,
diuresis due to acetazolamide. However, acidosisdid not alter the effect of aceta-
zolamide in reducing H+ output. The clean-cut difference can be explained by the
idea, noted above, that in acidosisproximal HCOa- reabsorption per seis activated;
but there is no auxilliary distal mechanism for H+ secretion independent of car-
bonic anhydrase.

TABLE 2 1. Catalyzed and uncatalyzed rates of COz hydration in renal acidiJication*
=:
I I I I I I I I I I I I
1 2 3 4 5 6 7 8 9
I I I I I I
’ I
Type Experiment and Reference Plasma Urine Out side
Total CO2-4-P
vobs cat
HCOi from col. 10 System, col.
HCOS-, PH Flow, 10 - (ll+ 12)
mu co2* mM (pK=6.17) ml/min Filt. 1 Reabs.
. - -- .-
pmoles/min
.4. Normal (335) 20 1.2 7.39 0.2 6.1 800 800 13 820 100 170 550
12 21 67
+C.A.I. (no infusion) 20 1.4 7.31 1.0 8.1 800 650 0 650 116 0 534
18 82
B. Normal (36) 23 1.2 7.45 1.2 7.1 920 910 10 960 100 290 570
9
11 30 59
+ C.A.I. (slow phosphate 23 1.4 7.38 3.0 7.8 920 670 0 670 116 0 554 l
z
infusion) 17 83 ‘(
C. NH&l, acute, 12 meq/ 10 0.8 7.27 0.4 5.6 4-Q 67 20 373 $
kg W) 14 5 81
+C.A.I. 11 1.4 7.10 10 0 324 !z
1.5 7.2 425 116
1
26 74
D. NaHsP04 infusion (49) 12 1.2 7.20 6.0 6.1 480 475 60 935 100 375 460
11 40 49
+C.A.I. 14 1.6 7.12 7.5 6.8 560 470 20 560 133 0 427
24 76
E. COZ, acute (15%) (49) 24 3.8 6.97 10 6.3 960 920 13 940 316 15 609
29 6 65
+C.A.I. (mannitol in- 26 4.1 6.97 12 6.6 1040 920 2 925 342 0 582

fusion) 37 63
F. Cont. acetazolamide, 12 1.1 7.21 0.3 5.8 480 480 55 550 92 30 428
12 hr after last dose (335) 17 5 78 c
+C.A.I. 13 0.4 7.3 520 13 520 116 0 %
22

G. K+ depletion (334) 30 1.8 7.38 0.3 6.3 1200 1198 5 12 1215 150 55 1010
12 5 83
+C.A.I. 30 1.8 7.38 0.7 7.5 1200 1155 0 5 1160 150 0 1010
13 87
H. Na citrate (oral) 29 1.4 7.49 0.7 7.9 1160 1000 0 0 1000 116 150 734
2 mwJ% (PF) 12 15 73
+C.A.I. 27 1.3 7.49 1 .o 7.8 1080 850 0 0 850 108 0 742
13 87
I. NaHCOa infusion, 15.6 38 1.7 7.52 9 8.0 1520 1120 0 0 1120 142 395 583
mq/kg W 13 35 52
+C.A.I. 36 1.6 7.53 12 7.9 1446 725 0 0 725 133 0 592
18 82
J. Hyperventilation alka- 12 0.85 7.32 0.1 7.6 480 476 0 0 476 71 101 304
losis (335) 15 21 64
+C.A.I. 12 0.90 7.30 0.4 8.1 480 375 0 0 375 75 0
20 80
IS. Max. phosphate infu- 22 1.5 7.35 10 6.2 860 350 60 1270 124 665 481
sion (337) 9 53 38
+C.A.I. 22 1.7 7.28 12 7.1 45 20 605 142 0 463
23 77
L. Max. phosphate infu- 12 1.7 7.0 6 5.8 480 340 70 883 142 408 333
sion acidosis (337) 16 46 38
+C.A.I. 12 1.7 7.0 7 6.8 480 40 20 475 142 0 333
30 70
M. Theophylline (375) 20 1.2 7.39 0.2 6.1 7 13 820 100 270 450
12 33 55

Theophylline +C.A.L 20 1.4 7.31 1.0 8.1 800 0 0 550 116 0 434
21 0 79
* C.A.I. means complete carbonic anhydrase inhibition. Italicized numbers indicate y0 of total. V,,, (col. 11) calculated from first-order
hydration kinetics; see text II, 1 and III, 5B. VObs cst (col. 12) is the rate (col. 10) abolished by C.A.I. The data are adapted to model form from
the experiments cited.

Distal Tubule
Lumen Cell Plasma
*K+
- HCO; (90)
CO2
+ HCO; (30)
FIG. 4. Same convention as Fig. 3, but for distal tubule. Vuno is not precise since fluid
volume is not known. The magnitudes of V ont and HCOJ’ transfer were estimated from effects
of inhibition on H+ and NH(I+ excretion (Table 21) and lack of effect of acetazolamide on distal
HCOa- reabsorption (423). Although the contribution of H+ secretion to distal HCOa- reabsorp-
tion is small, it is recognizable by luminal disequilibrium between H&O8 and CO2 (424).
The experiments of Figure 5 and Table 20 explain the once curious finding
that the acidotic animal cannot recover in the face of continual carbonic anhy-
drase inhibition, neither doesit progressively losebase. Such an organism is “dead-
locked in acidosis” (335). Recovery is impossible becausedistal acidification con-
tinues to be blocked; progressive loss of base is prevented by what appears to be
augmented proximal HCOa reabsorption lying outside the CO2 ---) H+ system
(col. 13 of Table Zl-compare rows A and F).
In the chronic acidosis of continuous carbonic anhydrase inhibition there is
no progressive lossof Na+ or K +. The pattern shown in the last period of Figure 5
persistsaslong as drug is given; however, the initial deficit of theseions, amounting
to about 10 % of body stores, is not repaired until drug is withdrawn. These ionic
patterns are fully documented (335) for experiments lasting up to 16 months.
Alkalosis, either respiratory or metabolic, mimics the effect of carbonic anhy-
drase inhibition (Table 2 1, rows H, I, J). This is reasonable, since added bicar-
bonate would ,effectively reduce [H+] in the cell and low CO2 concentration would
reduce both the catalyzed and uncatalyzed rate of H+ formation. The antipodal
relations of acidosis and alkalosis to carbonic anhydrase inhibition, one blocking
and the other mimicking, is a central theme in the physiology of all the organ sys-
tems under consideration (section IV). Enzyme activity per se is not seriously al-
tered by acidosis or alkalosis (section II, 4).

O&bet 1967 CARBONIC ANHYDRASE 699
0
003c 2 oonc 3 OOSL 4
4 4 4
O-IS I6 -24 24-32
OIAMOX m 31mg/kg per oa
FIG. 5. Failure of renal response to sustained carbonic anhydrase inhibition. Solid bars: 33
mg/kg acetazolamide orally at 0, 8, 16, and 24 hr. Hatched bars: control data. Top frame gives
drug concentrations. Mean of 4 experiments in a single dog. From Maren et al. (335).]
ROWS K and L of Table 2 1, taken from the buffer-loading experiments al-

ready cited (Table 20), make the additional point that’the measured catalyzed
rate in vivo (VbbsCat)is susceptible to increase (in this case, about fourfold) when
the cellular milieu is favorable. The change in this instance is the increasein buffer

capacity of luminal fluid, which facilitates the transfer of tubular H+, as formed
from CO* and water, into the lumen. Thus, the expression of distal carbonic
anhydrase activity is enhanced. As is shown below, the potential for acidification
based on enzyme or substrate within the cell is enormous; the limitation is in the
transfer step.
The final row (M) of Table 21 shows another situation in which I?& csf
appears to be enhanced, although the mechanism is not understood. The data
suggestthat aminophylline permits the transfer of more H+ than normal from cell
to lumen. Presumably there is somechange in permeability to H+. *Hence, a greater
proportion of luminal HC03 is dependent on enzyme-catalyzed H+ formation, as
shown by the augmented effect of acetazolamide (375).
A) MICROPUNCTURE AND STOP-FLOW STUDIES: PROXIMAL TUBULE. The major
role of the carbonic anhydrase system here is acid secretion serving the reabsorp-
tion of HCOa, after its conversion to H&Oa and CO2 . Stop-flow studies in dog
reveal a decreasein proximal Na+ reabsorption, after acetazolamide, of the mag-
nitude associatedwith the unreabsorbed HCOs ion (400). Micropuncture studies
in the rat show that the normal proximal lumen fluid pH is 6.8-7.1, whence
HC03 concentration is 6-12 mM (80, 184, 424). Since the volume -of fluid in the
proximal segment is reduced by at least 60 % in the first s of the tubule, it is
evident that at least 80% of filtered HCO;I is reabsorbed here. This figure ap-
proaches 100% if one extrapolates to the inaccessiblefinal segmentof the proximal
tubule. After CL 11,366 or acetazolamide, the concentration of HCOa- in the
proximal tubule risesto about 40 mM (80, 424), but since the volume relations are
not known, this type of experiment doesnot necessarily tell what fraction of HCOS-
reabsorption is dependent on formation of H+ from CO2 . Rector (423) has esti-
mated from his micropuncture data that it is 60 %. The stop-flow and micropunc-
ture experiments are compatible with the results of Table 21, from which the con-
tribution of CO2 to HCOa- reabsorption was estimated as3 l-63 %, chiefly through
the catalyzed rate.
The electrical potentials recorded from the proximal tubule [cited and dia-
gramed by Pitts (401)] show peritubular fluid about 20 mv positive to the lumen.
From the Nernst equation, HCOa .would then move down its electrochemical
gradient from lumen to blood until the lumen concentration is about 10 mM. But
very recently, it has been claimed that the transtubular potential in rat proximal
tubule is zero (166). Th ere is, then, a passive or active acidification mechanism,
apart from addition of H+ to luminal fluid. Stop-flow analysis suggeststhat Na+ is
the accompanying ion to proximal bicarbonate reabsorption from all sources(400).
A passive process(at +20 mv) could reduce the pH of luminal fluid in the- rat to
about 7.0; if there is no potential, it must be active. [HCOa reabsorption must be
wholly or in part the primitive mechanism for acidification in the Elasmobranchii,
where there is no carbonic anhydrase and where Vuncis not enough to explain
HCO,- reabsorption and H+ secretion- seeparts 2 and 3 below. Urine pH is 5.8
and rapid proximal acidification may be readily visualized (185).] Suggestive evi-
dence in favor of an active anionic processin the dog is furnished by inhibition of
HCO, reabsorption by maleate (35).

It appears to the writer that both the passive and active transport of HC03-
have been neglected and that they represent mechanisms at least as fundamental
and necessary to the organism as those for H+ secretion.
In the general scheme of acidification in the proximal tubule:
HCOa- + H+ + H&OS ti CO2 + HZ0
filtered --
into from reabsorbed
lumen cell from lumen
The details of the H&O 3 e CO* equilibria in the lumen, and the mechanism
whereby either species is returned to the blood, have received recent attention.
Walser and Mudge (508) suggested that if CO2 is the species reabsorbed, luminal
pH must be 6.4 or less to keep the H&O3 concentration high enough to drive the
uncatalyzed dehydration reaction at the known rate of bicarbonate reabsorption.
This type of argument is recounted below, with. the modification that the process
in question need only account for s of bicarbonate reabsorption (Table 2 1, rows
A and B). From Table 2 1, HCOS- reabsorption in the dog is 800 pmoles/min, of
which 270 pmoles/min appear to be due to H+ production. If x of the process
occurs in the proximal tubule, the rate for the model is 235 pmoles/min (Fig. 3).
The jirst-order rate constant for dehydration at 25 C is 13.7/set (173); extrapolat-
ing to 37 C yields 30/set or 1800/min. Estimating the volume of luminal fluid to
be 10 ml, we may solve for the concentration of H&03 that must be maintained to
drive reaction16 to the right:
18OO/min~O.01 .[H&Oa] = 235 pmoles/min (19)
[H2COa] = 13 /M
Thus, a H&O 3 concentration of 13 PM in the proximal lumen would assurethe

dissimilation of HCO3- to CO* at the observed rate of HCO 3- reabsorption
without any carbonic anhydrase present. The pH of the luminal fluid containing
13 /AM H&O3 and 20 mM HCOa- is calculated from the pK, of 3.57 (173) and
yields a value of 6.8. If [HCO3 -1 is 10 mM, the pH must be 6.5. However, 13
PM H&O3 is higher than that yielded by equilibrium with CO2 whose concen-
tration in plasma and, presumably, tubule is 1200 JIM. H&O 3 concentration at
equilibrium is J&O that of [CO21 (173), or 3 PM, whence the pH would be 7.4,
the same as plasma. This argument makes it essentialto know the pH of luminal
fluid, and whether CO2 and H&O3 are in equilibrium in the normal situation
and after carbonic anhydrase inhibition. Ideally, one would also want to know
if there is “luminal carbonic anhydrase.” These calculations make it appear
that, to reabsorb all titered HCO3- in the absence of carbonic anhydrase in
contact with luminal fluid, the pH of the fluid would have to be 6.5-6.8, and
there would be disequilibrium between CO2 and H&03. The following experi-
ments illuminate this problem.
The pH of proximal luminal fluid measured by quinhydrone electrode is
actually about 7.0 (80, 184, 185, 424). At the usual pCO2 of plasma, the HCO 3-
concentration would then be about half that of plasma. The question arisesas to

whether this is true fluid pH, i.e., whether this figure results from H&OS as
formed or after coming into equilibrium with CO2 in the electrode. Data are
given in the thoroughly documented paper by Rector et al. (424). Their in vivo
measurement was made with single- or double-barrel glass electrodes of tip
diameter 1-2 p. In their hands, proximal pH was 6.8-6.9 both in glass(luminal,
in vivo) and quinhydrone (in vitro) electrodes. In rats infused with NaHCOa ,
both measurementswere\ about 7.5. Paired sampleswere usually within 0.1 units
of each other. Proximal fluid thus appeared tocontain H&O3 and CO2 at equi-
librium. Table 22 gives the data in model form.
Rector et al. (424) thus followed the general argument of equations 16 and 19,
which, for proximal HCOa- reabsorption and luminal equilibrium between
H&O3 and COe, implies carbonic anhydrase in contact with these molecules.
In support of this, CL 11,366 reduced luminal pH, the expected result of inducing
disequilibrium between CO2 and H&O8 and elevating [H&OsJ above the
equilibrium value. Table 22 shows the data; it will be seen that the predictions
of the Waker-Mudge model are verified: in the absenceor inhibition of carbonic
anhydrase there is disequilibrium. But the increase in luminal [H+] is not great
enough to drive equation 18 far enough to the right to lead to the reabsorption of
all filtered HCOa-. The essential limitation in carbonic anhydrase inhibition is
still that of H+ production from the cell. However, luminal dehydration of H&O 8
appears to be an essentialfeature of proximal HCOa- reabsorption, and carbonic
anhydrase appears also to act at or near the cell membrane.
The arguments for and against cell- or luminal- or membrane-catalyzed
dehydration have a morphological counterpart since the enzyme was originally
stated to appear only in cell water (19, 99). However, here too are contrasting
data, with 20% reported in particulate fractions of renal and liver cells (233).
We have recently shown (325) that about 10% of enzyme in the renal cell is
in nuclei, mitochondria, and microsomes,in agreement with Karler and Woodbury
(233). Curiously, this was also the finding of Datta and Shepard (99), but they
thought particulate matter was contaminated by cell water. This point was
critically ruled out in the present experiments. It is impossible, at this time, to
assess the significance of the enzyme in the several fractions. As suggestedabove,
TABLE 22. Model of luminal CO2 equilibria
I Plasma = I Midmoximal Distal
HCOa-, rnM 20 10 30
Nonnd
.-- I I
l
tirbonic
anhydrase _
I
5 5 0.12
CO*, mM 1.2 1.2 1.2 1.2 1.2 1.2
H2COa 9w 3 3 20 15 3 ?
pH at equil. using p& = 6.17 7.4 7.1 7.6 6.8 6.8 5.2
pH in vivo using pK= = 3.57 7.4 7.1 6.8 6.1 6.8 ?
Adapted chiefly from refs. 423 and 424.

73
in dealing with substancesas diffusible asH&O3 and COZ, localization of enzyme

does not seem a vital issue.The most attractive aspect of Rector’s concept, con-
sidered quite apart from the site of the enzyme, is its statement of the fundamental
role of enzyme in proximal luminal function: to reduce H&O into very low levels
so that the H+ gradient from cell to lumen is small, thus markedly reducing energy
expenditure in H + transport serving HCO3- reabsorption. This latter function
would be carried out by enzymic dehydration or enzyme-mediated transport of
H&O 3.
In other micropuncture studies, Rector (423) showedthat proximal bicarbon-
ate reabsorption is reduced about 50% by 20 mg/kg acetazolamide. Since these
experiments were done during HCO3- diuresis, the enzymic contribution is high,
analagous to experiments of Table 21 that include bicarbonate and phosphate
loading.
The case for ionic HCOa- reabsorption receives support from the demon-
stration that the proximal tubule is permeable to this ion, in the direction blood
to lumen (16).
Proximal HCO 30 reabsorption is increased in NH&l acidosis, as shown by
micropuncture data in dogs. This is not the case in respiratory acidosisor alka-
losis (79). These findings agree remarkably well with data of column 13 of Table
21, which suggestthat some function outside the CO2 + H+ system subserving
HCO 3- reabsorption (active HCO 3- transport?) is increased in metabolic acidosis.
Figure 3 shows a provisional scheme for proximal tubular reabsorption of
HC03- , with particular emphasis on transport rates. This model attempts to
show how cellular hydration and luminal dehydration of CO2 might act together.
Features of the present scheme that differ from earlier ones of similar type (401)
are: emphasison separation of OH- and H+ in the cell; the quantitative aspects
of the contribution of H+ secretion to HCOS- reabsorption; active or passive
reabsorption of HCO 3- ion, as such, as a major component; reabsorption of
luminal H&O3 or CO2 into the cell, where there is enzyme-mediated dehydration
or transport.
B.DISTAL TUBULE AND COLLECTINGDUCT. It hasusuallybeenheld that HC03-
reabsorption is largely a function of the proximal tubule, while urinary acidi-
fication and NHd+ output are results of H+ secretion in distal tubule and col-
lecting duct (401). For purposes of exposition, this convention is used for Figures
3 and 4. Carbonic anhydrase is certainly involved in all these processesand the
enzyme appears in both proximal and distal segmentsin frog (317) and in rat
(232,409). The localization of function is not strict, however, for in the rat Rector
(423) hasrecently found some50 % of titratable acid to be formed in the proximal
tubule. Some NH 4’ secretion also occurs proximally in the rat (176, 204). Hayes
et al. (205) have made the interesting observation that acetazolamide does not,
while NaHCO 3 does, decrease proximal lumen NHd+ concentration in this
species.This effect, at first paradoxical, is just what would be expected, in view
of the finding of Rector et al. (424) that acetazolamide, by generating a disequilib-
rium between H&O3 and CO, in the proximal lumen, lowers luminal pH. In
the frog, however, both acidification (363) and NH 4’ secretion (507) are confined

740 T. H. MAREN volw?ze 47
to the distal segment. In the dog, stop-flow data suggest reasonable proximal
localization of HC03- reabsorption (400) as shown in Figures 3 and 4; this ana-
tomical question is not now regarded as primary.
Characteristic features of the distal tubule and collecting duct are neverthe-
less clear. Both stop-flow (400) and micropuncture (184, 423) data show that
steep pH gradients from cell to lumen -as much as 3 units-are developed:
NH4+ is secreted by proton attack on NH 3 (400); K+ is secreted by the common
mechanism with H+, in exchange for sodium (36). Depression of H+ secretion
by acetazolamide then decreases NH4 + and increases I<+ in distal fluid. General
details of these schemes are described lucidly by Pitts (401). The distal regions
are specialized to transport small amounts of H+ and Kf against large gradients
(Fig. 4), in contrast to the proximal segment, which transports relatively large
amounts of H+ for Na+ against a small gradient (Fig. 3).
It is of interest that the HCO 3- reabsorption rate in the distal tubule-which
is normally about g that in the proximal tubule -is generally (but not always)
unaffected by acetazolamide or CL 11,366. A decrease in reabsorption after
drug is sometimes seen when initial rates are high (423). This strongly supports
the thesis that the relative contribution of the enzymic rate (apart from the situ-
ation in acidosis) depends on the rate of HCO3- reabsorption or of acidification
(Table ‘21). Figure 4 suggests that HCO3- reabsorption in the distal tubule is
almost entirely mediated by the ionic process; whether it is active or passive is
not certain, but if the potential is +60 mv (166), HCO3- could achieve a luminal
concentration of 2 IIlM according to the Nernst relation. VObS cat in the distal
tubule is only 7 % that in the proximal tubule and is largely or entirely involved
in providing H+ at a rate to subserve titratable acid and NHJ+ output (compare
Figs. 3 and 4).
The normal pH of the rat distal tubule fluid is variously reported at 6.7
(184), 6.2 (423), and 5.6 (15) ; these were measured with quinhydrone electrodes
after equilibration with CO2 . The HCO3- concentration in this fluid is thus
< 5 mM. In NaHCOs-loaded animals, Rector et al. (424) measured the equilib-
rium pH 7.8, but the glass electrode or true luminal pH was 7.0. Thus, the situ-
ation is different from that found in companion experiments for the proximal
tubule, as cited above. In the distal tubule, at least under the stress of excess
HCO 3-, the disequilibrium predicted from equation 19 does seem to exist; intra-
venous injection of carbonic anhydrase, which passes the glomerulus, raises the
true luminal pH to the equilibrium value of about 7.8 (424). Why and how it is
that H&OS generated from H+ secretion has access to enzyme in the proximal
but not the distal tubule is presently a mystery; yet it is functionally reasonable,
since the overall secretion rates are certainly greater in the proximal region,
It is now clear that the excess pCO2 of alkaline urine over plasma is due to
delayed dehydration of H&O3 in the distal tubule. Ochwadt and Pitts (383)
found that the excess (about 40 mm Hg or 1.2 mM COZ) was abolished by the
intravenous injection of carbonic anhydrase. On this basis, the calculated pH
at the site of fluid formation would be between 4 and 5. In an excellent recent
paper, Guignard (192a) has returned to the ‘problem. Using the rat, he showed

that for a given level of HCO3- excretion the excessPC02 was greater for aceta-
zolamide-treated animals than those perfused with NaHCO3. This would agree
with the thesisof Rector, discussedabove, that proximal dehydration is catalyzed
by carbonic anhydrase. Thus, in the normal animal with alkaline urine distal
dehydration is delayed; in the inhibited animal dehydration is delayed through-
out the nephron. Guignard (192a) also showed that during moderate degrees of
NaHCO3 infusion, intravenous carbonic anhydrase could abolish the excesspCOz
of urine. This paper should be consulted for good data on acid-base balance in the
rat.
Table 22 gives models of CO2 equilibria in the different segmentsof the
tubule. In idealized form, and adjusted for conditions of normal acidAbasebalance,
this provides a summary of the present treatment.
2) Chemical kinetics and fractional inhibition in the mammal in vivo. The relations
between uncatalyzed and catalyzed hydration rates of CO2 and physiological
acidification rates in the kidney have been worked out for both mammal and
dogfish (320). This is summarized for the dog, taking advantage of up-to-date
kinetic and renal measurements. After this, inhibition kinetics, as they apply
to the kidney, are reviewed, and a comparison is made between rate and inhibition
kinetics as they apply to the question of enzyme excessand limitations of phys-
iological rate.
From the treatment and constants of section II, I, Vunc = kl [CO,], where
kl is 0.0434 see-1and [COJ is 1.2 mM (pCO2 = 40 mm Hg). Then
V UUC = 0.0434 l 1.260 l
(20)
= 3125 pmoles/liter per min
This figure may be applied to the kidneys of a lo-kg dog, which weigh
70 g. Tubular cell fluid volume is approximated at 30 ml, whence the uncatalyzed
rate *in vivo will be about 100 pmoles/min. Specifically, this quantity of H+
can b.e formed each minute in the canine kidney without carbonic anhydrase,
assuminga system that would remove it at least as rapidly. It is clear that calcu-
lations for other mammals will be identical, except for the different volumes of
tubular fluid. Yuric in vivo is then directly proportional to kidney size and so
roughly proportional t0 body weight.
For the potential catalyzed rate in vivo
turnover number E, renal . [S]
‘VE =
l
(21)
&n + cs3
The numbers used for equation 21 were obtained as follows. Turnover number:
the same figure as for canine red cells is used (4 X 10’ min.1) since renal and red
cell enzyme have been shown to have close to the same specific activity (325).
Using the terms of Tables 2 and 3, [&I/ e.u. for dog kidney supernate was
3.6 X lWg M, compared to dog red cell value of 2.4 X 10eg M and to HCA-C
of 2 X 10-g ~:This correspondence is part of the evidence referred to throughout
the review that virtually all enzymes examined to date (except rat liver, see
III, 5G) are kinetically akin to HCA-C. E, renal: in whole kidney there are ap-

proximately 400 e. U./g, most of which is in the cortex (Table 16). Over 90 %
of enzyme is in the supernatant fraction. The role of the particulate enzyme is
not clear; it is in any case susceptible to inhibition and almost certainly accessible
to the more diffusible of drugs presently used (325). Using the value cited just
above, the concentration of EO is 14 JUM.We had previously used a value of 10
PM (320), derived from the lower [E,ll e.u. for red cells. It will be evident that
this is not a critical difference, and, in fact, we do not claim that [EO]/e.u. is
accurate to more than about 50%. It is significant that the binding of acetazola-
mide to canine kidney, which gives an independent measure of [E,], is about 11 PM
(322). Equation 21 calls for E, in terms of weight; the dog kidneys are assumedto
have 70 ml of fluid available as reacting mass,whence E, = 1 pmole. Km: the
value for red cells (41 mu at 37 C) is used (Table 3) since there are no data for
dog kidney. Inspection of equation 22 below will show that variations in this figure
will not greatly modify VB. [s] : as in the expression for vunc,the value of 1.2 mu
is used on the assumption that tissue [Cod is equivalent to that of plasma. Sub-
stituting these values in equation 21 we have
4 X 107 min-1 . 10-s moles . 1.2 mM
VE =
41 mbf + 1.2 mad
= - l,OOO,OOO pmoles/min
for the theoretical or potential and unopposed hydration rate.

The magnitudes of the uncatalyzed (V”& and the catalyzed ( VB) rates
ultimately depend on quite different properties of the kidney. For V,,,, kl and
[S] are constant in all normal mammalian situations in which temperature and
pCO2 are reasonably well fixed. The uncatalyzed rate will vary directly with
the size of the kidneys or, more specifically, with the volume of the secretory
cells. Thus, the rate calculated in equation 20 is subsequently adjusted to tubular
‘fluid of the dog. VB, on the other hand, depends ultimately on the total weight of
enzyme in the kidney, since the other values in equation 21 are fixed by the con-
stants of the enzyme-substrate systemand the relative fixity of pCOz in mammalian
tissuesduring normal acid-base balance. The relationship between VunOand I&
in any species,cell group or altered pathological situation, then depends on the
relation between tissue size or tubular volume and weight of renal enzyme. This
may have a bearing on the relation between Vuncand VB in different. segments
of the nephron. It is evident from equations 20 (adjusted to tubular volume) and
22 that in the canine kidney VR/Y uric is about 10,000. Since other mammals
and birds have carbonic anhydrase concentration (Table 16) and tubular fluid
volumes/kidney size in the same range as the dog, this relation may be regarded
as a general property of the renal carbonic anhydrase system. This is of theoretical
interest, but not the true in vivo relation between the observed physiological
catalyzed rate (denoted l&s cat) and Vunc.This relationship emergesfrom analysis
of maximal inhibition, which follows.
A significant contribution of pharmacology to its sister scienceshas been the
development of the inhibitors described in II, 7 above in the particular context
that their use has led to the determination of &b8 cat-the in vivo catalyzed rate

in the kidney and elsewhere. In the normal, unanesthetized, unperfused dog, a

maximally effective dose of acetazolamide elicits the excretion of 150 pmoles
HCO a-/rnin and the elimination of acid and NHd+ from the urine. This is
shown in row A of Table 21. Dose-responsecurves for this and other drugs are
shown elsewhere (321, 323), and it is clear that this excretion pattern is unam-
biguous in defining complete inhibition in this species; it is achieved with any
of the drugs, and not exceeded, at a given dose for each. It is for this reason we
are confident in saying that the change elicited is the contribution of the enzyme,
and in terms of hydration rate of COZ, I&, cst. For the dog, it is about 170pmoles/
min.
The relation between v&s cat and Vuricis of the order of 2: 1 in the normal
animal (Table 21, row A). As emphasized above, l&r, caf can increase under
different conditions of buffer load so that this ratio I&s ,,t/Vunc can reach almost
6 : 1. (Table 2 1, row K). It will be seen in succeeding sections that this ratio is
always of this small magnitude, in contrast to the very large magnitude of VB/
V unce v./ vobs cat for kidney, in the normal animal, would be 106/l 70 or about
6000; there appears, then, to be a very large excessof enzyme or an almost in-
finite rate of acidification. However, the renal cell can scarcely be regarded as
an open system; plainly some constraint on the removal of product is expected.
The limiting event appears to be transport of H+. The very large concentration
of enzyme in the cell assuresnear-equilibrium conditions between CO2 and HCO a-
at the physiological removal rates for H +. How much is really in excessor reserve?
This can be answered by the consideration of inhibition kinetics to follow.
In section II, 7, for sulfanilamide, the fractional inhibition (i> was calculated
for the various magnitudes of renal response. No effect was elicited when i =
0.99. This is true for acetazolamide, CL 11,366 (321), ethoxzolamide, and meth-
azolamide (323). Dose-response curves are shown in these papers; for all
drugs’ 50% response correspondsto i = 0.9954997 (seealso Table 12). Max-
imal response-the value for HCOa- excretion of 170 pmoles/min referred to
above-occurs at i = 0.9990-0.9996.
It is apparent that despite the diverse chemical and physical properties of
these drugs, which ordain that they arrive in the renal cells by different routes
and at different concentrations relative to plasma, fractional inhibition is close
to the same for all drugs at the same renal effect. It is thus tempting to believe
that renal carbonic anhydrase, not only in cell water but in the particulate frac-
tions, is fully accessibleto all of these drugs. Given 10,000 parts of enzyme in the
normal kidney, 30-50 parts are needed for 50 % renal activity; W-200 parts
appear necessaryfor full activity. Renal carbonic anhydrase activity is not mani-
fest at all when only 4-10 parts are free. These conclusions may be compared to
those obtained from rate data above, where it was concluded. that YE/v&e crrt
is normally about 6000 and the question arose how much of this is really excess.
Since the dose-responsecurves begin at i = .99 (or, in the model cited, 100 parts
free out of lO,OOO),it is likely that the true excessis about lOO-fold. The enzyme,
then, appears to have the potential in vivo for the net hydration reaction at
about 540 of the theoretical unopposed rate of 10” peq/min (eqtiation 22) or

about 16,600 peq/min. The difference between these two numbers could rep-
resent forces of the back reaction or limitations imposed by the in vivo milieu.
If we recognize 16,600 peq/min as the net Vg, there is still an excessof 25.fold
over the highest known acidification rate due to v&s cat (Table 2 1, row K). This
excessmeans that enzyme is never rate limiting and permits near equilibrium
at low substrate concentration or at any known rate of removal of product. It is
significant that Rector et al. (425) found that the carbonic-anhydrase-dependent
port-ion of HCO3- reabsorption was unaffected by variations in pCOz as low as
10 mm Hg.
3) Comparative as$wts. A totally different pattern of renal electrolyte excretion
is mediated by carbonic anhydrase in the alligator (95). In this species,the chief
urinary ions are NHJ+ and HCO 3~ When carbonic anhydrase is inhibited
(50 mg/kg of acetazolamide), urinary HC03° is reduced and is replaced by
Cl-. NHd+ and N a+ are unaffected, and K+ is increased equivocally. The data
suggest that the alligator tubule cells hydrate CO2 to form HC03- , which is
exchanged for luminal chloride. Other data suggest that chloride conservation
is a prime function of the alligator kidney. It is interesting that the mimicking
and blocking pattern observed in the mammalian kidney can be found in the
alligator, in reverse fashion. Here, administration of NaCl (or NazSOg) is similar
to the effect of acetazolamide in decreasing HCO3- output. NaHCO 3 partially
blocked the effect of acetazolamide, presumably by increasing the level of [HCO3-]
in the cells so that Cl- reabsorption was less dependent on enzyme-mediated
formation of HCO3-. The analogies, at least in part, between these reactions
and those in the pancreas and other alkaline-secreting organs in vertebrates,
will become apparent later.
There remains the circumstance in which urinary composition is unaltered
by large dosesof acetazolamide or any carbonic anhydrase inhibitor. The chief
and perhaps only category of vertebrates in this classare marine fish, both elas-
mobranch and tel.eost. It was originally thought that the situation in these tW0
types was different because there was little or no renal enzyme in the elasmo-
branch (213, 317, 319), whereas the teleost (317) showed a considerable con-
centration of enzyme. However, neither type responded to acetazolamide or
other inhibitors (213, 3 17, 421) despite the finding of high concentrations of drug
in the kidney (327). The problem has recently been clarified (339), since the en-
zyme present in the marine fish can reasonably be accountable to a role of hemo-
poiesis,with nothing to do with renal function. The enzyme present, presumably
in blood-forming cells, is susceptibleto inhibition in vitro (339).
Consideration of specieswith no functional renal enzyme throws much light
on the basic role of renal carbonic anhydrase. In S. acanthias, which may for this
purpose be taken as typical of marine fish, urinary composition is fixed with
respect to pH at 5.8 (2 13 and literature cited), although acid secretion can be in-
creased by administration of appropriate buffers (86, 213). Urinary Hf output in
8. acanthias reached a maximum of about 1.6 pmoles/min in one study (213)
and 4 ~moles/min in another (86). The uncatalyzed hydration rate in the kidneys
of this speciesmay be calculated at 1.O pmole/min from kl [S], using kl for 14 C

and [S] equivalent to a pCOz of 5 mm Hg (320). Thus Vunc cannot fully supply
urinary H+. It is also clear that HCO 3- reabsorption in this species (2.4 pmoles/
min) cannot be associated with tubular CO2 + H+, since yUnc is again too low.
These experiments and calculations give further credence to the idea that active
HCO 3- reabsorption is a fundamental vertebrate process (320).
The injection of acids or bases does not alter urine pH. These are rapidly
eliminated by the gill, with only transient changes in acid-base balance of the
blood (213, 319, 437). Absence of enzyme is clearly associated with lack of urinary
acid-base lability. Such lability would be inimical to the seagoing elasmobranch
or teleost, since alkalinization would lead to precipitation of magnesium and
calcium salts; these are major urinary cations in marine fish (163). There are
other chemical and physiological points of interest in species that acidify urine
without carbonic anhydrase. It has been possible to study the effects of carbonic
anhydrase inhibition in the red cells, choroid plexus, and ciliary process of the
elasmobranch Squalus acanthias without the complication of the renal effect (319).
Comprehensive studies have still not been completed on functional renal
carbonic anhydrase through the vertebrate phyla. Particular gaps exist in the
classes Reptilia and Amphibia (III, 2). Dr. Leon Goldstein (personal communi-
cation) reports that the African clawed toad (X. Zaevis) does not give a renal re-
sponse to acetazolamide; enzyme appears to be present, but as in marine fish,
it may be in hematopoetic tissue (339). It is hoped that this discussion may stim-
ulate further work in the comparative areas.
G. The eye
Carbonic anhydrase inhibitors have their firmest place in medicine in the

treatment
. of glaucoma. Behind this lies an impressive mass and quality of literature
on the secretion of aqueous humor, the distribution and role of carbonic anhydrase
in the’ eye, and the pharmacology and distribution of sulfonamides in the eye
and their effects on flow of aqueous and intraocular pressure. Unfortunately,
there is some lack of agreement on the mechanism by which acetazolamide and
allied drugs reduce intraocular pressure both in normal and glaucomatous eyes.
A chief goal of this review is to resolve some of the problems and to arrive at a
tentative statement of the role of carbonic anhydrase in the eye and the effect
of its inhibition. This review is greatly aided by the existence of penetrating essays
on the subject by Becker (22), Kinsey and Reddy (253), and Davson (120). The
data discussed are largely from these workers and from Wistrand (522, 529),
Langham (272), Macri (304), and their co-workers.
The basis for this subject is the presence of carbonic anhydrase in the ciliary
processes of all vertebrate species thus far examined (Table 16); the bicarbonate
excess in the aqueous humor of the rabbit, which is the chief experimental animal
used in this research; and the effect of acetazolamide and allied chemicals in
reducing intraocular pressure, as well as altering the chemistry of the aqueous
humor. The main controversial points are twofold: I) Is bicarbonate formation
a central point in the formation of aqueous humor in primates as well as rabbits?

70I T. H. MAREN Volume 47
2) Is the reduction in intraocular pressure due to reduced secretion of aqueous

humor or to an effect on vascular dynamics within the eye?
The chemical composition of the aqueous humor of the rabbit shows that
the anterior chamber fluid has a HCO3- concentration of some 30% and the
posterior chamber fluid 70%, higher than that of plasma (reviewed in refs. 120
and 253). Mathematical analysis of aqueous secretion suggeststhat the primary
fluid, as formed from the ciliary cells bordering the posterior chamber, has a
still higher HCO3- concentration, about 90 mM, or 3-4 times that of plasma
(253). Not only in the rabbit, but in the two other speciesin which the posterior
chamber fluid has been analyzed (guinea pig and dog), there is an excessof
bicarbonate over plasma (Table 23). It is of interest also that in the EZasmo-
bran&ii, a class of animals that is generally considered to represent primitive
vertebrates and contains a ciliary processwith carbonic anhydrase, the HC03-
concentration in the aqueous is 2.5 times that in plasma in one species[Mustehs
canis (132)] and 1.1 times plasma in another [Squalus acanthias (3 19)]. It is for
these reasons that the reviewer believes that bicarbonate accumulation is a
TABLE 23. Anion composition of aqueous humor, and e$ect of carbonic anhydrase inhibition
[I-K&h-b-~io, [Cl-] Ratio,

w Aqueous/Plv,
Species Ref. Time, HP
Control C.A.I. Control C.A.I.
I I
Poster io r chamber
Rabbit (22) 6 1.70 1.39 0.88 0.94
(21) 0.5 1.78 1.45
Guinea pig (22) 6 1.52 1.30
Dog (526) 1 1.15 1.09 1.08 1.06
W6) 24 1.15 1.06 1.08 1.06
Monkey (27) . 1.25
Anterior chamber
Rabbit (22) 0.5 1.39 1.31
(272) 0.25 1.32 1.11
(272> 2.5 1.32 1.33
Nephrectomized rabbit (272) 2.5 1.34 1.28
(121) 2.5 1.18 1.20 1.01 0.99
(22) 6 1.35 1.11 0.94 0.96
Guinea pig (22) 6 1.42 1.19 0.90 0.93
Do&3 (526) 1 1.11 1.10 1.04 1.04
24 1.11 1.07 1.04 1.03
Cat (121) 2.5 1.22 1.32 1.07 1.03
Monkey (121) 2.5 0.83 0.80 1.12 1.14
(27) 0.76 1.11
Man (22) 24 0.83 0.94 1.08 1.02
Dogfish (319) 2 1.09 0.85 1.02 1.03
(319) 24 1.09 0.64 1.02 1.08
Lake trout (215) 24 0.97 0.56 0.80 1.14
* After complete inhibition of carbonic anhydrase (C.A.I.).

October 1967 CARBONIC ANHYDRASE 7=I
significant feature in aqueous humor formation of vertebrates. This is supported

in the following paragraphs by data involving carbonic anhydrase, and the position
is developed that HCO,- is formed from COS. These opinions are not held by
two of the most distinguished workers in the field; their reasons,in turn, are some-
what different from each other and will be summarized here. Davson (120)
emphasizesthe fact that many speciesdo not show HCOa- excessin the aqueous
humor; however, data from such species (man, monkey, horse, goat) are those
from the anterior chamber only. It seemsto this reviewer that even in such species
the prim& secreted fluid, as found for the rabbit by Kinsey and Reddy (253)
and reflected in posterior chamber analysis, might indeed be high in [HCOam].
Davson believes that carbonic anhydrase inhibition has several effects-a “gen-
eral poisoning of active transport,” and later “reduction in resistance to flow
or in the episcleral venous pressure, or both.” These points are discussedbelow.
Becker (22) baseshis thesison studies of human anterior aqueous in which there
is a slight HCOa- deficit and Cl- excess.He believes that the anionic secretory
processin primates involves Cl- rather than HCOg- and is analogous to cere-
brospinal fluid and nasal salt gland secretion. This is certainly possible, but, as
shown below, evidence is incomplete. It is difficult for the reviewer to believe
that some mammals have dominantly a mechanism for one ion and some for
the other; in connection with this argument the HCO auexcessin the elasmobranch
may have some weight in suggesting that the basic vertebrate pattern may be
one involving HCOa- formation from CO2.
A pivotal experiment on this question was done by Kinsey and Reddy
(252), illustrated in Figure 6. Radioactive HCOS- was injected intravenously
and the appearance of the W label in the aqueous was measured. The data are
interpreted in the following way. Assuming normal acid-base balance of the
rabbit and active carbonic anhydrase in the red cells, the label in plasma of
Figure 6A would be partitioned 20: 1 between [HCOS-] and [CO2]. At 1 min
after injection total CO2 counts were 121 units, or a HCO~YCO~ ratio of about
115:6. At the same time the posterior aqueous had a total CO2 count of about
140, and since CO2 diffusion is essentially instantaneous, that count is the same
as plasma, whence the partition is 134:6. This yields a HCO 80 ratio in aqueous
humor/plasma of 1.17, and at some parts of Fig. 6A it approaches 1.6, similar to
that of cold HCOa- shown in Table 23. This shows that in the normal animal
total CO2 is partitioned in its physiological relations within 1 min between plasma
and posterior aqueous. The data suggestthat HCOS- accumulation in posterior
aqueoushas a half-time considerably lessthan 10 sec. Figure 6B showsthe nature
of HCOa- accumulation; when 50 mg/kg acetazolamide is given intravenously
and 15 min later the protocol of Figure 6A is carried out, there is a profound
delay in accumulation of total 14cO2 in the posterior aqueous. These experi-
ments are, furthermore, susceptible to the following quantitative analysis: total
WO2 in plasma is equilibrated between gaseousCO2 and HCOa- in about the
normal ratio, .even during carbonic anhydrase inhibition (Table 18); note also
that labeled NaHWOa , as injected, has a pH near blood. The total label is 150
units, whence gaseousCO2 is 7. At zero time there is no label in the posterior

72I T. H. MAREN Vofumt 47
chamber; instantly after injection of label it will be 7 because of diffusion of

COZ. Since carbonic anhydrase is completely inhibited, HCOa- accumulation
in the aqueous must proceed either by diffusion or secretion of the ion itself or
by its uncatalyzed formation from COZ. We can test this question since the latter
rate is known-it is kl [CO,], where /?r is 2.7 x-&-l (II, 2) and CO2 *is 7 concen-
tratzon units. Thus HCO3- formation should proceed at the rate of 19 units/
min. At the end .of 2 min the total 14CO2 in the pos8erior chamber should be
38+7= 45 concentration units. The data yield 55. This is an important piece
of evidence in showing that HCO3- accumulation in aqueous is all due to for-
mation from C.02 and none from transport of the ion as such.
.
TOTAL Cl40 2
4 IZO-?rE, l .;. POSTER&m

0
E cts
zi IOO- x*Xx . ‘w-m
%bl 0
g 80- ix, -i .*
x
5 60- x %Ixx s xx% 0 l l
PLASUi ‘x x $” ‘1
zE 40- e 0
~*oemO~oO*oooo
g 20- ANTERIOR
0
” ofi [ 1 ! ~ I 1 I I I I 1 I r f t t 1 I I I I fj
0 I 2 3 i 5 6 7 8 9 10 II 12 13 I4 15 16 I7 I8 19 20 21 22
TIME (MINUTES)
TOTAL C’402 (AFTER ACETAZOLAMIDE)
I x PLASMA
y..J”
xYa
x~~xxxx l K
POSTERIOR . ax x&x x : . l l . l
x x x
ANTERIOR 0 0 0000
0 1 2 3 4 5 6 7 8 9 IO II 12 13 I4 15 16 I?
TIME (MINUTES)
FIG. 6. Accumulation of HCOa- in aqueous from blood CO2 and its inhibition by acet-
azolamide. 14C-labeled bicarbonate and CO, were injected at 0 time, and total 14C02 concen-
tration determined, in the fluids at times shown. A: normal rabbit. B: acetazolamide (50 mg/kg
iv) given 15 min befor 0 time. Interpretation given in text. Data from Kinsey and Reddy (252).]

October 1967 CARBONIC ANHYDRASE 73I
Table 23 shows the effect of carbonic anhydrase inhibition on the anionic

distribution between plasma and aqueous. The Donnan equilibrium requires a
ratio of 1.04 for univalent anions (120). If the potential of +7 mv in rabbit aqueous
(355) applies, the passive distribution ratio for anions according to the Nernst
equation would be 1.25. It is clear that this is not so and that active processes
are at work in the caseof HCOa-. In all the specieslisted, except monkey, guinea
pig, and dogfish, in which pressure does not appear to have been studied, the
intraocular pressurewas lowered at the time that these chemical measurements
were made. We are thus in a position to analyze the relationship between anionic
composition and pressure within the eye after acetazolamide. It seemsclear that
in the posterior chamber HCOS- concentration is lessenedby inhibition. In the
dog the effect is small, but it will be recalled that Kinsey’s analysis for the rabbit
(253) showsthat the freshly secretedfluid is 2-3 times as concentrated in HCO3-
as the measured fluid. Thus, it is not unreasonable to expect that the measured
drug effect is damped by inability to measurethe true secretion. Posterior chamber
analysis shows no evidence of chloride secretion; the highest ratio shown, 1.25
for monkey (Table 23), would fit passive distribution. There is a need for much
more data on primates, including posterior chamber HCO 3- concentrations
and the effect of acetazolamide. Data for the anterior dim&~, taken as a whole,
show no consistent changes in mammals after the drug. The rabbit and guinea
pig generally show a lowered anterior aqueous to plasma HCO3- concentration
ratio within 6 hr after acetazolamide, but this is certainly not true for the dog,
cat, monkey, or man. In at least one series, both normal and nephrectomized
rabbits fail to show this change (272). A difficulty in these experiments (22) is
that plasma concentration of HCO 3- is falling due to the renal effect of the drug
(except in the case of nephrectomy), tending to keep the ratio high and con-
ceivably obscuring the effect on the aqueous.It cannot be emphasized too strongly
that the underlying processis conversion of circulating or tissueCO2 into aqueous
HCOa:, and thus relations to plasma [HCO 3-1 are only useful when that con-
centration is relatively stable. That is the case for the shorter experiments of
Table 23. Theoretically at least, changes in plasma [HCOa-] should not be re-
flected rapidly in the aqueous; thus ratios could be misleading if not usedcorrectly.
The dogfish data have several salient features; the plasma HCOa- concentration
doesnot fall in the 2-hr time listed in Table 23 since there is no renal effect. At
that time aqueous [HCOS-] drops 15 %. After 6 and 24 hr plasma [HCOa-]
rises,tending to give a still lower ratio, since HCO a- ion as such diffuses relatively
slowly into the eye (3 19). In this speciesthe anterior chamber is very shallow, and
there is more or lessopen communication between the chambers. The 24-hr drop
in aqueous/plasma [HCO3-] ratio after acetazolamide in the fresh-water teleost
(2.15) agreesalmost exactly with that for the marine‘elasmobranch. In both cases,
also the chloride ratio rises. Further work on fish should include pressure meas-
urements in the elasmobranch and identification of the secretory tissue in the
teleost.
This writer feels that, except for the special case of the fish and the instances
where anterior chamber analysesmay agreewith those from the posterior chamber

(as in guinea pig and somerabbit experiments), anterior chamber chemistry does
not contribute greatly to the present problem. For example, the casemade by Becker
(22) that secretion in the human is fundamentally different from that of the
rabbit rests on analysesof HCO 30and chloride ions; yet the increasedratio in man
for [HCOa-] after acetazolamide is almost entirely due to the decreasein plasma
concentration (5 mM), which is greater than the decreasein the aqueous (1.4mM).
The decreasein chloride concentration in the anterior aqueous after acetazola-
mide is only 4 mu and of borderline significance. There is in fact no case, for the
eye, where chloride concentration drops significantly in the aqueous after ace-
tazolamide (Table 23 and references cited), as it does in cerebrospinal fluid. In
summary, and based on Kinsey’s analysis, the posterior chamber data for the
several species,the striking data for the fish, and the analogy to carbonic-an-
hydrasedependent secretions that clearly accumulate HCO 3- (as the pancreas),
the view is held that aqueous humor secretion in vertebrates is in part dependent
on a mechanism that converts circulating CO2 to HCOS- in the fluid.
Considerable attention has been given to the osmolar relation between aque-
ous humor and plasma. Levene’s (287) work and review show an osmolar excess
in aqueous of 3-5 mmoles/kg water in the rabbit; this is not changed by ace-
tazol,amide. Davson (120) considers that the osmotic contribution to flow is
negligible. The osmolarity of dogfish aqueous, however, is lessthan that of plasma
(935-962 milliosmoles/kg), but this must be studied in relation to the ambient
sea, which has’still lower osmolarity of 920 milliosmoles/kg. In this situation,
acetazolamide elicited some rise in aqueous osmolarity, which may indicate a
decrease of fluid production while osmotic equilibrium was approached (132).
The fresh-water fish also has hypoosmotic aqueous; nevertheless active processes
are supposedat work (215).
Arrival at this point through the maze of controversy is regrettably not
enough to answer the problem of how acetazolamide lowers pressure in the eye.
The reduction in HCO3- accumulation in the posterior chamber does appear
to be maintained on the basisof the 6- to-240hr data in Table 23, and there is
unquestionably a pressuredrop as long as drug is maintained. The drop must be
due either to a reduction in aqueoushumor formation or to a reduction in vascular
pressure opposing the outflow of fluid. It is necessary to marshal the arguments
and evidence in this important question.
Becker (22) maintains that the inhibition of carbonic anhydrase reduces
the formation of aqueous humor by 50% in the rabbit and that this reduction
is due to the decrease in the HCOa--accumulating mechanism discussedabove.
(The fact that Becker believes that Cl- accumulation is dominant in the primate
does not influence the present argument, which centers around the link between
carbonic anhydrase inhibition and suppressionof secretion.) Evidence pointing
to flow reduction may be taken from the following nine different types of experi-
ments. a) Alterations in the steady-state concentrations of certain substances,
which normally show a large difference between anterior and posterior chambers,
may be calculated to yield a figure that is proportional to flow. Two such sub-
stances are HCO3- and ascorbate. After carbonic anhydrase inhibition by ace-

October 1967 CARBONIC ANHYDRASE I
75
tazolamide, methazolamide, and dichlorphenamide in the rabbit and acetazolam-

ide in the guinea pig, calculations of the concentration changes of both of these
test substancesyield a figure consistent with a reduction of flow in the range 51-
60% (22). b) Tonography in man and rabbit yields flow data that show a re-
duction in flow of 45-60% after all of the active carbonic anhydrase inhibitors.
The tonographic method is by no means free oJ errors or possiblefaulty assump-
tions, so that this evidence is best recognized as supportive (22). c) Langham (271)
has directly measured the formation of fluid in the rabbit by blocking the outflow
from the anterior chamber with mineral oil and cannulating the posterior chamber
directly. The pressure of the eye was maintained at 35 mm Hg. Under these
conditions, 1 mg/kg methazolamide and 5 mg/kg acetazolamide lowered
fluid formation from about 5 to 2 d/min (270). The time course closely paral-
leled changesin pressureobserved in other experiments (269). d) The appearance
time of intravenous fluorescein into the anterior chamber of the eye, which has
been taken as an index of aqueous formation, is increased from 4 to about 6 min
after acetazolamide (272). e) It has been possibleto visualize the pumping action
of the ciliary processesin vitro*in terms of a shrinkage of the structure. The shrink-
age was reduced by acetazolamide and a reasonable dose-responsecurve found
between 10-* and 1W7 M. The phenomenon was elicited at pH 7.0, but not at
7.4 or 7.7. It ‘was suggestedthat enzyme-mediated buffering of acid (presumably
the author meant H&O2 + Cop) was critical at the lower pH, but not at the
higher range (33). f) Becker (25) found that iodide ion is transported actively
out of the eye in such fashion that its turnover is a measureof aqueousflow. In the
rabbit the value obtained, 1.5 % of the volume/mm, agreed well with other
methodsjust cited. Acetazolamide reduced this t0 0.8 %. g) Aqueous flow may be
measured by compressing the perilimbal area to a pressure of 50 mm Hg and
calculating the volume of fluid that enters the eye. Galin et al. (169) found that
in the human eye flow was reduced 32 %. h) Analysis of facility of outflow sheds
somelight on whether acetazolamide does directly reduce flow of ocular fluid and
whether any other processmay be involved. Relations in the eye are described by
where F = flow; C = facility of outflow, or l/resistance; P, = measuredpressure;

and P, = episcleral venous pressure. The pressure drop (the observed effect)
might then be due either to decrease in F or increase in C. Pv is generally held
to be constant, but Macri’s contrary opinion is given below (304). Since most
experiments show C to be unchanged (22), the fall in P, must be due to decrease
in F. Sears actually showed a decrease in C after acetazolamide, resulting in a
minimal change in P,, with F falling 27 % (464). On the other hand, an increase
in C was postulated by Davson (120) as the cause of lowered P, because s4Na
turnover studieswere interpreted as showing no change in F. The 24Naproblem
is discussedbelow. In another context, Galin and Harris (170) have reported
on four glaucomatous patients with low initial C, which increased after ace-
tazolamide. This appeared responsible for the satisfactory reduction in pressure;
there was no alteration in flow. The authors conclude that acetazolamide may, at

least in this unusual group, have a different action than on secretion; but the
‘improvement in outflow facility cannot be explained. It is hoped that the questions
raised in this paper will someday be answered. ;) The most recent, and in some
ways most compelling, evidence is that of Oppelt (384), who used the dilution of
labeled inulin, as it was perfused from posterior to anterior chamber, to measure
production rate of new fluid by the cat eye. Normal values averaged 15 &nin,
in good agreement with older methods. Acetazolamide produced a smooth dose-
responsecurve from 0.1 to 20 mg/kg, with the maximum effect of 50 % reduction
in rate at 10 mg/kg.
It is important to consider whether a decrease in fluid production of 50 %
could reasonably account for the observed pressure lowering. We may use the
expression of Becker and Friedenwald (26):
Av
1
= jf lw
PO
p
t
where V is the volume; K is a constant (0.02) incorporating physical properties
of the orbit; and P, is initial and Pt final pressure. If P, is 20 mm Hg and Pt is
15 mm Hg after acetazolamide, AV = 5.5 ~1. This decrease in volume would
occur in about 4 min if normal flow (3 pl/ min) is reduced by drug to 1.5 pl/
min. This is about the time at which the pressure drop is manifest (270, 521).
Seven points of evidence are cited to suggest that acetazolamide does not
in fact lower secretion, but elicits the drop in pressureby someother mechanism.
These are listed and evaluated. i> The changes in sodium turnover from plasma
to aqueous after acetazolamide are very slight-at most 10% (206). This matter
is discussedby Davson in detail (120), but the weaknessin the argument is that
sodium enters the aqueous by diffusion from the iris (possibly s-g of total)
as well as from the ciliary processand the proportions of these are not rigorously
known. Thus sodium is a poor substancefor this analysis, and it is possiblethat
the small changes seen after acetazolamide (22) are compatible with 50% in-
hibition of flow. However, using the technique of slow intraocular injection of
aNa, Davson and Spaziani (122) were unable to show any effect of acetazolamide
on the sodium clearance. They take this as evidence that the “effect of the drug
on intraocular pressure is due in part to a change in physical factors affecting
drainage.” k) The concentration of 14C-labeled bicarbonate in the posterior
aqueous of rabbits was measured after its injection into the blood stream (252).
Accumulation of label in the posterior chamber to a concentration greater than
that in plasma (the ratio cited in Table 23) was observed within 1 min of sampling
(Fig. 64. Acetazolamide (50 mg/kg) given 15 min before the injection of the
carbon label delayed accumulation of the label in the posterior chamber to
about 7 min (Fig. 6B). The significance of this in terms of the relation between
CO2 hydration and HCOa- accumulation has been discussedabove. However,
when the same experiment was done, except that acetazolamide was given 60
tin before the 1% injection, there was no effect, i.e., no lengthening of accumu-
lation time of HC03- in aqueous. Since at such time there is a maximal drop
in intraocular pressure [found in other experiments (22)], these observations

suggestedthat the reactions catalyzed by carbonic anhydrase are only indirectly

reflected in aqueous formation (253). But further consideration of these important
experiments leads to another explanation : the lack of effect at the I-hr time
might be due to the fact there is now a more marked respiratory acidosisand the
label in the plasma is now equilibrated at a much higher pCOs,, leading to the
possibility that more of the label can reach the aqueous rapidly by diffusion.
This would mask the effect of the drug, and therefore this experiment should not
be taken as critical evidence against the connection between HCOa- secretion
and pressure. In further work attention must be paid to the acid-base balance
of the animal. Z) All investigators agree that subconjunctivally administered
carbonic anhydrase inhibitors do not lower intraocular tension (22), .even when
it appears that the enzyme in the anterior uvea is > 99 % inhibited (191). How-
ever, such grossassaysdo not reveal whether drug can reach the intimate enzymic
site within the cell for they are based on homogenized tissue, Moreover, it has
been shown that intravenous administration of 10 mg/kg of the anionic drug,
CL 11,366, does not lower the intraocular pressure in rabbits, even though it is
found in reasonably high concentrations in the ciliary process (529). In this case
the drug did not reach the aqueous humor, and it is presumed that it did not
reach the secretory site. Why topically administered drugs-even lipid-soluble
ones-do not appear , to reach the secretory carbonic anhydrase site cannot be
stated. Dr. Norman Ballin recently attempted to lower intraocular pressure in
rabbit and man by ethoxzolamide in water or in peanut oil instilled every few
minutes, but failed. Drug may reach the secretory site, but transiently. Since
inhibition is reversible and an excessof drug must be present for physiological
inhibition, the blood stream may in this situation be uniquely suitable for delivery
of drug. This reviewer cannot agree that the negative results after local instil-
lation serve to dissociate carbonic anhydrase from the maintenance of intra-
ocular tension (191). m) Macri has challenged the position that acetazolamide
lowers intraocular pressure by reducing flow on the basisthat the fall in pressure
is really due to vasoconstriction of the ciliary artery (304) and that the formation
of aqueous humor, as measured by inulin turnover in the eye, is not decreased
by acetazolamide (305). Some of the problems implicit in his argument and data
are mentioned, but an ultimate and detailed analysis must be left to the reader.
Bill (42) has criticized Macri’s work on the isolated iris artery on technical grounds,
and it is certainly true that Macri’s intraocular pressures(31 mm Hg) are much
higher than normal. If acetazolamide and congeners are constrictors of the iris
artery, the action has nothing to do with carbonic anhydrase; Dr. Macri was
kind enough to send us his preparation, in which we found no enzyme. Macri
has not reported on the inactive analogues of acetazolamide. n> Experiments
in which 100 mg/kg of acetazolamide in the cat had no effect on inulin turnover
from the aqueous (and presumably no effect on flow) may be criticized on the
grounds of trauma to the eye, wide variations in flow rates, and the curious fact
that this and even higher dosesof drug were reported not to alter pressure, even
though in earlier experiments in the intact cat as little as 2 mg/kg was active
(305). o) Ytteborg (540) attempted to visualize flow directly in the aqueous veins,,

but found no change in glaucomatous eyes in which pressure was unequivocally

lowered by acetazolamide. If such measurementswere entirely reliable and sub-
jective, the evidence would be a seriousblow at the secretion theory. The author
of this ingenious study concludes, however, that inspection of visible aqueous
veins cannot furnish information about flow and that further work is indicated.
p) Another recent study yielding a radical result is that of Thomas and Riley
(490), who reported that 1% dichloroisoproterenol (DCI) instilled into the
eye *blocked the ocular effect of intravenous acetazolamide; other experiments
involving adrenalectomy and various autonomic drugs are also of interest. Since
DC1 blocks /3 adrenergic effecters, and /3 stimulators (isoproterenol and epi-
nephrine) reduce pressure, the authors raised the possibility that this receptor
system mediates, at least in part, the ocular hypotensive effect of acetazolamide.
This paper is not well documented; for instance, no actual pressuresare recorded
-only tonometer differences after acetazolamide (with and without DCI) are
given. Nevertheless, the questions raised must be answered, particularly in view
of Macri’s (304) contention that acetazolamide is a ciliary artery vasoconstrictor.
It is doubtlessly clear to the reader that the reviewer considers the points
i-p above lacking in one way or another. The original idea, probably derived
from Jonas Friedenwald (see ref. 22), is retained: that aqueous secretion is in
part an effect of the catalytic and noncatalytic conversion of CO2 to HC03-,
and the effects on flow and pressure that follow acetazolamide and its ,congeners
in health and diseaseare due to abolition of the catalytic pathway. Yet it would
be quite improper to discount the provocative experiments of Macri (304), Galin
(170), Ytteborg (540), and Thomas. and Riley (490), which point in another
direction. Confirmation and extension of such work are required.
With this basefor discussion,certain pharmacological data on ocular carbonic
anhydrase are reviewed. Wistrand (521) rigorously defined the intraocular pres-
sure lowering in the rabbit in terms of the distribution of acetazolamide. There
is a smooth dose-responsecurve between 1 and 10 mg/kg. Higher dosesof this,
or any other drug of this class, produce no greater effect than the reduction of
35 % of outflow pressure (intraocular-episcleral venous pressure). The onset of
effect takes about 5 min. The duration of effect is a function of drug concentration
in plasma, which moves toward equilibrium with drug in the ciliary process
and aqueous humor. For the lo-mg/kg dose the effect lasts 1-2 hr. The half-life
of drug in plasma was 27 min, and since plasma binding in this dose range was
about 80%, there must be some renal tubular secretion. In 5 hr, 96 % of drug
was recovered unchanged in the urine. Probably becauseof its great degree of
vascularity, the concentration of drug in the ciliary processrapidly attains that
of the unbound concentration in plasma. But based on other evidence (529;
discussion below), it is felt that the gross concentration of acetazolamide (or
other drug of this class) in the tissue is not the proper quantitative measure of
inhibition. What is sought is the concentration of free drug [Zf] available to react
with enzyme [&I, and it is reasonable to assumethat a minimum value for [Zf] is
the concentration in the aqueous, i.e., the concentration in diffusional equilibrium
throughout the tissue. From the data of Wistrand (521) one may approximate

October1967 CARBONIC ANHYDRASE 79I
this; 30 min after the administration of 10 mg/kg, when pressure lowering is

still maximal, the anterior aqueous contains about 2 pg/ml or 10 PM (the pos-
terior aqueous has somewhat less). Using the formulation of section II, i above
(Eq. IO), this yields, for fractional inhibition, i = lo/(10 + 0.06) = 0.994. Since
1 mg/kg has no activity and assuming that [Ifl would be 1 PM, it appears that
when i = 0.94 there will be no physiologically recognized inhibition. It is in-
teresting that in some tissues, as the kidney, there is little physiological effect
when i = 0.994 (323). The difference is that the ciliary process has about l/20
the enzyme concentration of the kidney; the molar concentration in the processes
is only about 0.4 PM (Table 16). Wistrand’s paper makesit clear why acetazolamide
and congeners are continuously effective in the treatment of glaucoma. As long
as drug is present in adequate concentrations in the tissue, the secretion is reduced.
He discusses,in this and a later paper (523), how the outflow resistancecan change
in compensatory fashion in responseto the decreasedsecretion, and this is why in
certain seriesof animal experiments a drop in intraocular pressure cannot always
be elicited. In glaucoma, however, outflow resistance may be more fixed, and
except in total closed-angle glaucoma, the drugs are effective.
The pharmacology of various other inhibitors, including the clinically used
drugs dichlorphenamide and ethoxzolamide, has been studied with reference to the
lowering of outflow pressure in a comprehensive seriesof papers by Wistrand and
his colleagues (summarized in ref. 522). This work has thrown considerable light
on the underlying physiology and biochemistry. It was shown that the action was
directly ,on the eye, since a unilateral effect could be obtained by injection of
acetazolamide into the carotid artery. For lipid-soluble and reasonably diffusible
drugs, such as ethoxzolamide, methazolamide, and sulfanilamide, the activity in
vivo, after intravenous injection into the rabbit, was a faithfui reflection of the
in vitro activity against carbonic anhydrase when certain pharmacological- data
such as plasma binding and half-life are considered (Table 13). These two findings
make it certain that we are dealing with a specific inhibition at the site of secretion,
or conceivably, but lessprobably, at someother site of carbonic anhydrase within
the eye. Lipid-insoluble polar drugs, which do not penetrate into the aqueous
humor (typified by CL 11,366), are relatively inactive in reducing intraocular
pressure.Failure to reach the aqueous is not in itself regarded as the causeof low
activity; rather this indicates a failure of diffusivity through the tissue and thus
failure to reach the enzymic site. It is significant that the three drugs, methazola-
mide, acetazolamide, and CL 11,366, have this order of activity in reducing intra-
ocular pressure,which is the same as the order of their diffusion into the aqueous
(Table 8). Oppelt has confirmed this observation (personal communication) using
the measurement of aqueous flow in the cat (384). CL 11,366 was about s as
active as acetazolamide (even though it is more potent in vitro, Table 4) and has a
delayed onset of effect. Wistrand also showed that the time of sustained reduction
in intraocular pressurewas a simple function of the half-life of the individual drug
studied. These data (529), together with an analysis of the renal activity of these
samedrugs (323), suggestthat methazolamide acts on the eye at a dose (2 mg/kg)
at which it has minimal renal and respiratory effects. This doseis also active clini-

720 T. H. MAREN VoLume47
tally. The samequantitative activity relations found by Wistrand for lowering of

intraocular pressure by acetazolamide, methazolamide, ethoxzolamide, and
dichlorphenamide (529) apply to the treatment of glaucoma (27).
It is of interest to consider that, for a drug that diffuses readily and is not
accumulated in tissues(494), fractional inhibition at any site is a function of the
unbound concentration in plasma and will therefore be the samethroughout the
body. However, as indicated above, pharmacological activity’ should occur at
relatively low fractional inhibition (and hence lower dose) in a tissue with less
original enzyme. If, for example, there are 1000 parts of enzyme originally present,
for the caseabove (; = 0.994) 6 parts will be free. But for 20,000 parts of enzyme
and the same inhibitor concentration, 120 parts will be free. On the simplifying
assumption that there is the same general relation between catalyzed and un-
catalyzed rates in the different tissues, a drug like methazolamide will then be
most active in the tissuewith the least enzyme. Experimentally, this does appear
to be the case (Table 13).
Since the fundamental reaction being discussedis CO2 + OH- + HCOa-,
it is reasonable to expect that in the eye, as well as in other systems, acid-base
changes would affect the reaction. Since we presume that HCOS- formation in
turn affects aqueous flow and thus intraocular pressure, acid-base changes should
alter intraocular dynamics. Langham and Lee (272) and Wistrand and Maren
(525) showed that metabolic acidosisdecreasedintraocular pressure, the expected
result if [OH-] in the above reaction was reduced by acidification of the secreting
cells. Conversely, metabolic alkalosisdid not alter the initial pressurebut markedly
diminished the effect of acetazolamide (525). This latter result was not confirmed
by Miller, but his data appear to show a modest fall in intraocular pressureafter
administration of NaHCO3, both with and without acetazolamide (355). The
resistance to acetazolamide in metabolic alkalosismight be expected if the increase
in plasma [HCOa-] favored its transfer into the aqueous or if the elevated [OH-]
increased the reaction rate so that it was no longer dependent on the catalytic
rate. Respiratory acidosisproduced only a small drop in intraocular pressure(525);
in this casewe are dealing with opposing forces, sincepC0~ and [HCO 3-1elevation
in plasma would favor HCOa- accumulation in aqueous and acidemia would
oppose it. In other experiments elevation of pCOz appeared to raise intraocular
pressure (194). Hyperventilation, which rapidly lowers pCO2 and raises [OH-],
lowers pressure (194). In all systemsstudied, respiratory alkalosismimics acetazof-
amide; this is discussedin connection with Figure 8 and in the summary (section
IV). The experiments with metabolic acidosis are also of practical interest, since
the long-term acidosisin the glaucoma patient treated with acetazolamide may be
regarded as a therapeutic advantage. The clinical observation that patients with
diabetic acidosis have “soft eyes” may be the result of this phenomenon, rather
than the alleged increase in plasma osmolarity.
The resting potential of the blood-aqueous barrier is 7 mv, with the aqueous
positive. The changes in potential after acetazolamide administration are trivial
(transient rise of 1.4 mv after 100 mg/kg in the rabbit, returning to normal in
20 min) and indicate that the electrical properties of the membrane are surprisingly
stable in the face of maximum carbonic anhydrase inhibition (355).

The sodium and potassium concentrations in the anterior aqueous of rabbit,

monkey, cat (199), and dog (526) do not appear to be significantly altered after
acetazolamide. Unfortunately, no measurementsof cations have been published
on fluid from the posterior chamber. It would be reasonable to expect that in
newly formed fluid inhibition of carbonic anhydrase would not lower sodium
concentration; to the extent that bicarbonate falls, chloride should replace it.
Further work on turnover of Cl- in the posterior aqueous would be of interest.
Attempts to localize carbonic anhydrase by histochemical means have not
been uniformly successful,in the eye or elsewhere.The macrochemical determina-
tion of enzyme has shown it to be present in ciliary body, iris, retina, and lens (14,
178). Carmichael et al. (67) attempted to localize the enzyme in the rabbit. eye
by meansof a zinc stain and by meansof a method described by Kurata (266) for
carbonic anhydrase, which depends on the precipitation of COCOS and then CoS
at the enzyme site. Both methods failed, asmight be expected: zinc is not sufficiently
dissociablefrom the enzyme and carbonate ion would form under conditions of
the test whether or not the enzyme is present. A more recent study (261), using a
further modification of the Kurata technique, claimed to demonstrate the enzyme
in the various structures of the eye of the mouse and rat. Whether this was due to
speciesdifferences or altered techniques is not clear. There is a similar unsatisfactory
situation with respect to application of the Kurata technique to the kidney and
other organs (II, IC above). Muther (368) hasreviewed the literature and suggested
that the stain is due to tissueelements that bind cobalt and is not necessarilydue to
carbonic anhydrase. Goren et al. (182) attempted to localize 35%acetazolamidein
the rabbit eye using radioautography. Their pictures show some localization in
ciliary processes,iris, and retina, but no cellular detail. Their conditions were not
optimal to distinguish enzyme-bound from free drug, and the scatter of beta rays
from % precludes an analysis of fine structure. The entire problem is presently
unsettled ; neither histochemical nor radioautographic techniques have been
perfected. The current work of Hansson ( 198) may resolve the histochemical
problem; he shows good localization of carbonic anhydrase in ciliary body and
elsewhere(section II, IC).
Electron microscopy o ciliary epithelium in the normal state and after
acetazolamide administration has been studied by Holmberg (221) and Pappas
and Smelser (391) in man and rabbit. An interesting discussion of the many
factors involved may be found in the Transactions of the Fourth Conference on Glaucoma,
Josiah Macy Foundation, published in 1960. Similar studies have been done on the
chicken (462). Among the changes observed after acetazolamide are ,increase in
vesicles and added complexity and infolding of plasma membranes. The signifi-
cance of these alterations is certainly not clear and they may be artifacts.
The role of enzyme in the lens is unknown. Acetazolamide does reduce the
pressure in aphakic eyes. In the rabbit, enzyme concentration in lens is 10 times
that in the ciliary process (14, 178), although this difference’ does not reflect the
actual concentration in the secretory cells of the ciliary body. The concentration
of enzyme (per wet wt) is greatest in the nucleus, diminishing outward to cortex
and epithelium in the ratio 5 : 3: 1. The gradient of Mg++ and Na+-K+-ATPase
occurs in just the reverse order, but the reviewer cannot agree that this throws any

light on the role of carbonic anhydrase (67). Animals receiving acetazolamide show
a concentration of drug in the whole lens (4 PM) about equivalent to its carbonic
anhydrase concentration (200 e.u./g X conversion constant 0.017 = 3.4 PM),
but there is no evidence of lenticular damage (213, 328). Neither the teleost (214,
308) nor the elasmobranch lens (319) contains carbonic anhydrase, although the
other tissuesof the eye have a similar enzyme distribution to that of the mammal.
A useful line of inquiry is the action of carbonic anhydrase inhibitors in the active
transport of K+ into fish and mammalian lens in the type of experiments of Kinsey
and McLean (251). Dr. Everett Kinsey found no detectable effect of lo-4 M
acetazolamide on transport of K+, Na +, Rb+, or Cl- into the rabbit lens in an 18.
hr culture period (personal communication). Perhaps a study of rates over a
shorter time would reveal positive effects. No cataracts have been observed in
chronic toxicity studies in dogs and rats (328), and humans treated for glaucoma
have been carefully observed, with negative results (B. Becker, personal com-
munication). Becausethere is so little acetazolamide in aqueous humor, it may be
argued that [If] in or surrounding lens was too small to inhibit completely, but
this would not apply to methazolamide, where again extensive clinical observations
have been negative. In one study (231), cataracts were reported in newborn rab-
bits after acetazolamide, but in similar and more extensive experiments this was
not confirmed (H. Sauers and D. Shanklin, personal communication).
There is no carbonic anhydrase ( < 1 unit/g) in the cornea of rabbit ( 177),
man, or bullfrog (unpublished observations). This is of much interest in the context
that the bullfrog cornea actively transports chloride and that the potential in vitro
is abolished by high (5 mu) concentrations of methazolamide (255). This is a
significant observation in showing one of the very few chemical or physiological
systems,other than carbonic anhydrase, susceptibleto this drug, albeit lo3 greater
concentrations are required. The situation is analogous to that in the frog gastric
mucosa (In, 5E).
The role of carbonic anhydrase in the retina is likewise unknown. One abstract
statesthat inhibition reduces K+ uptake in ox retina slices (113), which is similar
to data for brain and kidney (112). Leder (280) has attempted to localize the
enzyme by histochemical techniques. His results agree well with those of Korhonen
and Korhonen (261). The CoS stain is found in the limiting membranes, pigment
epithelium, and most strongly in the glial elements. Neurons were not stained.
Again the question of specificity of the Kurata technique must be raised. But this
finding is a striking analogy to the work of Giacobini on the brain (171) and
suggeststhat carbonic anhydrase may have a transport role in the retina. In
mammals there is no evidence that retinal function is injured by’long-term maximal
dosesof acetazolamide, but in the perch, rods and conesappear to be irreversibly
damaged and the fish become blind (308).
Several miscellaneousobservations in the comparative physiology of the eye
should be mentioned. Despite much chemical data on the aqueous of the elasmo-
branch. (132, 320) no flow or pressure measurementshave been made. A drop in
pressureafter acetazolamide has been reported in the lake trout (214), despite the
absenceof a ciliary body. The iris may be the secretory tissue;acetazolamide lowers

aqueous[HCO 8-1 in this species(Table 23). Seaman and Himmelfarb (462) have
speculated whether the avian pecten oculi also operates as a “marginal apparatus
for the maintenance of intraocular pressure.” This mysterious organ contains a
high concentration of carbonic anhydrase (234) and undergoes rather similar
ultrastructural changes to those in the ciliary process after acetazolamide (462).
There is somequestion whether the hen showsa drop in intraocular pressureafter
acetazolamide. Sears (463) found no effect at dosesup to 150amg/kg but Seaman
and Himmelfarb (462) obtained a drop at 75 mg/kg. Their initial pressurewas
18.6 mm IIg against Sears 15.6 mm Hg, which may be the critical difference; also
Seaman used tonometry whereas Sears placed a needle in the eye and measured
pressuredirectly. The matter is unresolved, but the two papers both show a lowered
responsein terms of drug dose compared with the mammal. Further work should
include the in vitro responseof ciliary processand pecten oculi enzyme to acetazol-
amide and measurement of aqueous electrolytes in this species.
D. Cerebroxpinal fluid and brain
This topic has certain facets in common with the previous section, but it will
be appreciated that there are many significant differences between the secretion of
aqueousand of cerebrospinal fluid and the respective roles of carbonic anhydrase
in the two situations. The anatomical basis for this section is the presence of
carbonic anhydrase in the choroid plexus of all vertebrates examined (Table 16),
and it is interesting that in this tissue the enzyme concentration is some 10 times
greater than in ciliary processand about equal to that in the kidney (44, 160). The
following treatment is divided into three topics: CSF formation and pressure, CSF
and brain electrolytes, and the role of carbonic anhydrase found in brain.
I) CSF formation and pessure. Although the early (1954-1960) work was ap-
parently contradictory and somewhat controversial, the sharpening and diversity
of techniques in the past 5 years have yielded experiments that show unequivocally
that carbonic anhydrase inhibition reduces CSF formation, with the usual sequel
of reducing pressure. The initial confusion arose from the fact that intravenous
acetazolamide causesa rapid transient rise in the apparent flow and pressure in
most species,due to inhibition of red cell enzyme and consequent elevation of
pCOz in tissues.Specific measurementsof pCO2 in brain tissueafter acetazolamide
have been made (51a, 352) and rises of 8-20 mm Hg have been found. Cerebral
vesselsmay be shown to dilate after as small a rise as 3 mm Hg pCO2; thus it is
easy to see how blood flow increases,compressing brain tissue and causing CSF
pressureto rise. CSF is forced out, giving the appearance of increased flow. The
subject is reviewed with pressuredata for man (332) and in an excellent study of
cerebral hemodynamics (141). The effect is great enough to increase p0 2 in
cerebral venous blood, even in patients with cerebral arteriosclerosis (183). It
was shown in these and other studies that the rise in flow and pressurelasted less
than an hour and that there was a later fall in both modalities (Table 24). More-
over, it was apparent that the rise in flow and pressureonly followed rapid intra-
venous drug; if slow infusion (526) or oral (332,526) or intramuscular (unpublished

TABLE 24. Experiments showing efect of acetazolamide on cerebropsinal fluid dynamics
Species Method Result: Effect of Acetazolamide Reg;zEnyd
Dog Direct, transducer. Initial rise; late fall absent or (362, 526)
slight.
24Na exchange time. Lengthened by about 100%. (161)
14C-inulin dilution. CSF production decreased 25- (97, 386)
50%.
Exit rate of 14C-inulin kout decreased 25-50yo. (443, 504)
dextran, RISA. ’
F Direct volume of out- Initial increase (20 min); late PF
flow. (2 hr) decline.
Cat P Transducer (362, 496) Initial rise (362) and late fall Note diver-
or Hz0 manometer (257) ; immediate fall (496). gence.
(257).
F Direct volume of out- Little or occ. rise; 50% fall in (3, 160, 254,
flow. 10 min. 324, 496)
F IGinulin dilution. Max. effect (50% drop) with 1 (es)
mg/kg CL 11,366.
Rabbit P Transducer. Immediate fall. (362, 496)
F Direct volume of out- Immediate fall (496)
flow.
F 24Na exchange. 40% reduction in turnover rate. (121)
F 14C-inulin dilution. CSF production decreased 40- (410)
50%.
F AA-V volume across 85% reduction. (512)
choroid plexus.
Monkey P Water manometer. Immediate rise; late fall only (257)
if initially elevated by alu-
mina gel.
Man P Water manometer. Immediate rise after iv but not Hydro-
oral drug (93,332); slight fall cephalus.
in normals and hydrocepha- c+% 14%
lies after 12 hr (332) 224)
Rat F 1311- and 14C-inulin Reduced. (427)
exit.
F&P Augmentationofurea Flow reduced; pressure un- (426)
effect; direct. changed
Dogfish F 14C-inulin dilution. Reduced 37%. WV
Drug given iv except as noted. Doses varied between 5 and 15 mg/kg except in refs.
160 and 254, where full response was obtained at 1 mg/kg in cat. Few dose-response curves
are available; see III, 5D.
data, Pharmacology Dept., Univ. of Florida) drug was given there was no rise,
with a later fall. It is also significant that in the dog methazolamide effectively
raises CSF pressure at 1 mg/kg, acetazolamide at 5 mg/kg, and CL 11,366 at
10 mg/kg (495,526), which reflects their relative rate of entry into the red cell (220).
A fall in flow or pressurewas elicited when drug was given orally or intramus-
cularly, or after the transient rise that followed intravenous injection, or imtiedi-

ately in a few species (Table 24; 44, 254, 257, 332, 496). Critics were properly
concerned that flow was measured directly in an open system against no pressure
head and that pressure lowering could also reflect alteration in the outflow chan-
nels; thus changes in formation or secretion of fluid were not rigorously proved.
However, measurement of CSF formation during carbonic anhydrase inhibi-
tion by a variety of new and more rigorous techniques shows a clean-cut decrease
and confirms the nature of the transient pressure rise. Table 24 lists the various
studies by species; historically, the first study of the newer type was by Davson
and Luck (121), who showed that 24Na turnover from plasma to CSF (but not to
brain) was reduced 40 %, which was interpreted to mean a slowing of fluid produc-
tion by at least this value. This is the same figure obtained by the open-flow (254)
and isotopic systems. The unique experiments of Welch are also entered (512) ; in
these, fluid formation is measured as the difference between the volumes of plasma
compartment of arterial and of venous blood flowing across the choroid plexus.
The effect of the drug, inexplicably, was greater than by other methods, whether
given by vein or applied to the plexus. Choroid plexus fluid in the cat was collected
directly at the formation site by Ames et al. (3), who found that acetazolamide
( 1O-3 M) applied to the secretory tissue reduced the formation rate 50 %.
The method that appears to define formation of new fluid most rigorously is
the ventriculocisternal perfusion system developed by Pappenheimer’s group (207)
in which the dilution of inulin as it passes from lateral ventricle to cisterna magna
indicates the addition of new fluid to CSF. Inulin or 14C-carboxy inulin are used
because they do not diffuse significantly into the brain (207) under these conditions.
Using this method, Oppelt et al. (386) obtained a rough dose-response curve in the
dog for both intravenous acetazolamide [in agreement with Cserr (97)] and
methazolamide. The maximum response was a 40-50 % reduction in flow. Half-
maximal response was obtained between 5 and 20 mg/kg of acetazolamide and l-5
mgJkg methazolamide. Under similar conditions CL 11,366 gave half-maximal
response at 1O-20 mg/kg (Oppelt, personal communication). These relationships
are reasonably close to those observed for lowering of intraocular pressure in the
rabbit (529; section III, 5C above), and probably for similar reasons of penetrability.
However, CL 11,366 reduces CSF flow in cat at 1 mg/kg iv; in these experiments
little or no drug could be detected in the CSF, but there was uptake into the choroid
plexus (48). Why the cat is more sensitive than the dog is in part due to the differ-
ence in plasma binding for this drug (cat, 80 %; dog, 93 %) at the observed plasma
concentration.
&err (97) showed that acetazolamide in the perfusion fluid also lowered CSF
flow. Full effect (50 % lowering of flow) was achieved by about 10 bg/ml in the
perfused fluid (Cserr, personal communication). This is more than is required in
CSF when introduced from the plasma side; unbound plasma concentration in the
first 30 min after 20 mg/kg iv in the dog would be about 20 pg/ml; CSF concentra-
tion would be about 2 pg/ml (328). Using CL 11,366 in the cat, Broder and Oppelt
(48) were also able to reduce CSF flow, but 100 pg/ml were required in the per-
fusion fluid. Since the effective intravenous dose (1 mg/kg) generates an unbound
plasma concentration of about 0.3 &ml and a CSF concentration of about 0.01

&ml, it would appear that the secretory cells are reached much more readily
from the plasma side. The lo-fold higher concentration required for CL 11: 366,
over that for acetazolamide in perfusion fluid, is probably due to the greater ioniza-
tion and lower tissue permeability of CL 11,366. These experiments are in contrast
to those for the eye, in which conjunctival instillation or aqueous injection of
acetazolamide or ethoxzolamide fails to lower intraocular pressure (III, SC). In
that case, however, the drug is almost certainly washed away rapidly.
The fractional inhibition (i) of enzyme in the choroid plexus may be calculated
for the minimum effective concentration of acetazolamide in contact with the
tissue (2 &ml or approximately 10 PM). Using equation II and the reasoning
developed in section II, 7, i = 0.994. Table 24 showsthat all methods in dog, cat,
and rabbit agree closely with each other and among other speciesin showing that
carbonic anhydrase inhibition reduces CSF flow a maximum of 50%. The cat is
curiously sensitive to the inhibitors in that it respondsfully to acetazolamide (254)
and CL 11, 366 (48) at 1 mg/kg-about soth of the dosenecessaryfor the dog.
Another speciespeculiarity, possibly of someimportance, is that the rabbit does
not show the immediate rise in CSF pressureafter intravenous drug that charac-
terizes all the other species.
There is no doubt, then, that acetazolamide and its congeners reduce CSF
flow; the effect on pressure is more complex and variable. The initial rise after
intravenous acetazolamide has been discussed;we are concerned now with the
later pressure fall that is consequent upon the reduction of flow. In the normal
animal or man, the reduction in pressureis usually delayed and of slight magnitude.
The immediate fall in the cat (496) has not been confirmed (254), although there
is a modestfall about an hour after the initial rise (257). The only other experiments
showing an immediate and large fall (to about 70 % normal in 15 min) are from the
rabbit, and these have not been published (362; Mithoefer, Mayer, and Stocks,
personal communication). Monkey (257), dog (526), and man (332) show little
fall in pressurefor several hours, but 25 hr after acetazolamide there is a small
pressurelowering in the latter two species.It must be recognized that CSF pressure
in the normal animal or man (90-144 mm H20) is not very much greater than
venous pressure. “Secretion” pressureis small-a difference from the eye in which
secretion pressure is half of total intraocular pressure of 250 mm H20. A further
point of relevance is that turnover of CSF is relatively slow-about 0.3 % of the
volume per minute compared to 1.5 % in the eye (118), so that lowering of secre-
tion might be predicted to have, if any, a delayed effect on the pressure.
In monkeys with acute experimental elevation of intracranial pressure, aceta-
zolamide elicited a lowering in about an hour. Unfortunately, there were no un-
treated controls (257). In hydrocephalic children who are reasonably stabilized,
there is usually a fall in pressure after oral acetazolamide (44, 144, 224, 332); it
is clear that this doesnot occur until 8-24 hr (332). In responsivecasesthe lowering
is maintained (44,224). Attempts to treat nine children in the neonatal period who
had rapidly progressive diseasewere unsuccessfulin all but one case (Jelks and
Maren, unpublished). This work was complicated by the difficulties of oral dosing,
and there were respiratory complications (tachypnea, elevated pCO2) in two

patients with associated pulmonary disease. The most recent and best clinical
study concludes that acetazolamide is probably superior to surgical treatment in
children with a slowly progressive disease (224).
One of the unsolved problems in this area is the quantitative relation between
flow and pressure. Although it is clear that acetazolamide reduces flow about 50 %,
the net pressure effect cannot be predicted either in the normal or pathological
state because of unknown factors of resistance dictated by the anatomy of the
CSF pathway, elasticity of the brain, and patency of the outflow to the veins.
As in other physiological systems, the effect of acetazolamide could be mimicked
by appropriate alterations in acid-base balance (385). Both metabolic and respira-
tory alkalosis diminished flow. The latter alteration, in which blood pH averaged
7.7 1 and pC0 2 10 mm Hg, produced a striking mean decrease of 46 % in the six
dogs tested. Addition of acetazolamide made a still further decrease to 25 % of
control rates. Using direct formation of fluid at choroid plexus in the cat, Ames et
al. (3) also obtained a decrease in flow when pCO2 was lowered to about 8 mm Hg.
Oppelt et al. (385) found no increase in formation with high pCO2, but with
greater changes Ames increased flow 60 % (3). This is similar to the kidney, in
which alkalosis mimics and acidosis opposes or blocks the acetazolamide effect.
Metabolic acidosis diminished the effect of acetazolamide in reducing CSF flow
(385), and it may reasonably be asked whether this might interfere with the desired
therapeutic effect in hydrocephalus. The effect of low pCO2 in reducing flow may
be mediated through cerebral vasoconstriction, perhaps directly on blood flow
through the choroid plexus. This may be a contributing factor, but the idea that
cellular pH is involved is supported by the fact that metabolic alkalosis produces
the same effect on CSF (385) and that the effect of inhibitors in all carbonic-
anhydrase-dependent systems is mimicked by low pCO2 (Table 28 and section
IV). The data suggest that the role of carbonic anhydrase# in the choroid plexus,
as in kidney, has to do with an acidic milieu or gradient at the secretory site. CSF
appears to be an acidic secretion, not only in the normal but in the face of long-
term systemic acid-base changes (414). The cat may be an exception (148); but
gradients of low pH and [HCOS-] are particularly convincing in the goat (157).
The relation of the carbonic anhydrase system to the cerebrospinal fluid control of
respiration (see work of Severinghaus, Pappenheimer, Loeschke, and Mitchell
reviewed in refs. 157 and 414) probably lies in this area of acid secretion, but has
not yet been explored. A critical experiment will be the type illustrated in Figure
6 applied to cerebrospinal fluid.
2) CSF and brain electrolytes. The ionic composition of CSF electrolytes in mam-
mals hasbeen reviewed by Davson (118). Further work has revealed that one of the
chief characteristics of this fluid-[Cl-] excessover plasma-is also found in fish
(319), so we may regard this as a stable vertebrate characteristic. [It must be
mentioned that the excessin cat choroid plexus fluid (10 mM) is lessthan that in
cisternal fluid (18 mu) ; but the ratio of [Cl-] in choroid fluid/plasma water of 1.08
(2) is still significantly higher than that of an ultrafiltrate (118).] The second
significant mammalian characteristic is a K + deficit in CSF, with regulation of
CSF [K+] independent of its plasma concentration (97). There is little species

TABLE 25. Electrolytes of venous plasma (P) and cerebrospinal jhid (C> of man: efect
of long-term carbonic anhydrase inhibition (44)
PH HCOa- I PC02 Cl- I Na+ El+

-- .. I
P C P c P c P c P c P c
-- --- P--P --
Untreated 7.43 7.37 23.7 21.4 35 37 108 123 154 147 5.3 3.8
Acetazolamide (avg 7.32 7.36 15.7 16.6 29 30 120 128 159 147 4.9 3.8
dose 66 mg/kg per
day)
Untreated group was composed of mentally retarded children. Treated group had
hydrocephalus; electrolytes are not abnormal in this condition (332). Average plasma con-
centration of acetazolamide was 190 PM and CSF 1.7 PM. Fluid was taken from lumbar space.
In each group N = 7. Standard errors are given in the original paper. Data are in miIlimoIes/
kg H20; pCO2 in mm Hg.
difference in CSF electrolyte profile among the various mammals. [Although it is

argued that fresh cat choroid plexus fluid with [K+] of 3.15 mM (compared with
plasma of 3.55 mM) is not significantly lower than that of an ultrafiltrate (2); this
ratio of 0.885 is in fact less than an ultrafiltrate ratio, which is usually taken as
0.96 (118), or at lowest 0.93 (2).] Table 25 gives data for man; these data were
selected because they show the effects of complete inhibition at equilibrium between
the two fluids. Normal CSF is slightly acidic in relation to plasma; this is altered
by acetazolamide. At the same time, the [Cl-] excess of 15 mu is reduced to half. A
small reduction of the chloride excess is observed in acute experiments in cat and
rabbit (12 1) but none in the dog (526). No reduction of [Cl-] was found in newly
formed cat choroid plexus fluid after local application of 10B3 M acetazolamide;
but it should be noted that under these conditions the [Cl-] excess in the control
fluid is only 10 mM (3), and about half of this is demanded by the Donnan ratio of
1.04 for (Cl- concn. in fluid)/(Cl- concn. in plasma H20) ( 118). The most significant
effect of acetazolamide on CSF [Cl-] was found in the dogfish (3 19) ; the normal
excess (concentration of Cl- in CSF minus that in plasma water) of 18 mM is
reduced to a deficit of 8 mM in 6 hr. It is possible that this clear result reflects
sampling close to the secretory site. These data suggest a role for choroid plexus
carbonic anhydrase in the secretion of Cl- and possibly of H+; further evidence
and relations on this point are now examined.
When carbonic anhydrase is inhibited for 24 hr in the dog, there is no change
in CSF HC03- concentration, despite a fall of 6 mM in the plasma (319). There
is then a marked rise in CSF/Pl,ste, ratio of [HCO3-1, from 1.01 to 1.31, and an
alkalosis of CSF relative to plasma. (It is notable that in the same experiments the
[HC03-] in the posterior aqueous fell 8 mM with a lowering in aqueous/Pl,,ter
ratio of [HCO 3-]). It could be argued that this relative CSF alkalosis after inhibi-
tion is due only to the fall in plasma [HCOZ-1. However, similar experiments with
acetazolamide in the dogfish, in which there is a rise in plasma [HCO,], yield a
greater rise in CSF [HCO 3- ] , so that 24 hr after acetazolamide the CSF/PlwBte,

ratio for this ion has increased from the control value of 1.OOto 1.63 (3 19). These
experiments, together with the fact that alkalosis mimics the effects of inhibition
on both CSF flow (385) and Cl- concentration (C. A. Goodrich, personal com-
munication), suggestthat H+ formation, subservedby CO 2hydration and carbonic
anhydrase, is a key event in CSF secretion.
Brain electrolytes have also been examined after acetazolamide administra-
tion. In the rat 6 hr after 20 mg/kg orally there was no change in brain [Cl-] and
an insignificant rise in brain [HCO 3-1. Th ere appeared to be a small decrease in
intracellular [Na+] and Na+ turnover (260). In a similar protocol, but u&g some-
what larger drug dosesintraperitoneally, the small rise in brain [HC03-] was
again found (256). Since this occurs with a falling plasma [HCO 3-], the result of
inhibition, as for the CSF/Pl,Bter ratio, is to raise the brain/Plwater ratio for this
ion. Again it is instructive to turn to the dogfish, where plasma [HC03-] moves
upward during inhibition. It was found that brain [HCOa-] also rises 6 hr after
acetazolamide (3 19). This does not appear to be secondary to the rise in plasma,
sincein companion experiments in which NaHCO3 was injected, plasma [HC03-]
rose to about 3 times normal with no change in brain [HC03-1. It is tentatively
concluded that brain [HCO3-] rises after carbonic anhydrase inhibition, as a
result of secretory changessimilar to those described for CSF.
The question of the electrical potential of cerebrospinal fluid has been ex-
amined by Held et al. (208). It bearson the present subject in showing that neither
Cl- nor HCO3- is passively distributed between CSF and plasma. The sum of Cl-
+ HCO, is constant, and the electrochemical differences for HC03- and Cl-
between CSF and plasma are maintained without net flux (157). Other work from
Pappenheimer’s laboratory (392) indicates that the pH of CSF and brain
extracellular fluid has an important role in the control of alveolar ventilation.
Small changes of the order elicited by acetazolamide, as shown in Table 25 (0.1
pH), .are significant. These are further clues that carbonic anhydrase in choroid
plexus and glia (171) has a role in secretion of H+ and Cl- in CSF and brain fluid
and that both respiratory control and flow of CSF are, to somedegree, the results
or hydration of CO2 in these structures.
K+ concentration in CSF is not altered by acetazolamide (Table 25) and K+
flux between fluid and plasma is unaffected (97).
3) Role of carbonic anhydrase found in brain. The concept of brain carbonic an-
hydrase arose when Ashby (9 and earlier papers cited) described the enzyme in
brain homogenates. Davenport found no effect on conduction rate in cat periph-
eral nerve after soaking in 2 mu thiophene-2-sulfonamide (103) ; this is readily
explained by absenceof enzyme in this tissue (379) and unpublished observations).
After large (200 mg/kg) intravenous dosesof this drug, which yielded concentra-
tions in brain of the order of 1 mu, there were no changes in spinal reflexes or
electrical activity of the cortex, including responseto strychnine (103). Almost 20
years later, however, Esplin and Rosenstein (149), using 20 mg/kg acetazolamide
in the samespecies,found that the effect was to depressselectively the spinal mono-
synaptic responseprecisely in the manner of CO2 gas. In both cat and monkey,
acetazolamide and CO2 decreased electroencephalogram (EEG) spikes in the

normal and after application of penicillin to the cortex (353). Sulfanilamide and
acetazolamide were found to reduce the electroconvulsive threshhold (ECT) in
experimental animals, and the results were correlated with the concentrations of
drugs in the brain (359). There has been a large clinical experience with acetazol-
amide in epilepsy; a seriesof 178 caseswas studied by Chao (74), with improve-
ment shown in 43 %. This paper also reviews the literature to 1961.
It has been confirmed that other drugs of this class, for example methazol-
amide, reduce ECT in mice and rats (186). Methazolamide, which penetrates the
brain better than acetazolamide, is more effective. There is a marked similarity
between the effects of acetazolamide and CO2 on brain excitability; this was inter-
preted as due to an increased concentration of carbonic acid within brain cells
(259). In this view, the role of carbonic anhydrase in brain cells is the catalytic
dehydration of carbonic acid.
However, all of the above findings must be reinterpreted in the light of the
evidence that CO2, not carbonic acid, is the end product of decarboxylation in
brain (374) and that carbonic anhydrase is not found in neurons but in glia (171).
The writer’s view is that the anticonvulsant effect is due to CO2 retention, second-
ary to inhibition of enzyme in red cells (III, 5.4 above). The connection between
brain penetration and anticonvulsant effect (186,359) is not causal; the samedrugs
that penetrate brain rapidly do the sameinto red cells (220). This thesisis certainly
supported by many parallels between the effect of CO 2 and acetazolamide: in
electroshock (259) ; on depression of spinal monosynaptic transmission ( 149);
the toxic and metabolic effects in patients with hepatic encephalopathy (413); and
the effect on EEG (353). It has also been shown that intravenous acetazolamide
causesa large rise in pCO2 recorded from the brain surface (352,353); thesepapers
by Meyer and colleagueshave been curiously neglected. The pCO2 effect is not
observed when indirect methods of measuring acid-base balance are used (260).
The CO2 effect in brain after acetazolamide is thus secondary to red cell inhibition,
and carbonic anhydrase in CCbrain”is confined to choroid plexus and glia, where
it has a secretory role, as elsewherein the body.
Certain aspectsof the anticonvulsant activity of acetazolamide and congeners
are of interest and may throw somelight on whether the mechanismjust suggested
is reasonable. Millichap (357, 358) put forth the thesis that the development of
seizure susceptibility in growing rats was related to the increasing level of brain
carbonic anhydrase between birth and 30 days. The newborn rat responds to
maximal electroshock with hyperkinetic behavior, but neither clonic nor tonic
convulsions could be induced. Brain enzyme was 25 % the adult level, and brain
total CO2 was 10 mu higher than adult level. In this situation, the hyperkinesis
was totally refractory to acetazolamide. It was also observed that the induced
minor (hyperkinetic) seizure in the 30day-old rat is resistant to acetazolamide,
while the major tonic-clonic discharge is abolished by the drug. Depending on the
grade of seizure, 50 % protection was associatedwith 86-99.4 % inhibition of brain
enzyme. Although these observations are of interest, they do not prove that brain
enzyme is responsiblefor the seizure discharge or that the anticonvulsant effect of
acetazolamide is related to inhibition of brain enzyme. It is likely that the high

level of total CO2 in the brain of the newborn protects against electroshock; it is
unfortunate that blood CO 2 was not determined at the sametime. However, the
level of brain enzyme at birth is by no meansinsignificant, and it is far greater than
the enzyme that remains active after moderate dosesof inhibitor. The argument is
thereby weakened, but the major defect, as noted above, is in the concept of brain
rather than red cell carbonic anhydrase as a regulator of CO2 equilibria.
Koch and Woodbury (260) believed that the anticonvulsant effect of acetazol-
amide was-due to CCretentionof metabolically produced H&O 3,” which reduces
the permeability of the cell membrane to Na +. The first part of this premise is
dealt with above; quite clearly CO2 is produced, not H&03. Evidence for altered
permeability of neurons to Na+ is not convincing, although a small decrease in
Na+ turnover after acetazolamide is reported (260); this is not in agreement with
Davson (119). Again, it does not seemnecessaryto invoke anything further than
CO2 retention in brain (352) to explain the data of Millichap (357) or Koch and
Woodbury (260). Unfortunately, the intimate action of CO2 itself is unknown.
Gray et al. (186) have studied the pharmacology of methazolamide with
respect to its anticonvulsant action. By the oral route the EDGO against maximal
electroshock was 2.4 mg/kg in the rat and 14 mg/kg in the mouse.Acetazolamide
was about s as active. NH&l enhanced and prolonged the anticonvulsant effect.
An important finding was that under properly controlled conditions no tolerance
developed to methazolamide. NaCl administration reduced the effectiveness of
methazolamide.
Gray and colleagues (187) found that carbonic anhydrase was virtually con-
fined to the supernatant fraction of brain tissue, but this shedsno light on which
cells contain the enzyme. It was alsofound that methazolamide reached the super-
natant fraction in concentrations sufficient to inhibit 99.9 % enzyme present.
Gray et al. (188) made a significant advance in this subject when they found
that reserpine given to mice, l-24 hr before methazolamide, entirely abolishesthe
anticonvulsant effect of the latter drug. In sharp contrast, the activities of diphenyl-
hydantoin and phenobarbital were not altered. The activity of methazolamide
could be restored by iproniazid or DOPA, and the conclusion was reached that
catecholamine depletion was the basisfor the effect. Further work (189) showed
that of several amines studied at a given dose of reserpine (0.125 mg/kg) only
norepinephrine was depleted from the brain -to the extent of about 20 %. It may
be that this is a labile and significant fraction. It was only when norepinephrine
was repleted in brain that the activity of methazolamide was restored. Thus, Gray
and Rauh (189) conclude that norepinephrine is specifically involved. A possible
explanation for the effect is that reserpinized animals have sufficiently depressed
metabolism so that CO2 accumulation was negligible even when carbonic anhy-
drase was inhibited. Gray’s work has been extended by Rudzik and Mennear,
who found that all amine depleting agentsand adrenergic blocking agents antago-
nize the action of acetazolamide (452). Furthermore, adrenergic stimulators, notably
metaraminol and ephedrine, potentiate the anticonvulsant activity of acetazol-
amide (351).
Recent experiments by Gray and Rauh (190) suggestthat the anticonvulsant

action of the carbonic anhydrase inhibitors dependson drug acting on the red
cells rather than in brain. Within one circulation time, the rat receiving CL 11,366
had marked protection to electroshock. Five minutes after giving CL 11,366 to
rats, the ~~60 was 4 mg/kg. By 1 hr this value was 20 mg/kg. In this interval the
drug would increase in the brain, but decreasein the plasma; it is the unbound
concentration of drug in plasma that determines its respiratory effect. For any of
the inhibitors, the anticonvulsant effect can be establishedwithin secondsafter an
injection, a time at which brain penetration for the more polar drugs is minimal.
Comparing CL 11,366 and methazolamide, there was close agreement between
anticonvulsant potency and elevation of venous pCO2 in the rat. It was felt that
in the particular caseof methazolamide in the mouse, there may be a direct effect
on the brain. Perhaps the most compelling evidence in favor of the thesisadvanced
above, that CO2 retention is the basisfor the anticonvulsant action of the carbonic
anhydrase inhibitors, is the finding (W. D. Gray, personal communications) that
reserpine abolishes the anticonvulsant action of CO2 in rats and mice, just as it
doesfor methazolamide. Since theseare the only two agents so affected, and since
their physiological actions are inextricably linked, the connection seemsinescap-
able. The author is pleasedto wield Occam’s razor, he believes effectively, after
many years of intimidation by this fashionable medieval instrument.
Nair et al. (373) found that the anticonvulsant action of acetazolamide is
enhanced by prior X irradiation to the head. The authors believe the basisfor the
effect is increased penetration of the inhibitor into certain regions of the brain
This writer finds the evidence unconvincing; the increase is only 20 %. However,
there is no other explanation of the effect.
Some miscellaneousexperiments on nerve and brain are mentioned briefly.
Various sulfonamides may produce a small decline in the resting potential of
peripheral nerves (379, 468) and an increase in stimulation threshhold (379). Such
effects are not always seen (103). Moreover, there is no carbonic anhydrase in
peripheral nerve (379, 502) and the observed effects are probably nonspecific due
to high concentrations ( 10e3M) used. The numbness, paresthesias, and tingling
in patients after acetazolamide and congeners is probably a central effect, but is
not understood.
Studies of ion movement in isolated brain or choroid plexus tissue after car-
bonic anhydrase inhibition remain to be done. Preliminary observations by Davies
et al. (112) suggestthat potassiumuptake by brain slicesis prevented by inhibition;
however, drugs were used in 10D2M concentrations and 02 uptake was inhibited.
It is essentialthat future in vitro aswell asin vivo work be done with varied dosesof
inhibitor and accompanied by estimations of residual enzyme activity of the tissue.
E. Stomach
The gastric mucosa was the first tissue(other than red cells) in which carbonic
anhydrase was found (loo), but its role here is probably the most elusive of all.
Table 16 showsthat concentration of enzyme in the whole rat stomach is about 500
units/g. If parietal cell or effective secretory volume is 5 % that of the stomach,

October 1967 CARBONIC ANHYDRASE
733
this yields 10,000 units/ml, which agrees with Davenport, translating his units to
ours. In molar terms, using the relations described in section 1 above, this is 200
PM. This figure has been recently validated by a study of the binding of acetazola-
mide in the rat stomach (369). The 160 of acetazolamide against perfused rat and
dog gastric mucosa (4 X 10-* M) is similar to that for dog blood and human red
cell carbonic anhydrase C. Also, and more significant for the in viva discussion to
follow, the 190 was 30 X 10-* M and enzyme was “completely” inhibited at concen-
trations of about 1 PM. An Easson-Stedman plot of dog stomach enzyme, analogous
to those reported for red cell (330) and liver (326), yields a & for CL 13,580 of
2.5 X W9 M and [&I/ e.u. of 2.1 X 10Bg M (unpublished data, Pharmacology
Dept., Univ. of Florida). These new results show that the kinetic properties of the
canine gastric enzyme are indistinguishable from those of the other “fast” enzymes
cited above (see HCA-C in Tables 3 and 6).
Davenport reviewed the literature from his initial discovery through 1946
(KM), at which time he concluded that carbonic anhydrase is not necessary for
gastric acid formation. Davenport anticipated the approach from both rate data
and inhibition; his reasoning was eminently sound but he was led astray by lack of
good 37 C rate constants, powerful enough inhibitors, and, to some extent, reliance
on data from isolated (in vitro) stomachs. Using Roughton’s old extrapolated value
forklof0.13sec -l, he calculated that VunO is about the same as the rate of acid secre-
tion in the intact rat. If we substitute the modern 37 C value for kl of 0.043 set-1
(320), Davenport’s argument shows that only 35 of the actual rate may be furn-
ished by the uncatalyzed reaction -not different in magnitude than the situation
for kidney or pancreas (320). The inhibitors used at that time were sulfanilamide,
which in most test systems cannot elicit a maximal in vivo response (321), and
thiophene-2-sulfonamide, which is reasonably powerful (& = 10B7 M). When
the latter drug did not reduce gastric H+ secretion at concentrations of 10m3 M,
it was concluded that carbonic anhydrase was not operating (104). However, the
in viva experiments in the cat were only mentioned in passing; the published ex-
periments on this critical point are based on the whole mouse stomach in vitro,
where H+ secretion (about 5 peq/hr) is probably less than the rate of the uncata-
lyzed reaction (106). Calculations on this point are given below in connection with
the same problem for isolated frog mucosa.
A position opposite to Davenport’s was maintained by Davies and his col-
laborators and reviewed in 195 1 (110). Using a higher gastric secretion rate than
Davenport, but keeping below maximal rates, Davies and Roughton (109) con-
‘eluded that the uncatalyzed hydration of CO2 was too slow to account for the
physiological process; they made the further important calculation that the
concentration of enzyme in parietal cells is enough to magnify the uncatalyzed
rate 15,000-fold for the cat or 1200-fold for the frog. (It must be remembered that
this magnification refers to the quantity of enzyme present; this magnified rate in
the present notation is VB -not the observed catalytic rate Vobs cat). In line with
this, they claimed to have decreased gastric secretion by the isolated frog mucosa
with 17 m p-toluenesulfonamide (111) All carbonic anhydrase activity appeared
to have been abolished. Appearance of perforations and ulcerations was thought

due to failure of the intracellular buffering reaction, CO2 + OH-, in the absence
of enzyme (110). Looking back on these experiments, their meaning is not entirely
clear (see discussion of this point in ref. 428 and also the calculations for isolated
frog mucosa below) but they served to sustain interest in the “carbonic anhydrase
theory.” The question of ulceration after acetazolamide has been reviewed re-
cently and the view put forward that inhibition of acid secretion in canine fundic
pouches is due to disruption of cell structure by acetazolamide in the presence of
acidic bathing media (5 13). Although such secondary damage may occur, it is
doubtful that it serves to explain the action of the inhibitors.
The first clear evidence that carbonic anhydrase was involved in mammalian
gastric acid secretion was given by Janowitz et al. (228), who showed that 20 mg/
kg acetazolamide reduced H+ concentration and output after histamine stimulation
of dogs with vagally denervated fundic (Heidenhain) pouches. These data are
often cited as a reduction of acid secretion by as much as 97 %; however, a figure
for percent reduction is not very meaningful, since it depends largely on the height
of the predrug secretion rate. In the data of Janowitz et al. (228) this ranged from
520 to 2910 peq H+/hr, while 3 hr after 20-l 20 mg/kg acetazolamide iv the rate
ranged from 100 to 340 peg H+/hr ( 12 experiments on 3 dogs). It is shown below
that this corresponds roughly to the uncatalyzed hydration rate of CO2( VUno) in
the stomach. Essentially similar results were obtained by Byers et al. (59), who also
used Heidenhain-pouch dogs but stimulated secretion with food, i.e. endogenous
gastrin. These data (59) d i f f er in one respect from others in that inhibition of
secretion was obtained with 5 mg/kg acetazolamide and was, in fact, more marked
than with 20-30 mg/kg. The possible reason for this is discussed below in connec-
tion with effects of acid-base changes on gastric secretion. Related experiments
through 1960 in dog, cat, and man are summarized in this paper. Rehm et al. (428)
showed a similar effect of acetazolamide in reducing H+ output of the histamine-
stimulated dog’s stomach arranged ona 20-cm2 segment for collection and electrical
recording in situ.
Powell et al. (415) used the intact dog stomach and the important feature of
maximal histamine stimulation, which yielded control rates of 20 meq H+/hr. This
is in excellent agreement with the figure of 200 peg/g per hr cited from Jacobson
for the maximally stimulated Heidenhain pouch (2 18). After about 60 mg/kg iv
of acetazolamide, this was reduced to about 4 meq Hf/hr. It is possible to estimate
roughly what the secretion should be after full inhibition on the basis of the un-
catalyzed hydration of CO2. This implies, and ultimately tests the idea, that the
overall reaction CO2 + Hf, however mediated in chemical sequence and cellular
structure, is the sole source of acid. As discussed earlier in this review (eq. 20) Vunc
at 37 C is 3 125 &min or 190 mM/hr. The chief uncertainty in moving from this
figure to in vivo events is the volume of parietal or other cells &here the reaction
occurs. The distribution of carbonic anhydrase may be a reasonable guide to this
cell volume, and although the enzyme is in high concentration in parietal cells,
it is also in other cells (100, 104), so it may not be unreasonable to take this volume
as 5-10 % of the 100-g dog stomach. If the volume is 10 ml, the uncatalyzed rate
would be 1.9 meq H+/hr-not far from the observed value of 4 meq/hr after

acetazolamide in Powell et al. (415), or 1.6 meq/hr from Berkowitz and Janowitz
(34). The lower values cited above from Heidenhain-pouch experiments of Jano-
witz et al (228) and Byers et al. (59) would be due to smaller tissue volumes in-
volved.
After carbonic anhydrase inhibition in the dog, gastric juice [Na+] rises, [K+ ]
declines, and [Cl-] is unchanged. Since in these experiments the volume of juice
decreases,the net result is lowered output of Cl-, K+, and H+, but a doubling of
Na+ output (34, 415). These data support the original idea of Teorell (487) that
there is H+-Na+ exchange in the gastric mucosa.
Acetazolamide increasespepsin concentration in gastric juice from Heiden-
hain pouches (415) ; a greater effect, in which total pepsin output was increased
some tenfold, was reported in va@lly denervated pouches (207a). The latter
workers attribute the effect to the mediation of gastrin, but this is not certain.
The best study from the point of view of dose responseand quantification of
control and inhibited H-t secretory rates is that of Em&s ( 145). He found the fol-
lowing: a) the response of the cat stomach to acetazolamide was the same for
gastrin and histamine stimulation; b) there was a smooth dose-responsecurve
from 10 mg/kg acetazolamide (no effect) to 50 mg/kg (maximal); c) at low control
levels of H+ secretion (about 0.8 meq/hr) maximum drug effect reduced secretion
about 50%. At high control levels (about 2.6 meq/hr) drug reduced secretion
about 80%; the residual secretion after complete inhibition was always 0.4-0.6
meq/hr. If one follows the argument above for calculating Vuno from the value of
160 mM/hr and 2 ml for secretory cell volume (10 % of stomach weight) the theo-
retical uncatalyzed rate is 0.32 meq/hr for the cat. It is important to note that the
completely inhibited rate cannot be increased by additional histamine (370).
Human studies agree with those cited for dog and cat. Acetazolamide was
given in dosesbetween 32 and 154 mg/kg as a I-hr intravenous infusion ; surpris-
ingly little toxicity wasreported, only breathlessness,anxiety, and tingling at > 114
mg/kg (230). Tested against feeding or histamine stimulation, there was about an
80 % reduction in acid output at dosesover 70 mg/kg (290, 349). Data for other
electrolytes (290) agree in the main with the findings of Powell et al. (415) for the
dog. The oral or intravenous dose necessary to inhibit acid secretion in man is
about 50 mg/kg (289, 349). This is the same as the dose found by Em&s (145) in
his careful quantitative study in the cat and somewhat more than is required for
the dog (59, 228).
Tsukamoto used the visualization of acid secretion by Congo red in the rat’s
exteriorized stomach. Submucosal histamine ( 1Oa4M) stimulated secretion; lo-20
min later 0.1 ml of graded concentrations of acetazolamide was injected submuco-
sally. Suppressionof secretion was observed at lOas M, with partial effects at lo-‘-
MY9 M and none at lo-lo M (497).
A synthesis of the gastric work of Em&s (145) and pharmacological data on
acetazolamide makes it possibleto estimate the important value for the concentra-
tion of drug in the stomach when maximum inhibition of H+ output is achieved.
Emas showed that the minimum dosefor full responsewas 50 mg/kg; onset of full
effect was at 30 min and this lasted for 120-180 min. There have not been extensive

pharmacological studies of acetazolamide in the cat, but there are enough data
(148, 328, 370) to yield the necessary information; there is in fact no obvious
difference between the disposition of this drug in the cat and dog. The plasma con-
centration at 30 min is about 80 &ml, decliningto about 50 pg/ml at 2 hr. Taking
the mean of these, applying a 50 % correction for plasma binding, and converting
to molar terms, the concentration of diffusible drug (If) for complete effect is 140
PM. We may also reckon this from the concentration in gastric juice after intra-
venous administration; extrapolating from earlier data in the dog (328), this value
would be about 18 pg/ml or 80 PM. This value is consistent with diffusion of the
nonionic species into the juice. It is of interest that in such an experiment the drug
concentration in gastric juice remains almost constant from 2 min on; this deserves
further investigation. From the above, however, we can be quite certain that gastric
carbonic anhydrase can be completely inhibited in vivo by approximately 1P4 M
acetazolamide. From the K1 of 6 X l@* M, the fractional inhibition (z’) is calculated
as 0.9994. These data are almost precisely like those for the kidney (32 1); a higher
dose is necessary for the stomach because the kidney concentrates the drug about
threefold above plasma, while gastric juice and presumably free drug concentra-
tion in stomach mucosa is about half that of plasma.
Much attention has been given to the effects of acetazolamide on electrical
properties of the stomach. Rehm et al. (428) showed that 20 mg/kg in the anes-
thetized dog reduced potential difference of the chambered gastric segment, but
only in the secreting stomach. These and other findings suggested that the electrical
changes were due to primary reduction in H+ secretion, and the model set forth
is that of an electrogenic H+ and Cl- transport system, each, being dependent on
carbonic anhydrase. A significant experiment that remains to be done is the effect
of acetazolamide on H+ secretion under conditions of constant potential difference.
Several studies have been done on the gastric mucosa of the frog mounted in
the Ussing-type chamber. The specific conditions and rates involved must be
recognized (2 18). The tissue is 1 cm2 and weighs about 0.1 g. Assuming that 20 %
of the mucosa in this species consists of secretory cells, the secretory volume may
be taken as 0.02 ml. Taking V uric at 25 C as 94 mhl/hr (320; section II, Z), the un-
catalyzed rate in the preparation will be about 2 pmoles/hr. The maximum ob-
served rate is 2.5 pmoles/hr (137, 218), so it may be tentatively concluded that
carbonic anhydrase is not utilized. The results bear this out, for acetazolamide in
high concentrations [ low3 M in (2 16) ; not given in (137) but probably follows
protocol of (2 16)] d oes not depress acid secretion. In a more critical recent study,
Hogben (218) sh owed that 10 mu methazolamide did not inhibit H+ secretion;
control rates were in all cases < 2 pmoles H+/hr. In this preparation and with
high concentrations of methazolamide and other inhibitors (137, 216, 2 18) there
is reduction of Cl- transport and transmucosal potential difference. This seemingly
specific effect on chloride and the unreasonably high (in terms of carbonic anhy-
drase inhibition) concentrations of drug cannot be explained, but are almost cer-
tainly unrelated to the effect of these drugs against the enzyme. An analogous
effect has been reported for the frog cornea, which has no carbonic anhydrase
(255; III, 5C). In both the mucosal and the cornea1 preparations, the NS-substituted

acetazolamideswere without effect on the potential. It is concluded that there is a

Cl- transport receptor in these tissueswith somestructural similarity to carbonic
anhydrase, but its affinity to the sulfonamides is much less(2 19).
Most schemesfor the role of carbonic anhydrase in gastric secretion visualize
the parietal cell as having machinery for the effective separation of H+ and OH-
ions (110, 428), with the former secreted into the lumen (linked to Cl-, as dis-
cussedabove) and the latter buffered by CO2 to form HCO,, which passesinto
the blood. at the serosalface. Bicarbonate formation is the carbonic-anhydrase-
dependent event; due to the large excessof enzyme (log), it is only rate limiting
when inhibited. Figure 7 shows Davenport’s model (105), which appears to fit
the data reviewed here. Anion exchange, Cl- - HCO,, or other experimen-
tally substituted ions, may be reasonably fitted into such a scheme (218). It is
significant that bullfrog gastric mucosa actively secretedBr-, I-, SCN-, NO3- and
SO4p (2 18). Acetazolamide lowers bromide secretion in man ( I).
Some recent experiments on acid-base equilibria offer additional insight
into the role of carbonic anhydrase in gastric secretion and may be reviewed in
the light of Figure 7. Byers et al (59) showed that HCl strikingly increased gastric
acid secretion in dogs; metabolic acidosis due to acetazolamide had the same
effect. This may be visualized as due to increased [H+] within the cell, driving for-
ward the reaction with the postulated carrier (as for instance in the scheme of
ref. 428) or simply increasing the gradient from cell to lumen. It is entirely anal-
ogousto the situation in the kidney. Intravenous NaHCO3 profoundly decreased
secretion of gastric H+, again as in the kidney. This is seenas the result of alkalosis
within the cell, including the critical area of H+ separation, leading to reduced
PLASMA PAR IETAL CELL GASTRIC

JUICE
-Metabolism -
I Active Transport
Cl-
Passive Transport
HnO
FIG. 7. Model showing the probable roles of carbonic anhydrase in the parietal cell. [From
Davenport (105).]

738 T. H. MAREN volu?nc 47
availability of H +. Unfortunately, there are no clear-cut data nor agreement on

the effects of respiratory acidosis or alkalosis (reviewed in ref. 59). The fact that
Byers et al. (59) obtained less reduction of gastric H-l- output at 20-30 mg/kg
acetazolamide than at 5 mg/kg was explained by the development of respiratory
acidosis at the higher dose. Relative refractoriness to acetazolamide in respiratory
acidosis is in part accountable to increased participation of the uncatalyzed re-
action, but there is probably also a direct effect of pH, augmenting acidification in
the manner described above for metabolic acidosis. Quantitative data on these
points are available for the kidney (323).
The question of using carbonic anhydrase inhibitors for the treatment of
peptic ulcers has been a recurrent one; most experienced physicians feel that it is of
limited or no value. Several reasons for this may be suspected from the foregoing
physiological and pharmacological data. It is clear that complete suppression of
secretion cannot be expected and that the rate of the uncatalyzed reaction is
enough to provide moderate H+ secretion, perhaps at the basal level. If acidosis
does in fact drive H+ secretion, the systemic effects of acetazolamide would effec-
tively oppose its inhibitory action on the stomach. There is some evidence on this
point (59). It is of interest that acetazolamide was able to prevent the acute onset
of ulcers in rats and guinea pigs induced by the differing techniques of electro-
shock, reserpine, and histamine. In this protocol, however, there was carbonic
anhydrase inhibition during the brief stress periods, without the complication of
acidosis (129a). Modest effects of acetazoladmide under clinical conditions have
been reported (168, 289, 349).
F. Pancreas
The pancreas is of particular significance for this review because it permits a

simple quantitative analysis of HCO 3- excretion in terms of its formation from
CO2. This is because the pancreatic fluid is easily collected and analyzed in terms
of rate and, as will be shown, all of HCO, that appears in the fluid arises from
the catalyzed and the uncatalyzed hydration of CO2.
Carbonic anhydrase was found in the pancreas by Van Goor in 1940 after
preliminary mention by Brinkman (104, 502). The concentration of enzyme in the
whole tissue is quite low compared to kidney and stomach (Table 16) and is about
that of the ciliary process, which also carries out HCO3- accumulation. However,
there are no estimates available for any tissue except stomach that yield the con-
centration of carbonic anhydrase in the relevant secretory cell. In the case of the
pancreas, it is claimed that the enzyme is located in duct epithelium but not in
acinar cells (388). This result, however, is based on a histochemical method (29)
that now appears not specific for carbonic anhydrase (367). General discussions
of cellular activity and functional anatomy of the exocrine pancreas may be found
in reviews by Thaysen [useful comparison with other exocrine glands (488)],
Grossman [physiology of secretin ( 192)], and contributors to a recent Ciba sym-
posium (229).
Tucker and Ball (498) g ave 500 mg/kg iv of sulfanilamide to dogs but failed

Octobtt 1967 CARBONIC ANHYDRASE 739
to alter secretin-stimulated pancreatic secretion. From the concentration of drug

in pancreatic juice and the K1, they calculated that inhibition was > 99 % and
concluded that the enzyme could have no role in this system. As for the stomach,
the advent of acetazolamide clarified the role of carbonic anhydrase in the pancreas
through the experimental work of the Mount Sinai Hospital Gastroenterology
group. Birnbaum and Hollander (43) showed clearly that this drug in dosesof
10-60 mg/kg reduces the volume and HC03 concentration of canine pancreatic
juice. The reduction in rate of HCOB- output was about 60 %, and these authors
concluded that the enzyme played an important role in pancreatic secretion (43).
Later studies in the dog showed that acetazolamide produces a rise in Cl- con-
centration in pancreatic juice, equivalent to the [HCOa-] fall. The concentrations
of NaS and K+ were unaltered. However, the decrease in flow (50-75 %) is so
large that there is a decreasedoutput of all ions measured (416). In man, results of
acetazolamide injection are similar; some differences such as minimal effect on
HCO3- concentration may be due to differences in protocol imposed by human
experimentation (135). The cat respondsprecisely as does the dog (7 1).
Quantification of pancreatic output in terms of the reaction C02+HCO 3-
was furnished by Rawls et al. (422); the same paper gives the effects of changes
in acid-basebalance on pancreatic secretion. Both topics throw light on the general
physiology of carbonic anhydrase and are considered in the following paragraphs.
Under the influence of maximal dosesof secretin, the anesthetized IO-kg ,dog
excretes an average of 43 pmoles/min of HC03- into the cannulated pancreatic
duct. The concentration of HCO3- is 127 m and of Cl- 37 mu. Acetazolamide
(10 I-%/kg iv > red uced output to 21 pmoles/min; HCO3- concentration drops,
and [Cl-] risesslightly. The rate data may be compared with calculated catalyzed
(VE) and uncatalyzed (V,,,) hydration of CO2; thesegive the potential rates in an
open systemwhere the back reaction is negligible. V# is calculated from the usual
Michaelis equation and is 10,000 pmoles/min. Vu*,, is calculated from the first-
order hydration expression and yields 30 pmoles/min. A small increment may be
included to account for the participation of OH- in the reaction. It is clear that
there is a large excessof enzyme; it appearsthat lessthan 1 % of that present would
provide the known formation rate of HCO3-. The observed catalytic rate
(V ohscst) may be taken asthat abolished by acetazolamide, or 22 I.cmoles/min. V&/
v ob8caf- 500, which iscomparable to ratios of several thousand for kidney (III, 5B),
red cell (III, 5A), and stomach (III, 5D). The residual rate after inhibition (2 1
pmoles/min) is remarkably close to the calculated uncatalyzed rate (30 pmoles/
min); thus it appears that all of pancreatic juice HCO 3- may be accountable to
the uncatalyzed (Vinh = J&,-J + catalyzed (v&s cst) hydration of CO2. Unlike
the kidney, there is no residual processof active HCOa- transport (323). All of
pancreatic HCO 3- is derived from the pool of CO2 in pancreatic tissue, which is
in diffusional equilibrium with blood CO2. It is significant that the samesituation
pertains to aqueous humor HCO3. (III, SC).
It is recognized that part of the so called “excess” enzyme is probably not a
true excessin that it may be necessaryto insure rapid equilibrium in the presence
of product or conceivably of low substrate concentration within the cell. However,

740 T. H. MAREN Volume ‘47
10 mg/kg acetazolamide produces 99.9 % inhibition of pancreatic enzyme (422)

and full in vivo effect, while 2 mg/kg produces little or no effect. In a protocol
similar to that of reference 422, 3 mg/kg of CL 11,366 reduced pancreatic secre-
tion; i was about 0.998. But 1 mg/kg was ineffective (unpublished work from Phar-
macology Dept., Univ. of Florida). It is interesting that both acetazolamide and
CL 11,366 appeared in pancreatic tissue at the concentration of unbound drug in
plasma, with no evidence of secretion. Thus [&J can be defined rigorously.
Sulfanilamide at 99 % inhibiton (498) produces no effect. Thus, while the ratio
&!r/ Vobe cat of 500 may not represent the true “excess” of enzyme, the actual num-
ber cannot be lessthan about 100. The analogy to the kidney (III, 5’) is striking.
It seemsquite clear that under normal circumstances the limiting event in
pancreatic HCO 3- secretion is not carbonic anhydrase but the rate of transfer of
HCO 30 into the ducts, after its formation from CO*. The transfer rate is limited
by the properties of the cell membrane that are taken asreasonably fixed; variations
in the transfer rate may be accountable to the different concentration of HC03-
achieved in the cell. When enzyme is present, this is due to variations in systemic
acid-base balance. In the absence of enzyme, the concentration of HC03- is
limited by its relatively slow rate of formation. These points are exemplified below.
Figure 8 showsthe ionic concentrations, asthey are imagined to exist, when secretin
is working in the presence of carbonic anhydrase and normal acid-base balance.
Secretin doesnot alter carbonic anhydrase activity (267). The sole change initiated
by secretin is the separation of OH- ion, leading to HCO3- accumulation at one
cell border. Given the constancy of cations in the juice, the potential of Cl- to
exchange for HCO a-, and the linkage of HCO, formation with flow, the effects
of acetazolamide and altered acid-base balance are readily understood. There are
a variety of stimuli that can “polarize” the secretory cells of the pancreas in the
manner of secretin; all appear to produce an increase in HC03- concentration
and flow (201). Secretin may be taken asrepresentative since it is a natural mech-
FIG. 8. Model of chemistry DUCT PLASMA

of the pancreatic cell activated by
secretin. Arrows indicate mecha-
nisms put into play by administra- SECRETIN
HO--
tion of hormone. Figures are mo- ’ H+ )
I
lar concentrations. In absence of + I I
secretin, electrolytes in juice are co2 [II H,CO,l= C O2 co2 III
roughly the same as plasma. Up- L’ 1
per limit of (HCOaf in cell or ,H C03~
juice is set by the unvarying cat- H CO; [1251w [135’1l ; -, HCO; 1241
ion concentration. All HCOa- in I
juice is derivedfrom CO2 hydra- I ’
cl- [301,
tion at ceil border. Mechanism of
Cl- removal from juice and cell is
not known nor are actual ion con- NA+ 11501 -
centrations in cell.
K+ bl -

anism (192). The change in [HCOa-] from resting to stimulated secretion is from
about 40 to 130 m.~ (201).
The administration of HCl precisely mimics that of acetazolamide, and the
two agents given together are additive (422). In the latter situation, HCO3 out-
put drops from the normal of 43 peq/min to 8 peq/min (Fig. 9). Pak et al. confirm
theseresults, and with a somewhat greater acidosisthey reduced pancreatic HCO 3-
output to 20 % of normal; addition of acetazolamide reduced this to 12% of nor-
mal (388).
It will be recognized that these results are exactly equivalent to those of ad-
ministering NaHCO 3 in renal experiments (323). Both pancreatic and renal secre-
tory cells appear to possess machinery for splitting water to H+ and OH-; COZ
reacts with OH- to yield HCO3-, which in the pancreas is excreted and in the
nephron reabsorbed. It is thus easy to see why the HCOa”-rich secretion of the
pancreas will be diminished by acid, and why acid urine will be neutralized by
alkali. In both cases,an unfavorable environment is created at the secretory border
of the cell, preventing the full accumulation of HCOa- or H+. Since CO2 + OH-
is also a key event in the separation of HCO, or H+, acetazolamide has the same
or an additive effect as the induction of the appropriate acid-base disturbance.
This is a general property of secretory systemscontaining carbonic anhydrase; it
will be recognized that they vary predictably according to the pattern reviewed,
i.e. pancreas and eye representing base secretion and kidney and stomach, acid
secretion. For such effects to be manifest, it is obvious that plasma elevations of
[HCO3-] and [H$3 are reflected in the cell, and there is some evidence on this
point for kidney (478). Among the various organs, both metabolic and respiratory
changesin acid-basebalance can produce effects similar to those of acetazolamide;
there are also situations in which respiratory changes have a greater effect than
the metabolic, due to relative impermeability of Hf and HCO, compared with
CO,. Each situation is subject to individual analysis,summarized in section IV and
Table 28. The pancreas is a suitable general model, as follows.
Figure 9 showsthe changesin pancreatic HCO, output induced by carbonic
anhydrase inhibition and simultaneous alterations in acid-base balance, along
with an idealized version of their etiology. The final picture to emerge from these
ten situations is reasonably simple and coherent : the secretin-stimulated pancreatic
cell (with no carbonic anhydrase active) is set to make HCO3 from CO2 and
water; the product is transferred, probably by diffusion into the ducts. Any in-
crease in the rate of formation (carbonic anhydrase) or concentration (exogenous
HC03-) increases the transfer rate. Dissipation of HCO3 by acid reduces the
transfer rate. Respiratory acidosisproduces little change in secretion becauseCO2
and OH- change in opposite directions, tending to keep HCO, formation con-
stant. Respiratory alkalosis, however, diminishes secretion, probably by repressing
polarization of cell fluids into acidic and basic components. It is shown in section
IV that low pCO2 generally mimics acetazolamide in secretory organs, regardless
of whether acid. or base is the collected fluid.
A review of various aspects of pancreatic electrolyte secretion is given by
Janowitz and Dreiling (229) and others in a Ciba Symposium held in 1961. From

data given there and the present treatment the following summary may be made:
The juice contains a fixed concentration of Na+ and K+ identical to that of plasma.
Secretin in some way elicits the polarization of H+ and OH- in the cell as shown
in Figure 8; the result is the elaboration of HCO,. Since cation concentrations ap-
pear fixed, [Cl-] d iminishes precisely as [HCOS-] increases. An important point,
incompletely understood, is that flow is directly related to HCO3- formation; the
same occurs in the aqueous humor. In both pancreas and eye (III, 5C) all the
HCO3- in the fluids is formed by hydration of CO2 : there is no evidence for trans-
port of HCO, as such from blood to duct, since the observed rates can be entirely
accounted for by the uncatalyzed and the catalyzed reaction. This is in clear con-
trast to the process of renal HCOa- reabsorption (323; III, B). Gaps in our present
knowledge include the cytological localization of carbonic anhydrase in the pan-
creas and the actual measurement of acid-base changes in the tissue.
The physiological results have suggested that carbonic anhydrase inhibitors
be used in patients with pancreatic fistulas and in acute or chronic pancreatitis.
Anderson and Copass report that 23 of 26 patients presenting with pancreatitis
obtained prompt relief of pain and other symptoms when 5004000 mg of acet-
azolamide was added to intravenous fluids. They also reviewed the clinical lit-
erature to 1966 (6).
G. Liver and biliary system
This topic has recently been reviewed (326), except for the new paper by
Pak et al. (388) and current work (Pharmacology Dept., Univ. of Florida)
that show the effects of acetazolamide and metabolic acidosis and alkalosis on
simultaneously collected bile and pancreatic juice. These data and some calcula-
tions that follow have led to modifications of our point of view (326) on the role
of carbonic anhydrase in the liver. New experiments in this laboratory indicate
also the necessity of making a distinction between bile produced in the liver and
that produced in the common duct. This is discussed at the end of this section. In
the treatment that follows, “bile,” if not otherwise described, denotes the fluid
collected from the common duct and includes hepatic and ductal fluid. All the
published works (326, 388, 5 15) are based on such collections. In Table 26, model
data on common duct are from reference 326 and unpublished work; data on
hepatic bile are from unpublished work (Pharmacology Dept., Univ. of Florida)
under similar conditions.
Table 16 shows that hepatic carbonic anhydrase is in a relatively low con-
centration, like that of other organs in which HCO3- is excreted rather than Ht.
Although about 80 % of enzyme is in the supernatant, particulate fractions of the
FIG. 9. Model for effects of carbonic anhydrase system and acid-base balance on secretin-
stimulated pancreatic HCOa- excretion. Figures at lower left of each box are rates of HCOa-
output in~mole/min for a lo-kg dog. Numbers in brackets are mu concentrations of HCOa-.
(HCOa-) in fluid at secretory border of the cell may be taken as 5-10 rnM higher than the cor-
responding concentration in the duct fluid. [All data from Rawls et al. (422).] In this model, it is
assumed that acid-base changes in the plasma are, at least qualitatively, reflected in the cell.
The substances added in excess of normal are indicated in lower right of each box.

t3LOOd
Normal situation. Full re-actlons Metabolic alkalosis + carbonic
[2 4
are given in Fig. (3, HC03 forma- anhydrase inhibition. Vunc can
tion in the cell is limited by product account for the observed rate, due
accumulation, since the transport F to Increase in OH’ and CO2. Note
step Is much slower than the enzyme that the contribution of the enzyme
rate WE). 36t here (E - F k 23) is the same as in
the normal state (A - 8 = 22).
I I
Inhibition of carbonic anhydrase. Resplratory acidosis. The increase
WI HO’ H* [24] The limiting event is now formation r I 321 HO- HC [261 in pCO2 does not increase the rate,
+ of HCO;, by uncatalyzed reaction t since the excess enzyme assures
B (V ,: - - G CO2 H* high rate at any pCO2. The situation
I C92 I uric
1 CA. is complex because several compo-
P nents are added.
46 HCO; + HCO;
n
Metabolic acidosis. In this and
succeeding figures, the added r Respiratory acidosis + carbonic
1”Ol HO’ H+ [16] acid or base is shown at the lower
Cl201 c271 anhydrase inhibition. The enzyme
+ component is the same (G - H 49)
right. HCO; is dissipated by H
C CO2 proton capture, lowering its as in the normal (A - B = 22).
1 CA. concentration and potential for
18 4 -HCOi + H+ 27 4
H2c03 transport.
Resdiratory alkalosis. There is
WI
1 HO-
t
H* M
Metabolic
anhydrase
formed
acidosis
slowly
inhibition.
+ carbonic
and dissipate
HCO- is
a,
[IOO] HO-
+
H+ Id
some decrease
than directly
of pCO2 to l/3 normal.
in rate,
accountable
but less
to lowering
As in all
I co2 carbonrcanhydrase dependent
D CO2
markedly lowering excretion.
lCA* . systems, this alteration mimics
1 28 nco; OH- inhibition, probably because it
8 HCO; + He H2C03 renders the cell less able to polar-
t ize Ht and OH-;
Metabolic alkalosis. HCO- and OH-

J gradients
an Increase
Althtugh
are incretsed, 3 eadlng to
In HC03 transport.
this may suggest that some
[851 bl Respiratory
anhydrase
alkalosis
inhibition.
+ carbonic
HCOi is
J formed slowly and gradient is small,
E HC03 is transferred as such, Vunc +
but rate is >O because celt is
do account for the HCOJ
I7 alkalotic which favors secretion.
d. 4
cp
w

cell all contain some activity. Enzyme concentration and distribution are the same
for rat and dog, but a crucial difference exists in that rat supernatant carbonic an-
hydrase is almost completely refractory to inhibition by sulfonamides (326). The
dog liver enzyme has kinetic properties similar to that of dog blood, dog kidney,
and human carbonic anhydrase C (326).
Bile, like pancreatic juice, contains higher [HCOS-] and lower [Cl-] than
plasma. Quantitatively, the gradients from plasma to bile are less than for pan-
creatic juice : [HCO 3-1 in bile is at most 60 mM and [Cl-] not less than 55 m.
Secretin increases [HCOa-] and [Cl-] by 10-I 5 TI~M, and also flow; taurocholate
increases flow but not concentration (388). The effects of acetazolamide or other
inhibitors on canine bile, first reported by Wheeler and Ramos (515), are not
greatly different whether carried out in the resting animal or under conditions of
secretin or taurocholate infusion (326, 388, 515). Acetazolamide at 65 mg/kg
produced maximal effects; 20 mg/kg is also active. The chief change seen, con-
sistently and under all conditions, is an increase in biliary [Cl-] of 15-30 m.
[HCOa-] generally falls; here the conditions of the experiment are important.
With no stimulation, acetazolamide produces no [HCOO] effect (388) ; in tauro-
cholate experiments there is a drop of 15 rrull in one series (388) but an inconsistent
effect averaging a 5-mu decline in another (326) ; with secretin the [HCO3]
drop after acetazolamide is about 15 m. The rise in [Cl-] always exceeds the
[HCOa-] drop (388, 515).
Some dose-response data (326) supplemented by new experiments give the
surprising result that the rise in biliary [Cl-] is dose related and reaches a maxi-
mum (about 30 ITU~ elevation) at 20 mg/kg methazolamide or 65 mg/kg acet-
azolamide. The drop in biliary [HCOa-1, however, is paradoxical, in being less
(or even absent) at these high doses than at 5 mg/kg methazolamide or 10 mg/kg
acetazolamide. This is not entirely understood, but might ultimately be related
to the greater respiratory acidosis at the higher doses. Table 26 shows some of
these relations in model form.
Unlike the effect on pancreas, acetazolamide causes no change or an in-
crease in biliary flow. Enzyme inhibition caused some rise in biliary [Nat] so that
the fluid may be hypertonic. KS concentration remains unchanged (326).
Consideration of the uncatalyzed rate of CO2 hydration (I&J in terms of the
liver yields the interesting result that I& exceeds the observed HCO3 output.
Vunc for the pancreas is 30 peq/min (422); it seems unlikely that the reaction
volume is less for the liver and, since kr and [CO21 are the same for any organ, we
may take 30 peq/min also as a minimum value for Vunc in liver. In the experi-
ments of Maren et al. (326) and Pak et al. (388) bile flow is 0.2 m.l/min and high-
est [HCOS-] is 100 TI~M, whence HC03 output does not exceed. 20 peq/min. The
lack of effect or minimal effect of acetazolamide on HCOS- concentration or output
is thus explained.
Methazolamide and CL 11,366 cause changes similar to those after acet-
azolamide administration. Methazolamide is not concentrated substantially in
liver or bile; acetazolamide is to a moderate degree. CL 11,366 is actively secreted
into the bile and concentrated in the liver. As with all other organs studied, cal-
culated fractional inhibition at active doses is over 0.99 for all drugs (326, 494).

The secretin-stimulated dog has biliary [HCOS-] of about 60 mu and [Cl-]

of about 70 mu. Metabolic acidosisby HCl infusion did not decrease[HCOaW] or
increase(Cl-] in bile; flow, however, is reduced, sototal HCO, and Cl- output is
reduced about half. Acetazolamide injected during the acidosisproduces its nor-
mal effect, i.e. a small drop in [HCOS-] and large rise in [Cl-] (388). Metabolic
acidosiswas also studied without secretin but with taurocholate infusion. The fall
in flow and HCO3- output was confirmed, but there was also a clear drop in
[HCO 3-1from 74 null to 43 mu and a rise in [Cl-] from 75 mu to 100 mM (Ellison
and Maren, unpublished data). Thus metabolic acidosisfaithfully mimicked car-
bonic anhydrase inhibition (Table 26), just as in the pancreas. There are no data
on pure respiratory acidosis.
Metabolic alkalosis during secretin stimulation causesa profound increase in
biliary [HC03-] to 93 mM and lowering of [Cl-] to 51 mu. In this situation aceta-
zolamide produced only a small (10 mM) drop in [HCOa-] and rise in [Cl-] (388).
Metabolic alkalosiswas studied without secretin and most of the previous findings
were confirmed. The rise in biliary [HC03”] was 25 mu, but the fall in [Cl-] was
negligible. The effect of methazolamide (5 mg/kg) was completely blocked with
respect to either [Cl-] rise or [HCOS-] fall (Table 26). Again, these experiments
are similar to those on pancreatic secretion.
Respiratory alkalosis lowered biliary [HCOa”] about 20 mM, and raised
[CP] about 40 mM. Flow may decrease(326). The effects of low pCO2 are greater
than those of enzyme inhibition (Table 26), a quantitative distinction from all
other organs studied. The opposite influences of metabolic and respiratory alkalo-
sisare similar to those seenin the pancreas (Fig. 9) but are of considerably greater
magnitude.
It is concluded that biliary [Cl-], and to a lesserextent [HCOa-1, is profoundly
affected by systemic pCOz . This fits with the calculated lack of dependenceon the
catalyzed rate for HCO, output and the failure of acetazolamide to greatly alter
HCO3: output. A reaction dependent on the uncatalyzed rate should vary directly
with the pCO2 . On the other hand, an in vivo catalyzed reaction ( VObs&, since
it is generally slow compared to V” , can function at any pCO2 ; this has in fact
been shown for the kidney (323). Thus, criteria for a principal role of uncatalyzed
rate in the biliary systemare fulfilled, but catalyzed rate is alsoinvolved, sincethere
is a clear effect of the three sulfonamides on biliary [Cl-], presumably through
inhibition of the enzyme.
The data appeared explicable on the grounds that a certain group of cells in
the biliary system (perhaps the ducts-see below) carries out a process of Cl-
conservation basedon HCO3& formation without carbonic anhydrase, and another
group (within the liver) usesthe enzyme for the same process.The ducts of the
biliary tree outside the liver do not contain carbonic anhydrase (unpublished ob-
servations). This tissue, perhaps including its radicles within the liver, appears to
be a site for the uncatalyzed formation of biliary HCO3-. Isolated ductal tissue
secretesan alkaline fluid, as shown by Rous and McMaster 45 years ago (451);
continuation of their experiments appeared indicated and is briefly described
below.
Experiments were designed so that common duct (“ductal”) bile was col-

TABLE 26. Model for biliary anions in taurocholate-infused anesthetized dog (326, PF)
Hepatic Fluid
[Cl-] [HCWI
millimoles/liter
Normal 112 22 70 60 80 40
Methazolamide,
Methazolamide,
HC1
20 mg/kg
5 mg/kg 112
112
117
22
22
13
80
95
90
50
57
40
90
100
100
35
46
30
NaHCQ* 108 48 70 90 80 90
Low PC02 123 16 110 40 60 35
* This alteration blocks the usual effect of methazolamide.
lected as above, but, in addition, hepatic bile was collected directly from a main
lobe of the liver. The data are presented in model form in Table 26. Hepatic bile
had about the same[Cl-] (70-80 no) asductal bile, but [HCOa-] was much lower
(about 40 mu). This suggeststhat there is Cl- - HCOa- exchange between plasma
and hepatic bile, and that a second processof HCO,- accumulation occurs in the
duct tissue. Inhibition was produced by 20 mg/kg methazolamide or CL 11,366.
In common duct fluid there were the samechanges noted above, rise in [Cl-] and
slight fall in [HCOaV]. Pure hepatic bile, however, showed only. the ,[Cl-] rise,
with no [HCOa-] fall. Respiratory alkalosis again showed the usual mimicking of
carbonic anhydrase inhibition in the common duct fluid with some exaggeration
of the [HCOS~] drop; but there was an unexpected fall rather than a rise in hepatic
bile [Cl-] (Ellison and Maren, unpublished data). Further work would be desir-
able, but these experiments suggestthat the major Cl--conserving system is medi-
ated in the liver and is carbonic anhydrase dependent. However, it must be men-
tioned that the only evidence for participation of the enzyme in the hepatic system
is the effect of the inhibitors in eliciting Cl- output. The liver system is anomalous
in not showing the same electrolyte responseto low pCO2 as to inhibition; also
the HCOa” responseitself is minimal or absent. It is conceivable, although un-
likely, that there is a “Cl--conserving enzyme” in liver that is responsive to these
sulfonamides. The common duct appears to secrete HCOS- by a mechanism inde-
pendent of carbonic anhydrase; in view of pCO2 dependence, this is probably the
uncatalyzed hydration reaction.
A most surprising finding was the lack of effect of the sulfonamides on bile
electrolytes of the rat. This seemsclearly accountable to failure of the supernatant
fraction of rat (also mouse and rabbit) liver to be inhibited by these drugs at con-
centrations at least 1000 times greater than required against dog or other tissues
of the rat or rabbit (including gall bladder), or in fact any other known vertebrate
carbonic anhydrase (326). Exploration of this unusual enzyme would be of obvious
biochemical importance.
The rabbit and dog gall bladders contain carbonic anhydrase, about 90% of
which is in the supernatant fraction. Rabbit enzyme from both supernatant and
particulate fractions is inhibited by sulfanilamide, acetazolamide, and ethox-

zolamide. The Iso concentrations for all three drugs are about 3-5 times greater
than for dog red cell or HCA-C (unpublished observations). Bladder bile is gen-
erally acidic (473). There is an active mechanism for removal of HCQ- from the
lumen, which was explained as either ionic reabsorption per se or the result of H+
secretion into the lumen (514).
Dr. H. 0. Wheeler has given permission to cite the following preliminary
data, which suggestthat at least part of this processinvolves carbonic anhydrase:
using the isolated rabbit gall bladder preparation (5 14), ethoxzolamide ( 1F4 M)
reduced fluid transport from mucosa to serosaby 80 %. The simplest explanation
is that the processis analogous to HCOS- reabsorption in the proximal nephron
(III, 5B) or small intestine (seebelow) in which the primary processis H+ secretion
from carbonic-anhydrase-containing epithelium. It is likely that further work will
answer such questions as whether there may also be active HCOS- reabsorption,
and whether the very large reduction in inward flux indicates that in somefashion
Cl- reabsorption is also tied to the carbonic-anhydrase-mediated reaction.
H. Salivary glands
Van Goor (502) and Yoshimura et al. (537) show the presence of carbonic
anhydrase in the canine salivary glands. The parotid has about the concentration
of blood (seeTable IS), which is one of the highest found for any whole tissue.
Submaxillary gland has >i to ?& of this value, while the sublingual has about s5
of the enzyme concentration of the parotid; Yoshimura et al. (537) report a low
concentration in rabbit parotid but we find it the sameas for dog. Although Van
Goor (502) and Szabo et al. (479) reported enzyme in saliva, we could find none
in human samples.
The electrolyte physiology of the salivary glands has been given excellent re-
views by Thaysen (488) and Burgen and Emmelin (53). It is clear that at relatively
high rates of salivary flow, parotid and possibly submaxillary saliva have HCOa-
concentrations greater than that of plasma. This is well documented for the dog
but the rabbit may be an exception (537); Burgen and Emmelin (53) call attention
to the fact that parotid saliva of sheep has HCOS- concentration and other elec-
trolyte characteristics of pancreatic juice. Sublingual saliva, on the other hand, is
relatively acid with [HCO,-1 < 10 mM (53, 537). It is tempting to correlate this
with the very low enzyme concentration of the sublingual gland; possibly the values
reported are an artifact due to blood contamination and the tissue really has no
enzyme. Or this may be an example of a Cl--concentrating system dependent on
carbonic anhydrase (395).
Yoshimura et al. (537) carried out a careful study of several carbonic anhy-
drase inhibitors on pilocarpine stimulated parotid secretion in the dog. No sub-
stantial alterations were found in [HCOs-] or flow, but unfortunately the doses
used were minimal : thiophene-2-sulfonamide (10 mg/kg), sulfanilamide (110
mg/kg), and acetazolamide (7 mg/kg). Brusilow and Diaz (51) made a similar
study except that stimulation was done electrically at the parasympathetic afferent
to the parotid. Using 40 mg/kg acetazolamide iv they obtained a clear-cut reduc-

tion in [HCOS-1, from about 50 mM to 25 mM, at similar flow rates. Sodium con-
centration decreased,while chloride was inconstantly elevated. In man there are
conflicting data; Chauncey and Weiss (75) found no effect after oral dosesof 4-22
mg/kg acetazolamide. They collected saliva from the parotid, using vacuum cups,
90 min after drug. Niedermeier et al. (378) studied the composition of mixed human
saliva during chronic administration of acetazolamide (averaging about 1 g/day
in divided doses)and obtained a drop of about 7 mM in [HCOa-1. In both human
studiesthe control salivary [HCOS-] was 18-20 mM, substantially lessthan plasma.
NH&l produced the same effect as acetazolamide (378); the nature of these ex-
periments is inadequate to reveal whether the acetazolamide effect itself is second-
ary to the metabolic acidosis, or whether this is another example of metabolic
acidosisdue to NH&l mimicking the effect of carbonic anhydrase inhibition. In
this connection, Burgen and Emmelin (53) showed that elevation of plasma pCOz
or [HCOS-] raised, and lowering of pCO2 greatly decreased, salivary [HCOa-1.
Analogies to the situation in the pancreas may be made by reference to Figure 9.
Thus there is only one small study (51) showing clear effects of carbonic anhy-
drase inhibition on salivary electrolytes in the manner that would be expected
from the anatomical and physiological analogiesto the pancreas (488). The matter
nevertheless seemsreasonably secure but further work, particularly comparing
the three different glands and using sheep as well as dogs and humans, would be
worthwhile. At the present state of knowledge, the role of carbonic anhydrase in
parotid and probably submaxillary gland physiology can be regarded as qualita-
tively similar to that described for the pancreas (III, 5F above).
A recent study on the submandibular (i.e. submaxillary) gland of the cat is
of interest because it may point in a different direction, of carbonic anhydrase
playing a role in Cl- secretion as it appears to do in CSF. Submandibular secre-
tion in this species,like that of the sublingual gland, is presumably acidic with Cl-
as the dominant anion. Acetazolamide reduced the secretory rate and also the
transmembrane potential. A curious finding was a latency after intravenous injec-
tion; but after retrograde injection into the lingual artery, there was an immediate
effect (395). The effect is also explicable on the basisof inhibition of HCOS--Cl-
exchange, with CO2 the source of HCOa-.
It has been claimed that saliva (obtained from persons subject to calculus
formation) promotes the rate of formation of insoluble carbonates, probably
hydroxyapatite, which is the matter of oral calculi. This effect is inhibited by boil-
ing the saliva or adding sulfanilamide (348). The implication is that salivary en-
zyme plays a role in calculus formation, but these investigators did not give any
data on carbonic anhydrase in saliva, nor did they give the concentration of sulfa-
nilamide or useother inhibitors. This paper has been appropriately criticized, and
experiments have been carried out showing that carbonic anhydrase did not initi-
ate or hasten the deposition of apatite in vitro (11). Conceivably, enzyme is present
in certain people that form calculi; we have been unable to find it in normal
saliva, nor is it in any other body fluid. The entire question must be regarded as
open; it seemsunlikely that carbonic anhydrase is involved, but the kinetics of
apatite formation is an interesting problem for dental and oral physiology of the
future.

October I967 CARBONIC ANHYDRASE 749
I. 5’weut glands; taste
Richterich and Friolet (430) have reviewed the small literature on this subject
and studied the effect of acetazolamide on sweat electrolytes in man. By a histologi-
cal method, carbonic anhydrase has been demonstrated in the skin, but, as stated
earlier, such methods are questionable. Acetazolamide did not alter the electro-
lyte composition of thermal sweat or of sweat obtained by pilocarpine iontophoresis
in normal. man. In children with mucoviscidosis (cystic fibrosis of the pancreas),
sweat [K+] is elevated from 10 to 19 mM; administration of acetazolamide (about
12 mg/kg iv) brings this high value back to normal. This would suggestthat there
is carbonic anhydrase in sweat glands but that normally it plays little or no role
in the elaboration of the fluid. In mucoviscidosis, there appears to be augmented
exchange of Na+ against K+ dependent on the enzyme. The greater the initial
[Kt-1, the larger the fall after acetazolamide; a plot of the data reveals that at 8
mu [K+], the inhibitor has no effect. This is close to the normal value and suggests
that in the healthy subject the exchange is either unrelated to the CO2 system or is
adequately served by T;I’unc . The emergence of the enzyme-dependent process in
diseaseis a significant finding and should lead to further work of great interest.
It is widely recognized that somepatients receiving acetazolamide, and prob-
ably other inhibitors, complain of a curious taste on the tongue when drinking
carbonated beverages. Similar sensations are appreciated when the mouth is
rinsed with inhibitors. The matter has been under investigation by H. Hansson at
Uppsala (196) but no full papers have appeared. We found no carbonic anhydrase
in the canine tongue.
J. Refroductive system
I> Female mammals. Carbonic anhydrase was sought and discovered in the endo-
metrium of the pregnant rabbit by C. Lutwak-Mann after her observation that
the 6-day blastocyst fluid had high (70 mM) bicarbonate content (301, 303). Since
then there have been numerous studies in several species,particularly by the endo-
crine group at Worcester; the data to 1963 have been reviewed by Pincus and Bialy
(398) and their studies are cited from that paper. It may be stated at the outset
that neither the functional aspect nor the significance of the enzyme in the uterus
has been elucidated. Unfortunately, the physiological connection between the high
[HCOa-] in rabbit blastocyst fluid (303) and the presenceof carbonic anhydrase
has not been followed up or extended to other species.This has particular rele-
vance since there are profound speciesdifferences in the responseof uterine enzyme
concentration to various stagesin the sexual cycle or to the equivalent hormone.
This is unusual in carbonic anhydrase physiology, which is generally characterized
by great intraspecies constancy within the classof mammals and often among the
different vertebrate classes.Accordingly, this section is organized by species.
A) RABBIT; In this speciesthe blood-free endometrial mucosa contains (on
the scaleof Table 16) about 20 e.u./g (301, 302, 398). After pregnancy, pseudo-
pregnancy, progesterone, or other luteoids, there is about a 20-fold increase. in
carbonic anhydrase concentration. This made possiblean assayfor progestational

effect (302), which compared very favorably with measurementsof endometrial

weight or glandular activity, and permitted the evaluation of many drugs of this
type (398). It is important to note that progesterone does not alter the carbonic
anhydrase concentration of blood, kidney, liver, or adrenal glands (398). The re-
sponseto a single dose of progesterone (5-20 mg) given to estrousrabbits was evi-
dent at 2 days and maximum at 8 days (301). Estrogens inhibit the progestational
effect on carbonic anhydrase (302); a smooth dose-responsecurve for natural and
synthetic estrogens is observed (398). Other hormones, from testis and adrenal,
have relatively little effect (302, 398). From a number of papers published by
Pincus and his colleagues (398) and Yamashita (536) it appears that the rabbit
carbonic anhydrase test will detect progestational activity or inhibition elicited by
any physiological stimulus or chemical.
B) RAT. This speciesalso has carbonic anhydrase in the adult nonpregnant
uterus (398), although the original paper (301) reported none. In direct contrast
to the rabbit, however, progesterone (301, 398) or pseudopregnancy doesnot alter
the enzyme concentration. Estrogen decreasescarbonic anhydrase concentration
of the uterus, and immature rats have 3 times the enzyme concentration of the
adult. Changesduring the estrouscycle are unimpressive, but the enzyme is prob-
ably lowest during estrus and highest in diestrus. A limited number of experiments
of this type show that the hamster and guinea pig resemblethe rat (301). All three
speciescontain carbonic anhydrase in both maternal and fetal placentae (301).
c) MOUSE. Study of uterine carbonic anhydrase during estrus in mice revealed
just the opposite pattern from that of the rat; furthermore, differences among the
various stageswere much greater. In estrus, enzyme concentration was about 4
times greater than in diestrus; the suggestion that estrogens stimulate carbonic
anhydrase was borne out by a dose-responseline that may be utilized as an assay
for these compounds. Progesterone reverses this effect; the situation is precisely
opposite to that in the rabbit. Despite these surprising differences, the data appear
reliable and were found independently in two laboratories (306, 398).
In considering the variations in uterine carbonic anhydrase in these three
species,Pincus and Bialy find no explanation; the only common feature is the
estrogen-progestin antagonism (398).
D) OTHER SPECIES. The uterine mucosae of nonpregnant sheep have consid-
erable enzyme that does not appear to change greatly under different endocrine
conditions. The following animals were reported to have no enzyme in the non-
pregnant phase: cow, pig, mare, dog, cat. The question of enzyme in the pregnant
uterus of these animals has not been resolved due to lack of adequate material or
presence of blood (301). Pig placenta has a high concentration of enzyme, origi-
nally said to be restricted to the chorion (301), but later found throughout the
membranes (347). The situation in the human is unknown.
Uterine carbonic anhydrase is thus the sole enzyme site showing both large
fluctuations in activity and marked speciesdifference in responseto hormone. It
is impossibleto arrive at any decision regarding its role, based only on these diverse
cyclic changes. Since carbonic anhydrase is generally present in excessof physio-
logical needs, it is difficult to believe that the role of the uterine enzyme will be

October 1967 CARBONIC ANHYDRASE 75 I
revealed by these perturbations. Far more significant will be a study of electrolyte

composition and gas exchange across the uterus in response to the specific inhibitors.
2) MzZe mammals. Table 16 shows the distribution of carbonic anhydrase in
the prostate gland of the rat. There is clearly a large concentration in the posterior
(dorsolateral) lobe, which is confirmed by the careful study of Leiter (285). In this
paper the activity of seminal vesicle and coagulating gland of the rat was also re-
ported. Buruina (58) made the surprising finding of high concentrations of enzyme
in sperm of cattle, but we cannot confirm it (unpublished work, Pharmacology
Dept., Univ. of Florida).
Pincus and Bialy (398) also studied the enzyme concentration of prostatic
lobes and seminal vesicles of the rat under various endocrine conditions. The varia-
tions induced were small. Certain effects on Zn uptake were also reviewed here,
but the authors were unable to draw any conclusions from their data or the litera-
ture: The rat data, however, are by no means typical, for dog and human prostates
contain virtually no enzyme (285). Human seminal vesicle, however, contains
about 25 e.u./g, which may be of significance (see below).
Certain of Leiter’s rat data are of intrinsic interest despite the species specificity.
Castration had no effect on enzyme concentration. The distribution of acetazola-
mide in the organs of the genital tract was studied; after chronic administration,
there appeared to be a rough correlation between the order of drug concentra-
tion in a locale and the enzyme concentration. However, the drug and enzyme
were not compared in the same terms and this association is probably fortuitous.
An example may be given: dorsolateral prostate, 610 e.u./g or 11 PM (see II, 3);
drug concentration, 24 hr after last dose of acetazolamide, 180 PM. Other parts of
the gland seemed also to concentrate drug in considerable excess of enzyme. This
is notably different from kidney, red cells, and choroid plexus of the dog, which
show drug equivalent to original enzyme present under similar experimental
conditions (322). This is a suitable matter for further work, particularly since the
chemical nature of Zn in the prostate is not resolved. Clearly there is far more Zn
in the gland than carbonic anhydrase (345), and the prostates in species contain-
ing little or no enzyme (285) have considerable Zn. The metal, but not the enzyme,
appears in semen in high concentration (346). Does acetazolamide bind to other
Zn proteins. 3 Do the alleged histochemical methods for carbonic anhydrase dis-
tinguish between rat and dog prostate?
Leiter also studied the effect of acetazolamide on seminal fluid in man. The
drug.appeared in semen in about the concentration found in plasma; spermatozoa
count was unaffected. The normal semen electrolytes were: pH 7.6, [Na+] 120
mu, [K+] 20 mu, and [Cl-] 50 mu. There was no change after acetazolamide
(285). The data suggest that HC03 concentration would be about 50 mu. Since
prostatic fluid is acid (222), it seems likely (since in man there is enzyme in vesicles
but not in prostate) that the alkaline fluid is formed in the vesicles. The failure of
effect of acetazolamide was unexplained; in future studies [HCOg-] should be
measured. It is suggested that the seminal vesicles in man may be analogous to
the alkaline gland of the skate (III, .5M below). In this case it is quite clear that the
uncatalyzed reaction can provide alkaline fluid, even though enzyme is present in

some species. If the analogy is pursued, the failure of acetazolamide to alter semi-
nal electrolytes is reasonable.
3) Female birds. The presence of carbonic anhydrase in the hen oviduct (91)
is related both to the general problem of the enzyme in uterine physiology (above)
and to its role in calcification (III, 5R below). The administration of sulfanilamide
to hens inhibits shell formation in the egg (30, 38, 193, 461, 500). It is of interest
that the first of these studies was done with no apparent recognition that there is
carbonic anhydrase in the uterus or that sulfanilamide is an inhibitor; it is never-
theless a useful work in that the decrease in shell thickness (from 2 to 35 %) is
shown to be a direct function of the sulfanilamide in the diet (0.002-0.5 %). The
drug was without effect on blood calcium. Benesch et al. (30) were the first to use
sulfanilamide in the context of an inhibitor in this situation; 100-200 mg/ kg, which
produced a blood and egg concentration of about 0.1 mM, yielded effects ranging
from entire absence of the shell to a thin and pitted shell. This concentration of
sulfanilamide does not generally produce physiological effects. It is thus of great
interest that acetylation of sulfanilamide was also studied; the egg contents had 5
times as much acetyl compound as free NH2 drug. Since this metabolite is also
considerably more active against carbonic anhydrase (Table 4), it is quite certain
that the effect is due to the N4-acetylsulfanilamide. Sulfapyridine had no effect.
Bernard and Genest also obtained the thinning effect on egg shell with neopronto-
sil, but not with sulfathiazole, sulfaguanidine, sulfamerazine, or sulfadiazine. There
was a small effect of sulfapyridine at high dose levels (38), which may be due to
general toxicity (see effect of mercurials in ref. 365). Gutowska and Mitchell
(193), independent of the above studies, showed similar effects of sulfanilamide and
provided data on enzyme levels in egg, shell gland, ovary, and blood before and
after inhibition. With respect to the latter, the usual fiat must be observed, that
enzyme activity in the presence of an inhibitor greatly overestimates the true situa-
tion as it was in vivo. It is likely, from these data, that enzyme activity was totally
suppressed in the shell gland.
Tyler (500) showed that sulfanilamide greatly lowered the calcium and car-
bonate content of the shells; there was virtually a perfect correlation between the
absolute weight of calcium and the weight of a given shell. Para-aminobenzoic
acid had no effect on the shell, nor did it alter the sulfanilamide effect.
The only experiment utilizing acetazolamide (25 mg/pullet by a single im
injection) is that of Mueller (365), who found a 70 % decrease in shell weight and
thickness, restricted to eggs laid on the 2nd day after injection. Although the author
concluded that the shell effect was secondary to the diuretic action, the data scarcely
support this; mercuhydrin, used as a control for this effect, produced only a mini-
mal lowering of shell weight.
It may be concluded from these studies that carbonic anhydrase, by speeding
the conversion of CO2 -3 HCO,, facilitates the deposition of CaC03 in the egg.
Why the rate of hydration is a major factor in what would appear a slow process
is not entirely clear. But it is likely that the answer is related to the necessity for
Ca++ and COS” ions to be produced at the same rate; the enzyme removes any
limitation on formation of the latter, whence secretion of the two ions can proceed
stoichiometrically.

K. Avian salt gland
The discovery of the nasal salt gland in birds by K. Schmidt-Nielsen (457;

literature to 1960 described in ref. 456) began a significant new chapter in electro-
lyte physiology and at the same time added a dimension to the known role of car-
bonic anhydrase. Now it appeared that the enzyme is involved in the secretion of
a neutral or slightly acid (to plasma) solution, and a solution that is not subject to
change in composition.
After the identification of carbonic anhydrase (about 30 e.u./g) in the gland
of the herring gull by Dr. Schmidt-Nielsen and the author, and evidence that
acetazolamide could stop the secretion (154), Nechay et al. (376) carried out a
quantitative study of the effects of carbonic anhydrase inhibitors and alterations in
acid-base balance on nasal salt output in two species of sea gulls. The following
discussion is taken chiefly from that paper.
The plasma electrolytes of the gull are similar to those of dog or man; the
nasal gland elaborates a fluid of the approximate composition (mM): [Na+] 800,
[Cl-] 780, [K+] 40, [HCOa-] 10, pH 7.0. The resting bird, particularly in the
laboratory, does not secrete and the activity of the gland is an all-or-none phenome-
non. When stimulated by a salt load, cholinergic drugs, or electric current to the
nerve, clear fluid, always of this composition, is produced. Under the condition of
intravenous saline infusion, carbonic anhydrase inhibition ( 16 mg/kg methazola-
mide) reduced secretion virtually to zero; this is the only case known for these
drugs of such total physiological effect. However, 1 mg/kg was inactive; 4 mg/kg,
which decreased flow about 70 %, was used for most experiments. The fluid com-
position was unaltered. The methazolamide inhibition was mimicked by HCl and
by severe respiratory alkalosis; metabolic alkalosis (NaHCOs) markedly diminished
or blocked the effect of methazolamide. It may be recognized that this is precisely
the pattern observed in the dog pancreas (III, 5F). This is of great interest in view
of fairly similar histology and innervation, despite the considerable differences in
the composition of the two fluids. It is submitted that the salt gland also requires
an alkaline milieu at the secretory border (see Fig. 8), even though the ultimate
secretion is neutral.
Events may then be visualized as follows: The initial step in secretion is OH-
and H+ separation, a second is OH- buffering by CO2 , and a third is active salt
secretion, which is in some (unknown) fashion linked to the milieu provided by the
first two steps. Acid-base changes affect the first or second step, carbonic anhydrase
inhibition the second, and mecurial diuretics the third. If the gland is stimulated
electrically (489) or by large doses of methacholine (154), carbonic anhydrase
inhibition has no effect. In the light (?) of the foregoing analysis, step one appears
more effective after electrical or cholinergic stimuli than after a salt load; hence
the concentration of OH- is great enough so that OH- + CO2 proceeds adequately
by the uncatalyzed reaction. The same explanation applies to the experiment in
which methazolamide fails to work after NaHC03 . We must record, however,
the different view of Schmidt-Nielsen (154, 456, 489), who believes that abolition
of the secretory block of carbonic anhydrase inhibitors by methacholine or electri-
cal stimuli shows that the inhibitors act in the osmoregulatory reflex chain outside

the salt gland. It was clear that this crux could be resolved by studying secretion
and carbonic anhydrase inhibition in the denervated gland. This has now been
done by G. Whyffels and H. G. Langford (personal communication). After pre-
ganglionic section of the parasympathetic division of the VIIth cranial nerve in
the sea gull, both the secretory responseto salt load and its inhibition by aceta-
zolamide remained intact. As with all other secretory sites considered in this re-
view, the locus of action is the organ itself. It is also of interest that the retrograde
injection of acetazolamide from the nasal duct into the gland of the domestic duck
lowers the salt concentration, but not the flow (226). This suggestsa dissociation
between salt and water transport, and again favors a local effect of the drug, rather
than one on the neural pathway.
L. Rectal gland
Burger’s discovery of the secretory function of the rectal gland of Elasmobranchii

(54) has both a historical and physiological parallel to the story of the avian salt
gland. The carbonic anhydrase concentration in the rectal gland is the highest in
any organ of S. acanthias (309, 420). Rectal gland secretion is essentially 0.5 M
NaCl. Unlike the avian salt gland, secretion is more or lesscontinuous, but there
are wide oscillations in rate, depending on conditions and responseto osmotic
and drug stimuli (54).
Attempts to alter secretion by injecting methazolamide (25 mg/kg iv) in S.
acanthias that had received a salt stimulus consistently failed (420). There was ade-
quate drug in the tissue and in the secreted fluid for total inhibition (420); the
rectal gland enzyme is normally susceptible to sulfonamides in vitro (309). It is
recognized that such failure of responseis most unusual. The most likely explana-
tion seemedto be that the very large elevation of systemic pCOz and [HCOa-1,
which follows carbonic anhydrase inhibition in this species(2 13, 319), blocks the
local effect of methazolamide on the gland. A parallel situation is found in aqueous
humor dynamics, where metabolic alkalosisblocks the normal effect of acetazola-
mide in lowering pressure(525). Thus, to define the effect on the rectal gland, the
systemic effect must somehow be eliminated. Preliminary experiments of this type
were done by Palmer (389) using the isolated perfused gland. Acetazolamide
(4 X 1w4 M) added to the perfusate stopped the secretion. Further work on both
the intact and isolated rectal gland should be done.
The finding of relatively high concentrations of carbonic anhydrase in the
mammalian “appendix” (267; in rat and dog more properly the cecum) heightens
speculation on the possible origin of this organ, in vertebrate evolution, as the
rectal or salt gland of fish. It would be interesting to know if the modern primate
appendix retains any of its presumed vestigial function.
M. Alkaline gland
The literature is happily confined to a single paper (331), which is discussed

on the basisof certain principles that are particularly well defined in this exotic

OCtQbt?Y 1967 CARBONIC ANHYDRASE 755
and rather occult organ. The alkaline gland (formerly and erroneously called
sperm sac), discovered by E. K. Marshall, Jr., is a hollow paired organ in the
genitourinary apparatus of male Raj~ and probably other skatesand rays. The fluid
is alkaline (pH 8.7-9.5) and contains the highest known physiological concentra-
tion of salts of H&O* . Total [CO 2] is as high as 300 nm, of which 40 % is COa-
and the remainder HCO,-, with only a trace of gaseousCO2 . The anions Cl& and
Sod- are also concentrated, all against the electrochemical gradient. About 98 %
of the cations is Naf. Of the three speciesexamined, two had a high concentration
of carbonic anhydrase in the gland epithelium and one had none. The latter species
has total CO2 concentration (100 mu) and pH (8.7) of gland fluid in the lower
range; this alkalinization was carried out via the uncatalyzed reaction. When the
two speciescontaining enzyme (and higher CO2 and pH) were given 50 mg/kg
methazolamide im, total CO2 in the fluid decreased.
The clear demonstration by nature that the identical function can be carried
out by specialized tissueswith or without carbonic anhydrase is of great value. It
provides an obvious parallel to studies of normal and inhibited functions (see
Fig. 9). Moreover, the alkaline gland is an ideal example of a tissue whose cells
are polarized, with the luminal surface concentrating OH-, akin to the luminal
side of the pancreas (Fig. 8) and the blood side of the kidney (Fig. 3) and stomach
(Fig. 7).
N. Gill
Carbonic anhydrase has been found in the gills of all vertebrates and inverte-
brates in which it has been sought (3 19, 502). The pseudobranchial organ may be
considered part of the gill system; although its physiological role is unknown, it
also contains high concentration of enzyme (308, 502). We may consider the pos-
sible role of carbonic anhydrase in the two main functions of the gill - gas ex-
change and electrolyte transport.
Acetazolamide, in the doserange 30-50 mg/kg, produces a threefold elevation
in plasma pCO2 in the marine elasmobranch S. acanthias, the marine teleost M.
seorpius, and the fresh-water teleost A. nabulosus(2 13). The question that arosefrom
these experiments was whether the respiratory acidosis
. was due to inhibition of
enzyme in gills or erythrocytes or both: this in turn devolves to the issueof whether
dehydration of HCOs to CO2 at the gill surface is mediated by enzyme in the
tissue or in the blood. The question seemsfairly well resolved by experiments with
the selectively permeable drug CL 11,366, taking advantage also of the fact that
there is 5 times as much enzyme in gill as in red cell of S. acanthias (327). Using 1
mg/kg iv, drug-enzyme relations in the tissueswere such that there. was 99.8 %
inhibition in the red cells and a negligible amount in the gills. The elevation of
pCO2 was precisely the same as that after large dosesof acetazolamide (213, 319),
where both gill and red cell enzyme were inhibited. This result suggeststhat the
relation between red cell and gill in the fish, with respect to evolution of CO2 , is
the sameas that between red cell and lung in the mammal. In the overall economy
of the marine fish, however, HCOa” is removed solely at the gill surface (213, 4363
since the marine kidney cannot handle an alkaline load (III, 5B above). Quantita-

tive aspects of this removal, indicating the relative roles of dehydration and diffu-
sion of HCOg-, remain to be studied. It is of interest that the gill membranes do
not concentrate or secrete an anionic drug, in contrast to the kidney of the same fish
(327).
Carbonic anhydrase in fish gill appears to have its chief, if not exclusive, role
in electrolyte transport. After his observation that acetazolamide lowered plasma
[Cl-] in the fresh-water fish Perca Jluviatilis (308), Maetz, with Garcia Romeau,
carried out an important study of sodium and chloride uptake by the gills of the
goldfish Carassius auratus (309). In some experiments 20 mg/kg completely in-
hibited uptake of Cl- from the external solution’ of 400 PM. Both Cl- and Na+
fluxes were reduced in both directions. The overall conclusion is that the inward
movements of Naf and Cl- are independent events; Na+ exchanges with NHI+
and Cl- with HCOa. Carbonic anhydrase in gill is involved in both steps, since the
hydration of CO2 yields Hf both for NHJf and HC03 accumulation. The paper
presents a tentative scheme of much interest, as well as analogies to events in the
stomach and kidney.
The situation in the marine teleost, which would presumably deal chiefly with
outward Na+ and Cl- movement, has not been well studied. Maetz found a mean
elevation in plasma [Cl-] in Serranus after p-sulfamylbenzoate, but the data have
too much scatter to be convincing (308). Hodler et al. (213) found no change in
[Na+] or [Cl-] in pl asma of the sculpin M. scorjGus, or M. octodecims&nosus, after
large doses of acetazolamide. Rigorously controlled flux data are needed to solve
this fundamental problem
The marine elasmobranch represents still another unsolved problem: here the
presence of the rectal gland and the osmotic effect of plasma urea make it (at least
theoretically) unnecessary for the gill to be an organ for salt excretion. Indeed,
S. acanthias has a slight osmolar (but not saline) excess over the sea, and conceivably
sodium and chloride movement have elements in common with fresh-water fish.
This idea is supported by the lowering of plasma [Na+] that followed acetazolamide
(319) and the papers by Burger et al. (55, 55a) describing net influx of Na+ across
the head of S. acanthus. Further work on ion flux across dogfish gill is desirable.
The pseudobranch has been the subject of numerous speculations, both as to
its, overall role and the significance of its high carbonic anhydrase content. The
matter has been thoroughly reviewed by Maetz (308). He also studied the effect
of pseudobranch ablation on vision, on the function of the swim bladder, and on
the enzyme concentration of other organs in the marine and fresh-water perch.
There was no change in any function measured. He concludes that the entire
matter is a mystery. It seems likely to the reviewer that the pseudobranch, like
the gill, serves as a salt transport organ, as well as a passive membrane in gas ex-
change.
0. Swim bladder
Leiner discovered the very high concentration of carbonic anhydrase in the

gas gland of teleosts [see review by Van Goor (502) for Leiner’s extensive survey of

the enzyme in fish]. This was confirmed by F%nge (152), who showed that both
secretory mucosa and rete mirabile contained enzyme. Similar data are given by
Maetz (308). The general physiology of the swim bladder has just been reviewed
by Fange (153).
Taking the works of Fange (152) and Maetz (308) together, it is clear that
systemic carbonic anhydrase inhibition (by p-sulfamylbenzoic acid, sulfanilamide,
and acetazolamide) profoundly reduces the accumulation of both 02 and CO:! in
the swim bladder. At the same time, enzyme activity in the gland plus rete mirabile
was reduced or abolished. Appropriate controls with inactive sulfonamides were
used by both investigators.
The physiology and chemistry of the\swim bladder have been clarified by two
penetrating studies, that of Ball et al. (13) and of Wittenberg et al. (531). From
their schemes it is easy to assign the role of carbonic anhydrase and explain the
effect of inhibition on the CO:! accumulation process. The primary event is gly-
colytic generation of lactic acid in the gland (13), followed by liberation of COZ
into the bladder. The source of CO2 is circulating HC03 (531); thus, dehydration
of H&Q is a critical step in the rate of CO2 accumulation. The very high partial
pressure of CO2 (275 mm Hg) is accountable to the rete mirabile acting as a
countercurrent multiplier (Kuhn, Scholander, reviewed in ref. 53 1).
The role of the enzyme in 02 accumulation is more subtle. It is significant
that this tissue does not show the Pasteur effect, i.e. lactic acid formation is not
inhibited by oxygen. It was initially supposed that 02 is evolved from oxyhemo-
globin by acidification or elevation of pCO2 , but this was disputed by Scholander
(see ref. 13). Wittenberg (532) showed that 02 could still be secreted when 97 %
of the blood hemoglobin was in the carboxy form; he believed that this ruled out
blood hemoglobin as the primary source of bladder 02 . He advanced the notion
that a carrier for oxygen (tissue hemoglobin) within the cells of the gas gland and
rete mediates the secretion. The high 02 tensions are then generated by the counter-
current system. The place of carbonic anhydrase in this *process can only be in-
ferred; the suppression of 02 secretion by inhibitors (152, 308) most simply seems
due to an unfavorable H+ or CO2 concentration at the secretory site, still possibly
involving the dissociation of 02 from a hemoglobin. It seems unlikely that the
multiplier system is susceptible to such an altered environment, but the matter is
open to further study. Unfortunately, the notable advances in general under-
standing of the gas gland since 1955 have not been accompanied by questions or
answers regarding carbonic anhydrase.
The idea of primary acid secretion is supported by the finding of acid effluent
blood from the gas gland (531), as well as direct measurement of acid formation
by the isolated surviving gas-gland epithelium (217). The latter study is of particu-
lar interest, since. both mucosal and serosal sides produced acid; this is quite unlike
the stomach (or presumably the kidney or pancreas) in which acidification at one
border is matched by alkalinization at the other. The fundamental distinction is
that, in the gas gland, acidification is the result of lactate formation, whereas in the
other cases (Figs. 3, 7, 8) only the ionization of water is invoked.

758 T. H. MAREN Volwne 47
P. Intestine
There has been little work on this topic, which could, nevertheless, be of con-
siderable physiological and medical interest. The concentrations of enzyme in the
various segmentsof gut have been published in terms of their relative activity (267).
Our own data show (e.u./g as in Table 16) for perfused rat tissue: duodenum 77,
jejunum 26, ileum 19, and colon 345. This agreeswith the work of Kuriaki and
Magee (267), who also report that the “appendix” has a high concentration-
translated into our units, about 300 e.u./g. The speciesused (rat and dog) lack a
true appendix; authors presumably meant the cecum. Investigation of the primate
appendix would be of some interest in connection with the (possibly) homologous
rectal gland in Elusmobranchii.
Thaysen has reviewed the electrolyte composition of the secretion of the in-
testinal segments(488). There is reasonableagreement among the usual laboratory
species.The jejunal secretion is acid (pH 6.8, HCOs” 15 mu) but all other seg-
ments excrete alkaline fluid with [HCOa-] ranging from 40 to 90 mn~
Only two papers have appeared on the effect of acetazolamide on electrolyte
transport in the intestine. Parsons (393) used segmentsof rat gut in situ, through
which bicarbonate-saline was circulated. As suggestedby the composition cited
above, jejunum normally reabsorbed HCOa, and secreted Cl-, while ileum and
colon added HCOa”to the circulating solution and reabsorbed Cl-. Acetazolamide
(given in water from the previous day and added in 2 mu concentration to the
circulating fluid) decreasedjejunal reabsorption and ileal and colonic secretion of
HCO,. In all three regions absorption of Na+, Cl-, and water was reduced. Kin-
ney and Code (250) made a considerable extension of thesefindings using the ileal
segmentof dog in situ and calculating flux ratios of chloride ion. Insorption (lumen
to blood) of Cl- was found to be an active processand was reduced about 50 % by
5 mM acetazolamide in the lumen. Exorption of Cl- was unaffected. HCQ- con-
centration of ileal secretion was reduced by acetazolamide, in agreement with
Parsons (393), but the amount of enzyme-dependent Cl- moved inward was con-
siderably greater than HCO a- moved outward. This may be an example of NaCl
transport dependent on carbonic anhydrase, like the salt gland and the rectal
gland and possibly the CSF. The authors of this important paper conclude that
acetazolamide is acting other than by carbonic anhydrase inhibition, but the re-
viewer seesno evidence in this direction. It is, of course, a possibility in view of
Hogben’s recent work on the frog stomach and cornea (III, 5E).
Further studies, including the effect of acetazolamide on the high colonic
[K+], would be of interest and possibly of practical use in understanding and
management of Kf loss after colostomy or tumor in the bowel (297).
QI. nnerear
Data on this subject are confined to the paper by Erulkar and Maren (148).
All segmentsof the cat cochlea and the saccusendolymphaticus had exceedingly
high concentrations of enzyme, reaching levels of 3000 e.u./g wet tissue(Table 16).

Acetazolamide reduced the very high [K+] of endolymph (usually about 120
mu) to about 15 mu. The perilymph was unaffected. It is supposedthat K+ secre-
tion is in someway linked to carbonic anhydrase activity, possibly analogousto its
action in the distal renal tubule. It was also of interest that the pressureand volume
of the endolymph appeared to be reduced after enzyme inhibition.
R. CalciJication
The role of carbonic anhydrase in formation of egg shell has been discussed
(III,54; it remains‘ to present data on calcification in certain classesof inverte-
brates and the explanation for the enzyme in mammalian bone.
I) Moltusca. Enzyme distribution was studied by Freeman and Wilbur (165),
who made the interesting observation that some specieslack carbonic anhydrase;
presumably, they deposit carbonate at a low rate. Distribution data for this and
other invertebrate phyla have recently been reviewed and new measurements
added by Polya and Wirtz (411, 412). The first experiments using inhibitors were
done by Wilbur and Jodrey (516), who found that benzothiazole-2-sulfonamide
at 60 e almost entirely suppressedcalcium (46Ca) deposition in the inner shell
surface of the oyster Crassostrea Virginia. No effect was obtained at 18 PM. Aceta-
zolamide showed a modest (about 40 %) reduction in calcium deposition for all
concentrations studied, 30-375 PM. The authors recognized that the benzothiazole-
2sulfonamide effect might include factors other than carbonic anhydrase inhibi-
tion. This drug (60 PM) also suppressedcalcium deposition by 47 % in the oyster
mantle, under conditions of high but not low control rates.
Further studies using five inhibitors were done on the fresh-water snail Physa
heterostropha( 164); in this caseonly growth was measured.Under control conditions
of rapid growth, acetazolamide, benzenesulfonamide, j+toluenesulfonamide, and
sulfaniiamide caused about a 40% reduction in growth. Acetazolamide had no
effect in slow growth experiments, but benzothiazole-2-sulfonamide completely
suppressedgrowth under all conditions; this drug has a separate and toxic effect
for the oyster.
2) Arthropoda. Carbonic anhydrase was found in the body and mantle of the
barnacle Balanusimprovisus,and the effects of several inhibitors on shell growth were
studied. As in molluscs,the shell is virtually pure CaC03 . Benzothiazole-2-sulfona-
mide (0.9 PM) caused almost complete cessationof growth; acetazolamide (29 PM)
produced about the sameeffect. When the inhibitors were introduced in the adult
stage, their effect was reversible; but if they were given before metamorphosis,
shell growth was permanently arrested, even when inhibitors were withdrawn (94).
3) Coelenterata. A more distant physiological system from the red cell than the
building of coral reefs can scarcely be imagined, although the relation of both
systemsto CO2 chemistry was foreseen in the remarkable initial paper on carbonic
anhydrase by Meldrum and Roughton (350). All speciesof the subclassHexacoral-
lia contain carbonic anhydrase, and inhibition under certain conditions slowsthe
deposition of 4Va in the coral (180).
Like the shells of other invertebrates discussedabove, coral reefs are pure

760 T. H. MAREN volume 47
CaCOa . A significant feature of all reef-building corals is their symbiosis with

certain algae. The action of carbonic anhydrase (presumably in the coral but not
the plant) has been approached by Goreau in the light of these and other findings,
and the following discussion is based on his work ( 180, 18 1).
Table 27 shows in idealized form the effects of light and dark and carbonic
anhydrase inhibition on calcium deposition in coral. Variations in this pattern
among different species requiring more or less light, or enzyme, are noted in the
original (180). The data suggested to Goreau that the photosynthetic reaction had
the same overall effect as carbonic anhydrase, since both removed CO2 (or HCOa-)
from the system. Calcium and HCO 3- ions are furnished by the sea, and the algae
(through photosynthesis) and the coral (using the enzyme) conspire to drive the
following overall reaction to the right.
C.A.
Ca++ + 2HC03 - CaC03 +CO ‘2 + Hz0 (W
It is of interest that in this system, just as in mammalian physiology, the role of

carbonic anhydrase and effect of acetazolamide is set by the quantitative position
of CO2 . In the presence of maximal photosynthetic effect (rate of 100 in Table 27)
and CO2 removal, enzyme inhibition only reduces calcium deposition by about
half. When photosynthesis is reduced, the role of the enzyme appears much more
critical and inhibition reduces calcium uptake by coral virtually to zero.
4) Mammalian bone. This matter has recently been reviewed by Ellison (143)
with the significant conclusion that carbonic anhydrase has no role in calcification
of mammalian bone, but that the enzyme, which is present in the epiphyseal area,
is confined to osteogenic cells destined for hemopoiesis. The evidence is based on
earlier studies in which large doses of acetazolamide failed to repress calcification
or to hinder epiphyseal development in growing rats and dogs (328), as well as
the finding that enzyme is absent from the shaft and from cartilage (143). It may
be significant that only calcified structures containing virtually pure CaCOa are
dependent on carbonic anhydrase.
5’. SickZing in red cells
Much interest was aroused by the report of Hilkovitz (2 10) that acetazolamide
reduced sickling both in vitro and in vivo. The in vivo data were not very convinc-
TABLE 27. Eject of acetasolamide and of photosynthesis on calcium deposition in coral (180)
Relative Rates of Wa Uptake
Light Dark
z noz z noz
Control 100 25 4-o 15

Acetazolamide, 1 mM 40 N N N
2 indicates presence of the normal symbiont algae of Zooxanthellae sp. N means not de-
terminable, probably less than 5 on this scale.

ing, and an alleged relationship between inhibition of sickling and red cell concen-
trations of drug [the latter obtained by the reviewer in earlier animal work (328)]
was based on apparent random variations in drug levels at a time when they were,
in fact, reasonably constant. It does appear to be true that acetazolamide reduces
sickling in vitro, using the 240hr coverslip technique (2 10). We obtained 50 %
reduction in sickling by addition of 6 pg/ml acetazolamide to the blood of three
patients; there was no effect with the control substance, CL 13,850. Acetazolamide
had an effect in blood with and without oxygen and also using sodium metabi-
sulfite in the preparation (unpublished data, Pharmacology Dept., Univ. of Flor-
ida). But at least three papers have appeared showing that there is no effect in
vivo; the failure of ethoxzolamide (209) was particularly convincing because of its
high activity and very rapid diffusion into red cells (220). The matter has recently
been reviewed by Basu and Woodruff (18). They gave 7 mg/kg of acetazolamide by
mouth to three patients with sickle-cell anemia and four with sickle-cell-hemoglo-
bin-C disease. There was no change in erythrocyte survival time, reticulocyte
count, or fraction of cells sickled. The therapeutic failure has unfortunately stifled
work on the possible in vitro effect and mechanism. It is felt that the systemic
metabolic acidosis disposes toward sickling and thus negates or opposes the possible
direct action of the drug on the red cell. It is not possible at present to draw up any
reasonable scheme that would explain a reduction of sickling through inhibition
of carbonic anhydrase. Possibly there is interaction between acetazolamide and
hemoglobin, which should be explored.
T. Adrenal and thyroid glands
There would not appear to be any obvious role for carbonic anhydrase in
endocrine glands, nor is the presence of the enzyme well documented. Certain
claims, which seem inevitably to follow the arrival of a new drug, will be briefly
dismissed.
Telegdy (485) reported that acetazolamide (0.5 mu) increases the synthesis
of corticosteroids by ox adrenals in vitro, and he concluded that inhibition of car-
bonic anhydrase activates adrenal enzymes necessary for steroid synthesis. In a
further flight from reality, he suggested that this is the basis for the decrease in
brain excitability after acetazolamide. The argument is not pursued here since
there is no carbonic anhydrase in the adrenal cortex of rat or dog or presumably
any other species (unpublished observations).
The thyroid has received more attention. The studies showing complete lack
of morphological activation or antithyroid effect of acetazolamide (328) were
entirely ignored by later workers (167, 264, 486), who were bemused by the “sul-
fonamide structure” of the drug. Acetazolamide, in fact, does not have the stigmata
of an antithyroid drug since it possesses neither an arylamino group nor a potential
-SH function, usually typified by -NH-C-NH-. Tenney’s (486) data show a
II
S
small decrease in metabolic rate in rats receiving acetazolamide and little else of
consistence. Gabrilove et al. (167) showed a moderate decrease in thyroid 1261

uptake in normal and hyperthyroid patients after the astonishing dosesof 85-125
mg/kg by vein. Similar experiments in the rat did not yield a consistent relation
between dose and responseand were never reported in full (534). In these experi-
ments the possible effect of CO2 retention on oxygen consumption was not ruled
out. Krieger et al. (264) and Woodbury et al. (233, 534) claimed to find carbonic
anhydrase in the thyroids of several species,including rat and dog, after due atten-
tion to perfusion of the gland. Acetazolamide (400 PM) reduced iodide uptake in
rat thyroid slices, but sulfanilamide and other weaker inhibitors had a greater
effect (264).
This confusing picture prompted further attention to enzyme in the gland.
Dog and rat thyroids were thoroughly perfused with saline in situ and immediately
removed, homogenized, and analyzed by the standard method of this laboratory
using the barbital-buffer system (330). In this system the red cells of these species
have about 3000-7000 e.u./ml. The thyroids of both specieshad 6-9 e.u./g. (This
is equivalent to about 2-3 e.u./g in the standard carbonate system, to which
Table 16 is adjusted.) This is x0, or less,that amount found in any organ whose
function is established. Since it could be due to 0.1% red cells in the tissue,we con-
clude that the thyroid gland per se lacks carbonic anhydrase (unpublished data,
Pharmacology Dept., Univ. of Florida). Even if there were a trace of enzyme in
epithelial tissue, it could scarcely be of physiological importance, since no well-
documented caseof thyroid dysfunction in man or animal has ever been associated
with long-term carbonic anhydrase inhibition.
U. Teratological efects
The initial toxicological studies of acetazolamide (328) failed to reveal any

lesion in the newborn; although rabbits received drug throughout the critical
period of pregnancy, the bulk of the work in rats and dogs was confined to the last
third of pregnancy since the drug was designed at that time as a diuretic for the
toxemias: Using a more critical dosageschedule and specific techniques of modern
teratology, Layton and Hallesy (279) discovered a remarkable abnormality in
offspring of rats and mice that received acetazolamide, 0.6 % in the diet, during
pregnancy. Most commonly, digits 4 and 5 were absent from the right forelimb.
The anatomical localization of this lesion is unique in descriptive and experimental
teratology. The question of whether specific carbonic anhydrase inhibition is in-
volved is probably answered by the fact that ethoxzolamide (517), dichlorphena-
mide, and methazolamide (W. M. Layton, personal communication) all yield the
abnormality. We currently believe (517) that the lesion is secondary to carbonic
anhydrase inhibition in the mother or embryonic membranes,since drug restricted
to days 10-11 of pregnancy elicit the lesion but enzyme does not appear in the
embryo until day 13 (III, 4). Acetazolamide and ethoxzolamide passfrom mother
to fetus and to membranes. It is hoped that further studies, correlating the various
electrolyte abnormalities in the mother with the appearance of the deformity, will
reveal its etiology.

Landauer (268) injected 2.5-20 mg acetazolamide into the yolk sac of chicks
on the 24th or 96th hr of incubation. At day 17 the chief abnormality was a short-
ened or crossed upper beak. Sulfanilamide produced a different but related lesion.
The author concludes that the effects are probably not due to carbonic anhydrase
inhibition; use of other sulfonamides and control substances is indicated. It was of
much interest that acetazolamide protected against the sulfanilamide lesion.
V. Baderia
The unexpected finding was made by Veitch and Blankenship (506) that car-
bonic anhydrase was present in many strains of Neisseriasp. and at least one strain
of Streptococcus. Forkmann and Laurel1 (162) made the significant discovery that
acetazolamide inhibited growth in many Neisseria strains; this was not overcome by
p-aniinobenzoic acid. A further fact of interest is that raising the concentration of
CO2 in the ambient air abolished the action of acetazolamide. These data suggest
that in somefashion HCOa- formation is essential to growth of these organisms.
As in other systemsdescribed in this review, this can be brought about by carbonic
anhydrase acting at low substrate (Con) concentrations or by the uncatalyzed rate
at high substrate concentrations.
Sandersand Maren (454) showed that carbonic anhydrase from thesebacteria
was inhibited in vitro by the various sulfonamides, similar to data found for mam-
malian enzymes, although there were some quantitative differences in K1 values.
The control substance CL 13,850, N%butyl acetazolamide, (315) was inactive
against the bacteria and the enzyme. NLacetylsulfanilamide, which is generally
inactive against bacteria but is a reasonably potent carbonic anhydrase inhibitor
(Table 4), suppressedgrowth of the Neisseria species.A rough correlation was found
between inhibition of enzyme and inhibition of growth for the samestrain of bac-
teria. An exception was the poorly diffusible CL 11,366, which was potent against
the enzyme but had little or no antibacterial action. The complete reversal of anti-
bacterial effect of acetazolamide (and other active drugs) by pCOz aslow as 25 mm
Hg was confirmed. Since 21 strains of pathogenic meningococci were found sensi-
tive to several drugs, the possibility of therapeutic utility was foreseen. The most ac-
tive drugs were CL 13,580and ethoxzolamide (10-100 pg/liter suppressedgrowth),
probably due to the combination of high activity against the enzyme and good
diffusibility. Even in this favorable circumstance, however, abolition of the effect
by CO2 in the concentration in which it is found in tissuemight mitigate against
clinical activity.
IV. GENERAL SUMMARY AND CONCLUSIONS
The massof detailed observations in this field has not, happily, obscured a
number of principles that emerge on the chemical, pharmacological, and physio-
logical sides.An attempt is made here to summarize each of these and their connec-
tions as briefly as possible.

1. Chemical
Carbonic anhydrase may be thought of as an idealized low-molecular-weight

soluble metalloprotein. In the vertebrate kingdom, so far as is known now, its
properties are reasonably constant, although there is no doubt that different species
and organs have enzymes with slightly different structural properties. Its turnover
number is perhaps the highest of any enzyme, and there is the unusual feature that
the physiological reaction catalyzed (CO:! e HCOs-) proceeds at very significant
rates without the enzyme. Both the active and inhibitor sites are in intimate rela-
tion to the Zn atom, although the precise reactions are unknown. The enzyme
appears to serve as a proton exchanger, and the new finding of certain nonphysio-
logical substrates suggests that it is a general acid-base catalyst.
2. Pharmacological
This field has been enormously advanced by the discovery of a class of drugs,
the aromatic sulfonamides (R-SOzNHg), many of which have no known actions
apart from inhibition of carbonic anhydrase and have dissociation constants of
@I) as low as low9 M. The structural specificity for inhibition is absolute, since re-
moval of a proton from the nitrogen atom completely destroys activity; further-
more, drugs with the given configuration invariably inhibit the enzyme, the only
differences being those of affinity, which cover a 106-fold range. Drugs of widely
differing chemical and physical properties have been prepared, making it possible
to study physiological inhibition when drug reaches the organ by secretion, or by
diffusion, or to exclude drugs from certain tissues while permitting them to cohcen-
trate in others. The drugs also vary in their general properties of absorption, distri-
bution, excretion, and metabolic fate.
The analysis of physiological effects must also take into account certain funda-
mental findings in enzyme kinetics. Inhibition is always reversible, both kinetically
and in terms of the in vivo effect. The question of whether inhibition is competitive
has not been answered with certainty; for the hydration reaction, noncompetitive
kinetics are followed, and there is now evidence that for dehydration it is competi-
tive. This would follow structural analogy, since HCOs, but not CO, , may be
written to resemble-SO2NHz . However, the ratio of Km/K1 is of the order of 10s-
107, and substrate in vivo < K,,, ; thus, inhibition kinetics may be followed in vivo
as if substrate did not interfere.
Dose-response curves in the intact animal show that maximal effect in all
organs can be reached, for example, with acetazolamide at 5-20 mg/kg. Higher
doses have no further action, and it is assumed that full inhibition has
been achieved. The residual physiological effect is due to the uncatalyzed reaction
and in some cases (i.e. kidney) a process such as active HCOs? absorption, which
lies outside the CO 2 S HCO, interconversion. When the fractional inhibition (Q
in tissues is plotted against the physiological response, it is found that there is no
drug effect when i = 0.99 and that the maximal effect is reached when i = -0.999.
This is consistent with rate data that also show that enzyme is present in at least

loo-fold excess. Analysis of the effects of %omplete” inhibition has been the cardi-
nal factor in defining the varied physiological roles of this enzyme.
3. Physiological
The enzyme has, broadly, two physiological roles. One is in red cells, where it
subserves the hydration of metabolic CO2 in the tissue capillaries and its dehydration
in the capillaries of the lung or gill. The second is concerned with transfer or accu-
mulation of H+ or HC03 in organs of secretion. It is important to realize that, in
this context, carbonic anhydrase may also have a role in the elaboration of a neu-
tral fluid by its effect on intracellular CO:! equilibria. Table 28 shows representa-
tive situations. When the rates of these transport processes in vivo are studied, it is
found that they exceed those of the uncatalyzed hydration of CO2 or dehydration
of HC03-; thus, the enzyme has a clear place in the secretory processes. The re-
quirement for in vivo catalysis is satisfied by acceleration of the nonenzymic rate
TABLE 28. Summary of relations between inhibition of carbonic anhydrase and alterations
of acid-base balance in secretory organs
1 2 3 4 5 6 7
..
Mimicking of C.A.I. Blocking of C.A.I.
Physiological Metabolic Respiratory Metabolic Respiratory

Product Separated EfrIof.’ . . -- -
Acid- Alkg Acid- Alka- Acid- Alka- Acid- Alka-
osis OSlS 1OSlS osis losis osis losis
----_ -- --
Kidney Hf + 0 + 0
Stomach H+ + 0
CSF Cl-, H+ + 0 - 0
Pancreas HC03- 0 + 0 0
Liver HCOs- 0 + 0 0
Eye (rabbit) HC03 0 + 0 0
Salt gland Na+, Cl-, K+ 0 + 0 0
Key: 1 means effect of acetazolamide or other inhibitor at full dose, Key: + means that the
of suppressing the normal secretion (~01s. 2 and 3). A full ( .i ) or acid-base change
partial (L> effect in the same direction by acid-base changes blocked the normal
alone is shown in ~01s. 4 and 5.0 means that the alteration did not (col. 3) or second -
mimic inhibition. - means the experiment has not been done. ary (i.e., fluid for-
mation) effects of
acetazolamide. 0
means no blocking;
the col. 3 effect is
either unchanged or
enhanced. - means
the experiment has
not been done.
Relevant experiments and references are given in the appropriate part of section III.

by 2- to 5-fold, and it may be imagined that under the stress of unusual demands it
would be some 20.fold. The amount of enzyme present in secretory tissues is
enough to increase the rate about lOO- to lOOO-fold. Carbonic anhydrase is fur-
nished in excess of physiological needs and has never been found to be rate limit-
ing.
In secretory organs, the effects of carbonic anhydrase inhibition can be blocked
or mimicked by appropriate changes in acid-base balance. The pattern of these
experiments reveals much about the primary process. Table 28 summarizes data
of this type, discussed in section III, 5. This comparison of the different organ sys-
tems permits the following generalization: in tissues secreting H+, or HCOs, addi-
tion of the product through metabolic alteration blocks, and addition of the antipodal
ion mimics enzyme inhibition. Thus, for the kidney, metabolic acidosisblocks and
metabolic alkalosismimics. For the pancreas, the opposite pattern holds. For organs
producing a neutral solution, the pattern of blocking and mimicking reveals the
ionic balance within the cell that is struck by enzyme action. Thus, for the salt gland,
an alkaline milieu seemsnormal since metabolic acidosis mimics inhibition. The
responseto respiratory alkalosis is most significant, since it mimics carbonic anhy-
drase inhibition for all types of secretion. The reason for this is not entirely clear,
but the data fit the concept developed here: that secretory cells are polarized with
respect to H+ and OH-. Hyperventilation would abolish this by rapidly swamping
all parts of the cell with OH-. The fact that respiratory acidosisis lesseffective may
be due to the fact that such changesare of lessermagnitude. The data of Table 28
provide a unifying basisfor the physiological roles of carbonic anhydrase: the rapid
adjustment of the intracellular and extracellular concentrations of the seven molec-
ular speciesinvolved in the equilibria between Hz0 and CO2 .
I record with pleasure the many useful discussions and critical comments of my col-
leagues: Dr. Kenneth C. Leibman, Dr. Thomas F. Muther, Dr. W. Walter Oppelt, Dr. Roger
F. Palmer, and Dr. David M. Travis. My best thanks are also due Mrs. Aija Gotti for assist-
ance in the preparation of the manuscript. Various sections were reviewed and improved by
the suggestions of Dr. Robert P. Davis, Dr. John T. Edsall, Dr. C. Adrian M. Hogben, Dr.
Arthur B. Otis, and Dr. F. J. W. Roughton.
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Carbonic Anhydrase: Chemistry,

1. Introduction: Scope and Definitions. ................................... 597

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C. Methazolamide (Neptazane) ............................... 650

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I. INTRODUCTION: SCOPE AND DEFINITIONS

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Second-order rate constant for CO3 + OH- + HCO3-.

K a’1 The observed ionization constant of H&03.

V UUO Velocity of the uncatalyzed hydration reaction, kl [Cog].

mally, this implies

Velocity of the catalyzed hydration reaction

V’ oat The similar expression for dehydration from H3COa .

V mh Velocity after inhibition of carbonic anhydrase. If inhibition is com-

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Concentration of free inhibitor. Also written [lfw.]

Before reviewing the individual types of chemical reactions in this section, it

The seven speciesinvolved in thesereactions (including OH- and HZO) attain

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It is not possibleto say whether equation 3 or 4 is catalyzed by carbonic anhy-

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This encompassesa wide variety of methods, including simple indicator

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D. Recent advances: whole-cell methods

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2. Kinetics of the Uncataly~ed Reaction

A remarkably incisive review by Kern (240), as well as the treatment by Edsall

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The advantage of this schemeis that it encompassesboth H&OS and HCO,- in

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3. Chemical and Physical Properties of Carbonic Anhydrases

With a few interesting exceptions, the work on carbonic anhydrase derives

A. Primate red cell carbonic anhydrase

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TABLE 2. Properties of human red cell carbonic anlzydrases

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B. Bovine red cell carbonic anhydrase @CA)

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TABLE 3. Kinetic constants of red cell carbonic anhydrase of various species”

Temp, C HorDt Dog ox Horse Human Dogfish

Ref. except where (247 > (338)

* Red cell hemolysate or crude extract. t Hydration or dehydration. $ Based on

C. Bovine and human lens carbonic anhydrase

Sen et al. (465) separated two components of carbonic anhydrase in partially

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D. Dog red cell and renal carbonic anhydrase

E. Othr vertebrate carbonic anhydrases

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Comparison of properties of human red cell enzymes (Table 2) and those of

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IV. Esters and amides of

B El e D 0; 10 2;2 (102) same for kid-

Copyright © 1967 American Physiological Society. All rights reserved.