The Microbiologic Assay of Vitamins in Clinical Chemistry

Donald D. Van Slyke Lecture, 1958

Harry Sobotka

HE STUDY OF PRESENCE AND QUANTITY OF VITAMINS in the body fluids has gained ever-increasing importance for clinical medicine. Most vitamins have been discovered in the course of investigations of the deficiency disorders to which their absence gives rise, and some have their names actually derived from the diseases caused by their absence. The practice of their quantitative analysis is not confined to these conditions, but it has been found that the amount in which they occur in various fluids and tissues, and the rate at which they are absorbed, bound, retained, excreted, or otherwise metabolized, bears significant relationships to a variety of pathologic states and is apt to illuminate their etiology (1). Rather than trying to give a formal definition of vitamin, we shall shortly survey the principles of their biochemical function. Let it be said at the onset that many of the water-soluble vitamins of the B type have been correlated with certain enzymes or groups of enzymes; they form indeed the prosthetic group of these enzymes, the coenzyme (Table 1). Their character of vitamins is based on the inability of the animal organism to synthesize the non-protein moiety of vital enzymes. In these instances, knowledge has advanced to the point where we may answer with more or less assurance the question, What connection is there in a given avith.minosis between the morphologic lesion and physiologic dysfunction, their metabolic etiology, usually
the Department of Chemistry, Mount Sinai Hospital, New York, N. Y. Delivered before the New York Metropolitan Section of the American Association Clinical Chemists, February 11, 1958. Received for publication February 17, 1958. From Copyright #{174} 1958, by Thi
Asic

of

AssoowrzoN

07

CLINIOAZ.

CESNISTS,

INC.

93

94
Table 1. vITAMINs
AS COFACTORS IN

SOBOTKA
ENZYMATIC

Clinkal
REACTIONS
function

C1i.inkfr

AND MErABOLIC SYSTEMS

Via.,in

Enzymatic

Thiamine Riboflavin
Nicotinamide

Cocarboxylase (Ilycolysis (cytochrome

Dehydrogenases

oxidase)

Pyridoxal Pantothenic

acid

Transaminase, amino acid decarboxylase, tryptophan metabolism Coenzyme A, 2-carbon transfer, citric acid cycle, conjugation of bile
acids Fatty acid metabolism Nucleic Nucleic Oxidative acid metabolism acid metabolism; decarboxylation, methyl transfer,

Biotin Folinic acid Cyanocobalnmlne Thioctic acid

choline

synthesis

conjugation

of thiamine

centering on an enzymatic defect, and the primary cause, namely the absence or inavailability of the vitamin? Methods of chemical analysis have been applied to the water-soluble vitamins, the vitamins of the B group, and are serving as control for the animal test-e.g., in the industrial preparation of these factors. Wherever the chemical constitution of a vitamin has been clarified, chemical analysis has of course become the primary checking method in production and in the clinical laboratory. I shall discuss today microbiologic methods for a number of deficiency factors, which have been developed in our laboratory and elsewhere for dilnicochemical problems. More than 10 years ago, I had become interested in the microbiologic assay of deficiency factors. We developed a method for the deteTmination of inositol in patients serum by means of a strain of Saccharomyces carisbergensis, based on Atkin s modification of Woolley s method (2). In the course of subsequent work on thermophilic algae and bacffli we had to use synthetic media; these experiments led first to the study of nutritional requirements and trace elements and eventually to the development of microbiologic assay methods with thermophific and mesophilic bacilli (3) and with protozoa.1 Table 2 gives a survey of the vitamins studied, of the deficiency symptoms produced by their absence, and of other pathologic conditions where they have been suspected to be involved. We are using various microorganisms, bacilli, cocci, and protozoa,
I
staff and

am greatly
experimental

indebted

of our vitamin assay laboratory especially my collaborator Dr. Herman

aspects

Seymour Hutner for his counsel as protistologist, to the for their devoted assistance, and I wish to thank Baker for his ingenious approach to the theoretical of this work. to Dr.

Vol. 4, No. 2 Table

ViAn.4n

MICROBIOLOGIC 2.
WATER-SOLUBLE Condition w4t d4cioncy

ASSAY OF VITAMINS
VITAMINS AND HUMAN

95
Diszasz

Area

of invoZveawnt

Thinmine Riboflavin Nicotinamide Pyridoxin group Pantothenic acid Biotin Cyanocobnlnrniu Folic acid group Thioctic acid Inositol

Beriberi Cheiosis Pellagra Infantile

convulsions

Pernicious Pernicious
.

anemia anemia

Neurologic conditions Dermatologic conditions Dermatologic conditions, dementia Diabetes, infection, toxemia of pregnancy Neurologic; endocrine malfunction Dermatologic conditions () Neurologic and hematologic Neurologic and hematologic Hepatic disease Deficiency of fat metabolism

but our technics deviate in several points from those of traditional bacteriology. The media we are using are strictly synthetic. The selection of grade and brand of the inorganic constituents used in bulk are based on extended comparative studies and are designed to exclude known and unknown trace elements at or above those minute concentrations, where they still could influence the outcome of the test. Similar considerations apply to the organic constituents of the media, which are rigorously screened and confined to compounds of known constitution and good uniformity such as oligosaccharides and amino acids. We can not accept, for example, proteins such as serum albumin, however well they may have been purified, since their constitution is unknown indeed, and the nature and amount of bound, adsorbed, engulfed, or otherwise connected factors remains either undetermined or variable. Such factors may themselves be vitamins, or lipids, or, most important, trace elements. The latter are introduced individually and each of them is tested for its function as a required factor for the microorganism in question. In many instances we are lucky to be able to discern the specific purpose of their presence, usually as the known component of an enzyme system. As with bulk ingredients, we have assured ourselves of the purity of the compound we introduce from traces of other trace elements, so to speak-trace elements of the second order. Most of the trace elements are heavy metals; the absolute requirement of them in the medium places us in a quandary: Too high concentration.s will prove toxic to the microorganism, too low concentrations wifi not support growth beyond a rather low level. The way out of this dilemma consists in the addition of a chelating agent which forms

96

SOBOTKA

Clinical Chemistry

complexes with the various heavy metal ions. In this manner the ionization of the various cations is repressed and kept below the toxic level. At the rate at which ions of a given species are incorporated in a growing bacterium, some atoms dissociate from the complex and become free cations in proportions governed by the dissociation constant of the complex. When, on the other hand, free ions are liberated by lysis of the microbes, they are chelated to maintain the equilibrium and no toxic concentration of the free ion can accumulate. Substances like citric acid serve this purpose well enough, but, as they themselves may enter into the metabolism of the microorganism, they threaten to vanish, and their chelating effect with them. The use of nonmetabolizable chelating agents, especially of ethylenediamine tetracetic acid (Versene) and the related Versenol has removed this difficulty. The numerical values of the dissociation constants of their complexes with various trace elements render these compounds ideally suited to act simultaneously as buffers for a variety of metal ions. What has just been said about nonmetabolizable chelating agents holds equally for nonmetabolizable pH buffers. Superior to phosphate and other conventional buffers, which could be incorporated or consumed as nutriment, substances have been selected such as transaconitic acid, triethanolamine, and dihydroxyethylethylenediamine which are not metabolized and insure stability of pH. METHODS The media may be made up from the individual ingredients each time when required, or the solid components may be well mixed in quantities, totalling several kilograms and then used by dissolving appropriate amounts of such a solid mix with the addition of the few liquid components. The medium is then distributed, usually by automatic pipet holding, say, 5 ml., into a number of squat glass Erlenmeyer flasks of 10 or 25 ml. capacity. At this point a volatile preservative may be added, which will later completely evaporate on autoclaving. Rising quantities of ingredients to be tested for their effects on bacterial growth are added to the flasks. In the case of clinical analysis, systematic dilutions of suitably treated blood, serum, urine, cerebrospinal fluid etc. are added, usually in the ratio 1:3:10:30:100, etc., with extra dilutions interpolated in a critical range. Finally, all flasks are properly marked and covered with glass or aluminum caps. All these opera-

Vol. 4, No. 2

MICROBIOLOGIC

ASSAY OF VITAMINS

97

tions are carried out with a minimum of exposure to atmospheric contamination, but without flaming and without the use of cotton plugs, as these have been found to contain biotin, certain lipids, and other unwanted factors. The flasks are then placed in a Pyrex glass tray of 20 by 36 ems. and are then sterilized at 118#{176} 30 mm. in the autoclave to avoid confor tamination with spore formers. A ring of condensed water between flask and cap protects the contents against contamination. Upon removal from the autoclave, the flasks are inoculated, using the customary sterile precautions. In the case of thermophilic cultures to be incubated at 55#{176} or higher, the bottom of the tray is covered with a layer of 0.5-1.0 cm. of water to keep the volumes in the flasks constant. The tray is then covered with an inverted tray of identical dimensions, and both trays are sealed together hermetically by plastic tape. They are now placed in an incubator of the proper temperature or, in the case of chlorophyll containing algae, in a box with overhead illumination which has been selected for optimal intensity and wave length. We do not find it necessary to accelerate algal growth by shaking. After a given incubation period, e.g. 24 hours or 5 days, the tests are read by determining the optical density (O.D.). The contents of the individual flasks are read in cells of dimension 12 by 12 by 48 mm. in a Welsh Densichron. The reading of bacterial cultures rarely exceeds unity. The algal cultures run to optical densities of over 3.0; for higher values appropriate dilutions are made and the O.D. of the original solution is obtained by computation. Together with each group of unknown samples, a standard test, consisting of a series of dilutions of the pure vitamin, is set up, inoculated and incubated under identical conditions. From its optical densities one prepares a standard curve (Fig. 1). In a successful run, the O.D. s of the unknown sets will fall on curves of similar shape and amplitude. The position along the abscissa at which an unknown curve coincides with the standard curve permits the calculation of the vitamin content of the unknown sample.
RESULTS

We shall now relate some of the results obtained in specific stances and refer to special technical points as we go along.

in-

98

SOBOTKA

Clinical Chemistry

B 4
F

ci3
/

/ / /

I
Fl%. 1. Growth grac#{252}ss with (B) the cultures. The ordinates adjusted

10

30
ijg./mI.

100

300

1000

3000

of Loctobaosflua iciohmannsi (L), Ochrornonaa malhcrnensis (0), ugiena increasing amounts of vitamin B12, The ordinate gives optical density of dotted line (L) gives the curve for Lactobaciilus ieschmann#{248} with the to the level of Eugiena. CYANOCOBALAMIN

A vitamin that is effective in quantities 1000 times smaller than any other vitamin (with the exception of biotin) is cyanocobalamin, or vitamin B12, the extrinsic factor of Castle. The quantities in which it occurs in the human body fluids and tissues are proportionately minute and many orders of magnitude below the detectability by the most delicate physicochemical methods. Its natural history has not been fully disentangled, but we know that its absorption through the wall of the gastrointestinal tract depends in general on the presence of the intrinsic factor, a specific mucoprotein. The part it plays in hematopoiesis, or, more widely speaking, in nucleic acid synthesis, appears to be of a multiple nature and interlocks with the role of the folic acid complex. A tentative scheme of these interrelations is depicted in Fig. 2. As has been so well demonstrated in the cases of mutants which have lost a specific enzyme, one finds that either a vitally important enzyme, when absent in a microorganisms own arsenal, may be added, or one may compensate for its absence by supplying a product

Vol. 4, No. 2 Diet or Intestinal Folic Acid (Ascorbic Folinic Acid

MICROBIOLOGIC Flora Vitamin Acid) B1,

ASSAY OF VITAMINS Gastric Intrinsic Mucosa Factor

99

-Bit-Complex

in Blood

Uracil(Methyl

I
1
Donor)

-+Thymine Desoxyribose

Thymidine (Desoxyriboside) and Phosphate

Thymidylic

Acid

(Desoxyribotide)
Nucleic Acid (Blood Cells) Fig. 2. Function of vitamin B12 and folic acid in nucleic acid synthesis.

in the metabolic chain, following the step for which the enzyme is missing. An example: One may determine vitamin B12 by its effect on the growth of Lactobacillus leichmannii, Euglena gracilis, Ochromonas maihamensis and a vitamin B12-less mutant of Bacillus coli. L. ieichmannii requires vitamin B12 primarily for the synthesis of desoxyribosides, whereas (a) the formation of folinic acid and (b) the methylation of uracil may be taken care of by alternative enzymes. Hence, the organism will get along without vitamin B12, if provided with sufficient nucleosides. In contrast, Euglena and Ochromonas, require vitamin B12 at two or more spots in their metabolism; thus, it becomes impossible to replace its enzymatic functions by addition of an end product. Hence, the high, one may say multiple, specificity of vitamin B12 for these organisms. One may discern in Fig. 2 the involvement of our enzyme system in the methylation of uracil. The absence of B12 may be overcome by addition of relatively high concentrations of methionine, a methyl donor. In this instance the subnormal concentration of an enzyme is made up for by the excessive concentration of one of the reactants, following the mass action law. As a practical application of this ob

100

SOBOTKA

Clinical Chemistry

servation the assay organism may be used for the quantitation of methionine. When we discuss vitamin B12, we must consider the numerous congeners of the true vitamin B12, in which nature or directed biosynthesis in the laboratory have replaced the benzimidazole group in the nucleotide portion of the molecule by other cyclic groups such as adenine, for example, or have omitted the nucleotide portion altogether, as in factor B. These various factors are inferior substitutes for the true vitamin, and the extent varies to which they may replace its functions; their effectiveness as a vitamin B12-substitute in higher animals is negligible, but assumes different values for different microorganisms (Table 3). The presence of these congeners, which one may call pseudovitamins or paravitamins, in body fluids will produce discrepancies in the response of diverse assay organisms. Among them, Ochromonas maihamensis is in our experience the most finicky and thus the most specific for the true vitamin. By virtue of its high specificity, the response of this phagotrophic organism approaches the response-and that means the requirementsof the mammalian organism. It has been our preference in clinical studies. There is one more hurdle to be taken before practical applicationthe varying amounts of the vitamin in the body fluids and in the tissues in bound form. The binding factor may be identical or related with the intrinsic factor, but we have discovered distinctly different binding factors in various microorganisms. Since only the free vitamin elicits response in the assay organism, the fluid must be hydrolysed to deconjugate the complex and to destroy the binding factor. This is accomplished by heating at 118#{176} 30 minutes at pH for 4.5, conditions which do not affect the vitamin itself; the presence of cyanide or bisulfite is necessary to prevent oxidation.
Table
Substance

3.

REL&rIvE

vIvAMmB12
Lb. Ieicbmannii

RESPONSE

TO DIPFERENT Euglena granUle

COMPOUNDS Ochromonas maihamensis

Cyanocobalamin Factor A Factor B Factor C Pseudovitamin

B12 (4).

100 64 60.3 14 400

100 137 4.2 100 800

100 3.4 0 0 1.2

From J. B. Ford

Vol. 4, No. 2

MICROBIOLOGIC

ASSAY OF VITAMINS

101

I have already described some features for vitamin B12, especially the specificity the specificity for various pseudovitamins. vitamin B12 in human serum ranges from express this in more familiar dimensions, whole blood.
CLINICAL STUDIES

of the microbiologic assay of various organisms and The normal content of 300 to 1000 ,g./ml. or, to 0.03 to 0.1 11g.in 100 ml. of

In a study of the distribution of B12 between blood plasma and erytlirocytes (RBC) in untreated cases of pernicious anemia, we found that the RBCs hold on to their B12 and that the extremely low values of B12 in whole blood in pernicious anemia are due to its signal decrease in the plasma (5). The RBCs contain about two-thirds of the normal amount, but their contribution to the over-all titer is of course reduced by the low hematocrit figures in anemia (Table 4). In a study of our obstetric material we have compared the vitamin B12 content of mothers and their infants blood (6, 7). We found, in agreement with other authors, that the newborn baby holds on to its vitamin B12 at the expense of the mother, whose B12 sinks in the majority of unmedicated cases to levels typical for megaloblastic anemia. We have supplemented this investigation with determinations of folic acid (PGA). We have developed a thermophiic bacillus, a strain of B. coagutans, for the microbiologic assay of PGA and its congeners (8). Quantitatively similar responses are obtained by folio acid, folinic acid, presumably its active form, and by its polyglutamic acid conjugates (with due allowance made for their labifity). As these results are obtained without previous deconjugation, B. coagulans appears superior to Streptococcus fecalis which requires pretreatment with deconjugase, an enzyme supplied from chicken pancreas and which is
Table 4.
VITAMIN

B12

CONTENT

OP PLASMA, B12

RBC s

AND BLOOD

S/hO. VU.
Plas,no (1 ml.)

Subject

RBOe (1 ml.)

Blood (1 ml.)

Hematocrit

reading

Normal

620

170

425

44

Normal 2 Normal 3 Peru. anemia 1 Peru. anemia 2 Total gastreetomy

600 320 32 23 50

140 185 115 95 185

410 260 45 35 75

42 47 15 15 20

102

SOBOTKA

Clinical Chemistry

invariably accompanied by folic acid, thus necessitating large corrections for this added extrinsic folio acid. But the most striking advantage of the thermophiic bacillus is the complete absence of any contaminants viable at 55#{176}. greatly simplifies operations, This as it eliminates the need for the various precautionary measures which I have mentioned previously in the description of the general technic. PGA in the blood during pregnancy was found high in contrast to vitamin B12. An important role ascribed to this vitamin in pregnancy is its protective action toward progesterone in overcoming the inhibitory effects exerted upon this hormone by PGA-antimetabolites such as aminopterin. Toward the time of parturition, progesterone and folic acid fall simultaneously, and the fetus draws relentlessly on the maternal stores of PGA as well as of vitamin B12, both vitamins being needed for fetal growth, more specifically for nucleic acid synthesis. The differential thus established between maternal and infantile blood is even higher for PGA than for vitamin B12. These studies indicate the importance of vitamin supplementation during pregnancy (Table 5). The difficulties encountered in any attempt to disentangle the etiology of multiple sclerosis are well known. The vaccilating, unpredictable course of this disease makes it doubtful whether one should expect chemical stigmata to be perpetually evident or only during or before attacks and episodes of exacerbation. This question may be raised for any deviation from the normal in the chemical findings, whether one deals with proteins, lipoids, enzymes, or vitamins. The great variations in acuteness and degree of severity from patient to patient impel us to keep our sights and our hopes low in this battle. We have, nonetheless, studied the PQA and B12 level in the cerebrospinal fluid (CSF) of multiple sclerosis patients (9).
Table 5. B12
AND

PGA
B

CONTENT

OF

MATEun1

AND

INFANT

BLOOD

(g./,ni.) InjatU

PGA Maternal

(ng.Jmi.) Infant

Quartile

MasrnaZ

let 2nd Median 3rd 4th No. patients

25-120 120-190 190 190-270 270 104

35-250 250-390 390 390-600 600 70

1-4 4-7.5 7.5 7.5-22.5 22.5 67

1-9 9-40 44) 40-90 90 38

Vol. 4, No. 2

MICROBIOLOGIC

ASSAY OF VITAMINS

103

PGA in the CSF of normal controls ranges from zero to 30 mLg./ml., about the same as in blood. Values of B12 in CSF are substantially lower-0 to 30 g./ml.-as against 300 to 1000 ipg./ml. in blood. In multiple sclerosis the PGA values in the spinal fluid run as follows: Quartile First Second Third Fourth 11111g. 10-60 60-200 200-500 500-4000

This means that more than three-fourths of the cases (to be exact 34 out of 42) show elevations, many of them to enormous amounts. In the case of vitamin B12, 14 out of 41 cases showed elevated values in CSF, 9 of these to more than twice the upper limit of the normal range. Likewise, about one-third of the B12 values in serum were elevated. These findings point to a relationship with the neurologic effects of PGA therapy in pernicious anemia; first it seems to mobilize the available B12-stores with ephemeral clinical benefits but eventually it produces combined lateral sclerosis. Possibly the deviation in the level of these vitamins and of the pertaining enzymes influences the methyl transfer between lecithins and cephalins and thus plays some part in the demyelinating process. The tests may serve for the differential diagnosis of neurologic conditions brought on by disturbance of B12 metabolism, as in multiple sclerosis and combined lateral sclerosis and, on the other hand, other disorders in which B12 is not involved. Peculiar deviations in the B12 situation have been claimed in diabetes and diabetic retinopathy (10). Using a greater number of cases than Becker et at., we have shown that these claims are untenable (11). In the course of this study we made however the following observation. If a normal subject is injected intramuscularly with a load dose of 60 g. of vitamin B12, he excretes in the urine 10-40 g. (15-65 per cent) of the load dose within the next 8 hours (12, 13). In a series of 40 cases of cirrhosis, hepatitis, and other parenchymatous liver disorders, we find the excretion reduced below 10 g. with very few exceptions. The diseased liver is known to have lost its binding power and its function as a storehouse for vitamin B12, but this function has been taken over by the circulating blood to prevent depletion of B12 in the organism. The vitamin B12 load test has been compared with a number of other liver function tests and was found to be well corre-

104

SOBOTKA

Clinical Chemistry

lated with them, but more sensitive at the onset and during recovery of the patient. In another project, we utilized the vitamin B12 load test in patients with thyroid disorders. In thyrotoxic subjects we found the B12 levels in the blood low and the 8-hour urinary excretion at an abnormally low level. By contrast, myxedematous patients, starting out from a normal level, display a higher rise than normals after 4 and 8 hours and a higher excretion (Table 6). These findings suggest that B12 turnover is accelerated in hyperthyroidism and slowed down in hypothyroidism (14).
FAT.SOLUBLE VITAMINS

A few words must be devoted to vitamins outside the B group. Aside from ascorbic acid, these vitamins comprise the fat-soluble factors A, D, E, and K. They are involved in the metabolism of the more highly specialized tissue systems of vertebrates and other higher phyla with features such as a bony skeleton, a coagulable circulating fluid, a complex, strongly differentiated integument, and the like. Thus, their mode of action may be expected to be more diversifled and more complex. Microbiologists and biochemists alike evince a kind of abstemious preference for aqueous media; in the chemists eyes the growth of microorganisms in a medium is a heterogenous reaction. Thus, the introduction of such lipids as fat-soluble vitamins would further complicate medium and mechanism-an unpopular measure, especially in the absence of efficient and at the same time nontoxic emulsifying agents. Only in recent years have microbiologists turned to the study of organisms among the ciliate and flagellate protozoa that are phagotrophic and, thus, capable of devouring fat droplets. Paramoeba thrives on lipid media (15) and Paramecium is stimulated by sterols
Table 6.
BLOOD LsvziS AND ExcanTloN

or B12
tlhbjacic

IN LoAD

PasT

(50 ng.) IN Tnmom

NormaZ

ParIENrs

Thyrotozic

controls

Myaud.,natotzs subjects

No.cases

10
pLO.

7
VITAMIN B12 IN

4 1
ML.

Before
After

injection
4 hours

320
605

575
1210

600
1675

After 8 hours Urinary excretion in 8 hours (% of load dose)

410 8

825 17

1315 37

Vol. 4. No. 2 Table

Vitamin

MICROBIOLOGIC 7. Microorganisms
Microorganism

ASSAY OF VITAMINS
and Sensitivity of Bioassay Sensitivity
per ml.

105

PoSe acid
Cyanocobalamin Cyanocobalamin

Thiamine
Biotin

B6 group
Thioctic acid Pantothenic acid Pantothenic acid

B. coaguians (thermophil) Euglena graciii8 Ochroinonas cnalhamensia Ochromonas malkamenRis Ochro,nonas maiharnensia Tetrcjiyinena pijriformis Tetrahyrnena pijriformss Tetrahymena pyrifornus Lactobaciiius arabinosu.

10 1 1 10 1
300 30

mig. jeng. ng. m,ag.

qog.

i0 1012 1012 10.8

1012

10

3 mpeg. 10 mng.

10b0+ 108 +

108

(16, 17) ; Lab yrint hula has absolute sterol requirements (18). Microorganisms with unorthodox tastes of this type may eventually be developed for the assay of fat-soluble vitamins. At present their determination must be achieved by conventional biologic or chemical methods.
SUMMARY

The examples which have been given above in the cyanocobalamin and folic acid field illustrate the usefulness of the microbiologic assay of vitamins in investigate work. The application of these methods to routine determinations will follow, once the indications for their usefulness are established. Table 7 summarizes the vitamins for which we have developed assay methods, the microorganisms used, and their sensitivity. The methods described are much simpler than they may appear to be at first sight, and they require no expensive or special equipment. They can become in due course routine procedures and will contribute to one of the lesser known chapters of clinical chemistry.
REFERENCES
Sobotka, H., J. Mt. Sinai Ho8p. 24, 1231 (1957). Sonno, S., and Sobotka, H., Arch. Biochem. 14, 93 (1947). 3. Baker, H., Sobotka, H., and Hutner, S. H., J. Gen. Miorobioi. 4. Ford, J. E., Br-it. J. Nutrition 7, 209 (1953). 5. Baker, H., Pasher, I., Sobotka, H., Hutner, S. H., Aaronson, 1. 2.

9, 485 (1953).
S., and Ziffer, Soc. Exp. Btoi. H. Nolure 94,

1043 (1957). 6. Baker, H., Erdberg, R., Pasher, 513 (1957).

7. 8. 9. Baker, Baker, Sobotka, H., Ziffer, H., Hutner,

180,

I.,

and Sobotka,

H., Proc.

Med.

H., Pasber, I., and Sobotka, H., Brit. Med. J., in press. S. H., and Sobotka, H., Proc. Soc. Exp. Biol. Med. 89, N., and Baker, H., Internati. Neurol. Congress,

210 (1955).
(1957).

H., Christoff,

Brussels

106

SOBOTKA

Clinical Chemistry

10. Becker, B., I*ng, C. A., and Chow, B. F., 1. Chn, Nutrition 1, 417 (1953). 11. Bookman, J. J., Joelson, B. H., Toll, W. 0., Baker, H., and Dolger, H., Ass. J. Cisn. Nutrition 5, 26 (1957). 12. Baker, H., Pastier, I., Dolger, H., and Sobotka, H., Chn. Chess. 2, 328 (1956). 13. Baker, H., Brill, 0., Packer, I., Sobotka, K., Olin. Chess. 4, 27 (1958). 14. Ziffer, H., Gutman, A., Packer, I., Sobotka, H., and Baker, H., Proc. Soc. Ezp. Bsoi. Med. 96, 229 (1957). 15. Storm, 3., and Hutner, 5. H., Ann. N. 7. Acad. Sci. 56, 901 (1953). 16. Johnson, W. H., and Miller, C. A., J. Protozooi. 3, 221 (1956). 17. Miller, C. A., and van Wagtendonk, W. 3., J. Gen. Microbioi. 15, 280 (1956). 18. Vishniac, H. S., and watson, S. w., 1. Gen. Microbial. 8, 248 (1953).