Ann. din. Biochem.

11 (1974) 103

TECHNICAL BULLETIN No. 31

Determination of Uric Acid in Blood and in Urine

Prepared for the Scientific and Technical Committee of the Association of the Clinical Biochemists

R. W. E. WATTS
Division of Inherited Metabolic Diseases
M.R.C. Clinical Research Centre, Harrow, Middlesex

The methods which are currently used to assay uric acid. The difficulties inherent in these methods
uric acid in blood and urine depend upon either are four-fold: the tendency of uric acid to co-
chemical or enzymatic oxidation to allantoin. In the
precipitate with the plasma proteins; the possible
former, a chromogen is reduced concurrently with formation of turbidity in the final coloured solutions;
the oxidation to yield a chromophore which may be non-linearity in relation to the colour yield and
measured spectrophotometrically. In the latter, amount of uric acid present over the range of uric
uricase (urate: oxygen oxidoreductase EC 1.7.3.3.)acid concentrations commonly encountered; and
interference by other reducing agents in plasma or
is used to catalyse the reaction, and the concentration
of uric acid may be determined by direct spectro- urine. The potential interfering reducing agents
photometry (following the change in absorbence at include ascorbic acid, free thiols (for example,
292 nm during the reaction) or by measuring the thioneine, cysteine, glutathione), methylated purines
amount of oxygen consumed or the amount of (caffeine, theobromine, theophylline), gentisic acid
hydrogen peroxide produced. Alternatively, a (a salicylate metabolite), homogentisic acid, and
chemical oxidising agent may be employed, and very high concentrations of glucose.
colorimetric measurements made before and after Frabot (1904) observed that an alkaline tungstate
treatment with uricase. solution became intensely blue when uric acid was
added to it, and that this reaction could be used to

0 7 detect uric acid at a concentration of 1 part in

10000. The reduction of tungstate also forms the
H..... N basis of Folin-Benedict copper methods for blood

O~N
7 I:): [=0
N
I 0. + H 20 sugar determination in which the reaction is effected
by Cu(I) under conditions which minimise the effect
I I of uric acid. Cu(I) is omitted in the uric acid method,
H H and the conditions modified to give maximum
sensitivity of the reaction towards uric acid. Folin
Uric Acid and his colleagues (Folin and Macallum, 1912;
Folin and Denis, 1912) precipitated silver urate
from serum after acid deproteinisation, and deter-
mined the uric acid colorimetrically with phospho-
tungstate using sodium carbonate to achieve the
necessary measure of alkalinity. Benedict and
Hitchcock (1915) replaced sodium carbonate by
sodium cyanide in order to increase the sensitivity
of the method, and Benedict (1922a, b) applied the
phosphotungstate reaction directly to a protein-free
plasma filtrate. Peters and Van Slyke (1932) reviewed
these early methodologies and described a procedure
Allantoin for plasma based on that of Benedict (1922a, b),
using tungstic acid as the protein precipitant, with
arsenophosphotungstic acid (an aqueous solution
COLORIMETRIC METHODS containing sodium tungstate, arsenic pentoxide,
Manual colorimetric methods syrupy phosphoric acid, and concentrated hydro-
chloric acid) and sodium cyanide for colour develop-
The most widely used colorimetric methods ment. Brown (1945) modified this method by
depend upon the reduction of sodium tungstate by omitting the arsenic pentoxide; he also recommended
103
104
the inclusion of urea as a reagent to prevent turbidity
in the final solution, as had Folin (1930). Natelson
(1953) published a useful detailed account of
Brown's method and reviewed some of the earlier,
less wel1 known, precipitation and titrimetric
methods. Caraway (1955) reintroduced the use of
sodium carbonate in place of sodium cyanide, and
this method was further modified by Eichhorn et al.
(1961). Kern and Stransky (1937) had used a mixture
of sodium silicate and glycerol to overcome turbidity
in the final coloured solution, but the success of this
depended on the quality of the sodium silicate, and
Clarke (1963) recommended that it should be
abandoned in favour of urea. Archibald's (1957)
method, in which phosphotungstic acid is used both
for protein precipitation and for colour develop-
ment, compares wel1 (Alper and Seitchik, 1957) with
the enzymatic method of Praetorius (1949) and is
widely used particularly in the U.S.A. A more
extensive comparison of an enzymatic method-that
of Blauch and Koch (1939)-was carried out by
Kanabrocki et al. (1957) with regard to the techniques
of Folin (1922), Newton (1937), Brown (1945), and
Archibald (1957). It was found that the results of
Archibald's technique agreed most closely with those
of the enzymatic method.
Thus although uric acid has been measured colori-
metrical1y for more than 50 years by methods
based on the manual procedures developed by Folin
and his col1eagues, many modifications designed to
overcome the inherent difficulties have been, and
continue to be, published. For example, lung and
Parekh (1970) treated plasma or serum with tri-
sodium phosphate, used phosphotungstic acid as a
protein precipitant, and added a 'carbonate-urea-
triethanolamine' reagent to develop the colour. They
claimed that the trisodium phosphate destroyed
serum chromogens, that turbidity was eliminated by
avoiding the use of tungstate and sulphate, and that
triethanolamine improved colour stability. Pileggi et
al. (1972) described a one-tube method involving
only a single addition of phosphotungstic acid.
Patel (1968) incorporated EDTA and hydrazine
sulphate into the reagent in order to improve both
the specificity and the relationship between the
colour yield and the amount of uric acid present.
Wel1s (1968) reinvestigated the method, and recom-
mended a different procedure designed to achieve
similar objectives. He favoured a modification
(Eichhorn et aI., 1961) of the Folin and Wu (1919)
protein precipitation method and he adopted the
Folin-Denis (1912) phosphotungstic acid reagent
rather than the more toxic arsenophosphotungstic
acid. He also omitted cyanide, since this can itself
reduce phosphotungstic acid both directly (Folin,
1934) and indirectly via the reduction of disulphides
to thiols. Interference by thiols is particularly
important in the analysis of urine where cystine is
available for reduction to cysteine; this can be
avoided by adding phenylmercuric acetate (0.2 gil)
to the urine. Wel1s (1968) also investigated the time
course of colour development and the effect of
several other factors on the efficiency of the method:
the concentration of phosphotungstic acid, the time
interval between adding the alkali and the phospho-
tungstic acid, the temperature, and the pH and nature
of the alkalinising reagent. Detailed recommenda-
tions were made with regard to each of these points,
including the use of sodium glycinate buffer (pH
10.6) in place of sodium carbonate or sodium
cyanide, and the omission of urea from the reagent.
The evident need for constant revision of the basic
colorimetric method with reports of multiple modifi-
cations, sometimes small or even contradictory,
suggests that these means of measuring uric acid
are fundamentally unsound when applied to the
routine diagnostic analysis of blood or urine.
Automated colorimetric methods
Automated colorimetric methods of determining
uric acid depend upon either the reduction of
phosphotungstate or arsenotungstate, or upon the
reduction of a metal complex (for example, cupric
phenanthroline, neocuproine, or bathocuproine).
Both types of analysis can be performed with the
AutoAnalyzer Mark I or II and with the Technicon
SMA 12/60. The method used with the Du Pont aca
Automatic Clinical Analyzer is based upon the
reduction of Cu(II)-2,2'-bicinchoninate. Automated
methods involving metal complex reduction have
also been used with the Searle Analmatic and Aga
Autochemist analysers, and the manufacturers of
such instruments are usual1y wil1ing to provide
supporting literature. Methods which depend upon
the reduction of a metal complex, however, appear
to be as subject to chemical interference as the
phosphotungstate methods (Lum and Gambino,
1973). The difficulties associated with manual
methods which utilise the reduction of phospho-
tungstate are still potentially present when such
methods are automated and applied to a plasma
dialysate or to urine. Although dialysis removes a
constant proportion of the total uric acid, which
would otherwise be available for colour development,
the ability to control the reaction conditions closely,
and at the same time analyse large numbers of
samples relatively cheaply, accounts for the popu-
larity of automated colorimetric methods. The
results for plasma agree closely with those of manual
enzymatic spectrophotometric methods. However,
discrepancies are found when urine is analysed,

presumably due to the presence of larger concen-
trations of interfering reducing agents. Detailed
accounts of automated colorimetric methods based
on the standard manual procedures have been
published by Allan (1964), Crowley (1964), Musser
(1966), Nishi (1967), Crowley and Alton (1968),
Lavery (1968), and Wells (1970).
ENZYMATIC METHODS
Manual Enzymatic methods
The catalytic activity of uricase is highly specific
for the oxidation of uric acid. As outlined above,
quantitative assays may be based on direct spectro-
photometry, on measurement of the uptake of oxygen
or of the production of hydrogen peroxide, or
indirectly by utilising a chemical oxidising agent.
Differential ultra-violet spectrophotometry. Uric
acid has a characteristic ultra-violet absorption
spectrum (maximum at 292 nm and molar absorption
coefficient, 12500 em -2 rnol r! at pH 9.4; Liddle et al.
1959), and its determination by measuring E292
before and after treatment with purified uricase was
introduced by Kalckar (1947). Further studies were
carried out by Praetorius (1949), Praetorius and
Poulsen (1953), and Feichtmeir and Wrenn (1955).
Liddle et al. (1959) described a thorough validation
of the method as applied to blood and urine, with
modifications which avoided the need to plot
graphically the fall in absorbence; a biological fluid
blank was used to correct for changes in E292
during the reaction due to substances other than
uric acid. This method offers the advantage of
avoiding protein precipitation, it has a high degree
of sensitivity and specificity, and does not necessitate
constant standardisation with solutions of uric acid.
It does, however, require a good quality ultra-
violet spectrophotometer with silica cuvettes, and
highly purified uricase which is relatively expensive.
The studies of Mahler (1970) indicate that uricase
prepared from Bacillus fastidiosus (strain NCIB
10372), is suitable for use as an analytical reagent.
Similarly, that derived from Candida utilis is recom-
mended by Gochman and Schmitz (1971). The large
scale production of the enzyme from microbiological
sources of this kind could yield a cheaper product
than animal uricase, thereby removing one of the
main obstacles to the general adoption of these
methods.
Liddle et al. (1959) encountered no problems due
to the presence of uricase inhibitors in biological
fluids even when the powerful inhibitor 6-chlorouric
acid, a metabolite of 6-chloropurine, was present in
urine. If inhibition should be apparent, Liddle et al.
recommended adding more uricase. Simmonds
105
(1967) introduced a simple chromatographic step in
order to separate uric acid from drug metabolites
which might act as substrates or inhibitors or uricase.
The enzymatic oxidation of 6-thiouric acid, which
occurs at a rate of about 1 % of that of uric acid,
might introduce a significant error if uric acid were
determined in its presence. Inhibition of the enzyme
by 6-thiouric acid or 8-thiouric acid might also
present potential sources of error (Simmonds, 1967).
Measurement of oxygen consumption. The rate of
oxygen uptake during the enzymatic oxidation of
uric acid can be measured with a polarographic
oxygen electrode. Von Bunemann and Kruse-Jarres
(1973) included this method in a systematic compari-
son of six months of measuring plasma uric acid:
macro- and micro-phosphotungstic acid methods
with commercially available test kits; a standard
phosphotungstic acid method after deproteinisation
with uranyl acetate; enzymatic methods with
differential ultra-violet spectrophotometry; measure-
ment of oxygen consumption; and measurement of
hydrogen peroxide formation. The oxygen uptake
method, however, was inferior to most of the other
methods.
Measurement of hydrogen peroxide formed. Some
of these methods combine the specificity of differen-
tial ultra-violet spectrophometry with the use of
simple colorimeters or spectrophotometers which
operate in the visible range. Others use a fluorimeter
as the final detection system to achieve higher
sensitivity.
The underlying principle is that the reduction of
hydrogen peroxide by either catalase (H202:H202
oxidoreductase EC 1.11.1.6) or peroxidase (donor:
H202 oxidoreductase, EC 1.11.1.7) is coupled to
another oxidative reaction which yields a coloured
product.
Thus Kageyama (1971) coupled catalase to the
uricase reaction, and measured, colorimetrically, the
dihydrolutidine formed by condensation with acetyl-
acetone:
Uricase
Uric acid + 2H20 + 02 ------+
allantoin + C02 + H202
Catalase
H202 +
CH30H-----+
HCHO + 2H20
HCHO + acetylacetone + NH3 ------+
3,5-diacetyl-l, 4-dihydrolutidine + 3H20
Serum was used without deproteinisation and there
was no significant interference by haemolysis,
bilirubin (up to 10 mg/IOO ml), ascorbic acid, or
glucose. The author's data showed excellent agree-
ment with the differential ultra-violet spectrophoto-
metric method, and better reproducibility and
106
stability. With both serum and urine, there was
good recovery of added uric acid, and linear calibra-
tion curves up to 20 mg/1oo mJ. The Boehringer
Corporation (London) Ltd. now market a test
combination ('Urica-quant') based on Kageyama's
(1971) method.
Several authors have coupled the peroxidase-
catalysed reduction of hydrogen peroxide to the
uricase reaction, with o-dianisidine as the chromogen.
Lorentz and Berndt (1967) deproteinised serum
samples with hot acetic acid, and carried out the
uricase reaction; the addition of o-dianisidine and
peroxidase gave a colour which was stable for as
long as 30 min. Sulphuric acid was added to convert
the faint brown quinone diimine into an intense
purple dye and the final absorbence was read with a
filter photometer. Domagk and Schlicke (1968)
deproteinised serum with ethanol, and carried out
the uricase and chromogenperoxidase reactions, but
read the absorbence at 436 nm due to the quinone
diimine, which reached a maximum after 30 to 120 s,
The authors claimed that there was linearity of
colour yield up to 20 mg of uric acid/loo ml.
Kabasakalian et al. (1973) measured the hydrogen
peroxide formed in the uricase reaction by fol1owing
the hydrogen peroxide-peroxidase-catalysed oxida-
tive coupling of 2:4:6-tribromophenol and amino-
antipyrine. The chromophore was extracted into
n-butyl acetate, and measured colorimetrically.
Comparison with the results of differential ultra-
violet spectrophotometry gave recoveries of
92-101 %, and calibration curves were linear up to
uric acid concentrations of 20 mg/loo ml. The
authors reported that ascorbic acid, bilirubin,
cysteine, glucose, and salicylate did not interfere,
and claimed that the accuracy, precision, and
sensitivity compared favourably with enzymatic
methods. No appraisal of the value of this method
in the clinical context has yet been published.
Godicke and Godicke (1973) coupled the uricase
reaction to the hydrogen peroxide-peroxidase-
catalysed oxidation of 3,5-diacetyl-1, 4-dihydroluti-
dine to the corresponding dimethylpyridine. Since
the diacetyl1utidine was strongly fluorescent, in
contrast to the oxidised product, it was possible to
develop a fluorimetric assay using a filter instrument
with a mercury lamp. The results correlated wel1
with those obtained by differential ultra-violet spectro-
photometry, and the sensitivity of the method was
such that 25 picomoles of uric acid could be measured
in as little as 10 pJ of serum.
Chemical oxidation 'difference' methods. Correc-
tion may be made for non-urate chromogens in
phosphotungstate reaction methods by destroying
aJl the uric acid in a duplicate sample with uricase,
so as to derive a blank value which is subtracted from
the result obtained on an untreated sample. A
relatively crude preparation of uricase is suitable for
this purpose.
Manual methods which utilise this principle have
been described by several authors including Blauch
and Koch (1939), Bulger and Johns (1941), Schaffer
(1944), Buchanan et al. (1945), and Caraway and
Marable (1966). Although they do overcome the
problem of non-urate chromogens, which can be
especially troublesome with urine samples, these
methods double the number of determinations which
have to be made and double the potential sources
of analytical error. They make no contribution
towards solving the problems of co-precipitation with
plasma proteins, development of turbidity in the final
coloured solution, and the need for ensuring that the
colour yield obeys the Beer-Lambert law over a
wide range of concentration. The relative specificity
of these methods is probably outweighed by the
increase in work load for laboratories which rely on
manual methods. They have however been adapted
for use with AutoAnalyzers where the number of
samples presented for colour development is a less
serious limitation. As manual methods, they are
less accurate than differential ultra-violet spectro-
photometry, but have the advantage of using simpler
instrumentation.
Automated enzymatic methods
Differential ultra-violet spectrophotometry. Steele
(1970) described the application of an enzymatic
spectrophotometric method to the AutoAnalyzer.
The dialysed specimen was divided into two streams,
uricase being added to one but not to the other, and
the net differences in absorbence at 292 nm were
monitored. Changes not due to uric acid were thus
cancel1ed out, this being an advantage over the
earlier method of Barron and Bouley (1965), in
which the specimen was analysed a second time after
incubation with uricase. Steele pointed out that
dialysis reduced the non-specific absorbence at
292 nm, and that approximately 30 % of the urate
actual1y dialyses in this assay. Specimens could be
processed at a rate of 30 per hour, with a total
sample volume of 0.6 ml. The method appeared to
give excel1ent results with both urine and plasma,
and agreed very wel1 with the manual method of
Liddle et al. (1959), the merits of which seem to be
preserved.
Kinetic assay. Lum and Gambino (1973) described
an automated kinetic assay for uric acid using
uricase of high specific activity prepared from
Bacillus fastidiosus. They compared this method with
a manual differential spectrophotometric method
(Praetorius and Poulsen, 1953) and with automated

methods based on the reduction of Cu(I1)-2,2'-
bicinchoninate or phosphotungstate. There was
excellent agreement between the two enzymatic
methods, but the kinetic technique had a smaller
coefficient of variation than the manual method. It
is unfortunate that the authors did not select the
method of Liddle et al. (1959) as their reference
method, and that they did not study urine as well as
serum. However, it is clear that the automated
kinetic assay has a high order of both precision and
accuracy and could also provide for the analysis of
a large number of samples.
Measurement of hydrogen peroxide formed.
Gochman and Schmitz (1971) devised an Auto-
Analyzer procedure in which hydrogen peroxide and
peroxidase effect the oxidative coupling of 3-methyl-
2-benzathiazolinone hydrazone and N, N-dimethyl-
aniline to produce a stable blue indamine dye. The
good reproducibility of the method was demon-
strated by the findings for normal men and women.
The results quoted for 30 men and 30 women were:
men, mean 5.32 (S.D. 0.73), range 3.4-6.4 mg/loo ml;
women, mean 3.75 (S.D. 0.80), range 2.2-5.0 mg/loo
mI. The correlation of the results obtained by this
method with those of manual differential ultra-violet
spectrophotometry (Liddle et al., 1959) was excellent.
The method was also shown to be resistant to many
reducing substances which can interfere with reac-
tions depending upon the reduction of phosphotung-
stic acid.
Chemical oxidation 'difference' methods. Morgen-
stern et al. (1966) described an automated method
which depends upon the reduction of phosphotung-
stic acid before or after treatment of duplicate
specimens with uricase. Crowley and Alton (1968)
compared a method of this type, and an automated
colorimetric technique which made no allowance for
urate chromogen, with a differential spectrophoto-
metric method. They found that the results of the
two automated methods agreed very closely with one
another, and in view of the small differences felt
justified in recommending omission of the uricase
step for most purposes. It is worth emphasising,
however, that these authors considered only the
analysis of serum, in contradistinction to urine.
MISCELLANEOUS METHODS
Coulometric Titration
Troy and Purdy (1970) described a coulometric
method for the determination or uric acid in serum
and urine. The total reducing substances were
measured with coulometrically-generated iodine
before and after treatment with uricase, It was
found that the results agreed satisfactorily with those
107
obtained by measuring the change in absorbence at
292 nm on treatment with uricase. However, this
is a manual method requiring special apparatus
which is unlikely to be available in most clinical
chemistry laboratories.
Fluorescence assay using scopoletin (7-hydroxy-6-
methoxycoumarin)
Bloch and Lata (1970) described an ingenious
fluorimetric method for measuring picomole quanti-
ties of uric acid in serum, urine, or liver samples. The
fluorescence of scopoletin (excitation maximum,
348 nm; emission maximum 465 nm) is abolished by
oxidation with hydrogen peroxide in the presence
of horse radish peroxidase. Uric acid may also be
oxidised in this way, but when both substrates are
present in the same system the oxidation of scopoletin
does not begin until all the uric acid has been
destroyed. The time lag before the fluorescence of
scopoletin decreases is a measure of the amount of
uric acid present. The method would not appear to be
suitable for the situation where large numbers of
assays are needed, and where the sensitivity of
automated colorimetric or enzymatic methods is
satisfactory.
Chromatographic determination of uric acid and
related purines
Orsulak et al. (1968) described a system for the
separation of uric acid, hypoxanthine, and xanthine
in 200 iLl samples of deproteinised plasma on
ECTEOLA cellulose. The purines were eluted with
sodium tetraborate buffer and determined by direct
spectrophotometry. This method is only likely to
be of value in circumstances where it is necessary to
determine all three purines simultaneously. It may
also be noted that satisfactory specific enzymatic
spectrophotometric methods are available for deter-
mining hypoxanthine and xanthine in biological
fluids (Chalmers and Watts, 1968; 1969).
SURVEY OF SOME INTER-LABORATORY QUALITY
CONTROL DATA
Whitehead et al. (1973) summarised the findings
of the U.K. National Quality Control Scheme in
which pooled serum samples were analysed at
monthly intervals for particular constituents over a
period of two-and-a-half years. The data for uric
acid are reproduced in table I. The results show, as
might have been predicted from the foregoing
discussion, that the AutoAnalyzer techniques gave
gave more reproducible results than manual colori-
108
Table I. U.K. National Quality Control Scheme: results of serum uric acid determination by different types of method
and in different laboratories (Whitehead et al., 1973).
AutoAnalyzer
No. of analyses 112
Mean (mgjlOO ml) 5.56
S.D. 0.35
"
V.c. % 6.33
metric methods, with the precision of the manual
uricase methods being intermediate. The mean results
of each of the three methods were within one
standard deviation of each other.
The data do not provide any information on
the merits of individual methods, because of the
way in which they have been grouped together under
broad headings. Thus, the uricase methods presum-
ably included both 'difference' and 'differential' ultra-
violet spectrophotometric methods; AutoAnalyzer
techniques could include those which depend on the
reduction of metal complexes, as well as enzymatic
procedures involving the measurement of hydrogen
peroxide. In practice, most AutoAnalyzer techniques
currently used in Britain are likely to be based on
colorimetric reactions involving the reduction of
phosphotungstate or arsenophosphotungstate.
The Wellcome Group Quality Control Programme
distributes freeze-dried serum samples and collects
data at fortnightly intervals from laboratories using
a wide range of analytical methods. However, most
participants use either an automated phosphotung-
state or arsenophosphotungstate technique or one
of the corresponding manual methods. The results
suggest that automation improves precision, but the
numbers available for each method are too small
for any detailed comparison to be made. The cupric
phenanthroline, neocuproine, and bathocuproine
AutoAnalyzer methods which are grouped together,
appear to have good precision in respect of data for
the cycle April-September 1973. The relevant
results are summarised in table 2, which demon-
strates the small number of laboratories which use
methods other than those based on the reduction of
phosphotungstate or arsenophosphotungstate, and
therefore shows the limited value of these data as a
basis for appraising modern methods.
The findings of these quality control surveys
relate to pooled or reconstituted serum samples and
are not necessarily relevant to the analysis of urine,
which may contain different types and concentra-
tions of potentially interfering substances.
Manual colorimetric Manual uricase
123 26
5.45 5.26
0.55 0.44
10.20 8.44
RECOMMENDATIONS
In reviewing the relative merits of the many
published procedures for determining uric acid, the
following factors must be considered: the need for
a reference as wen as a routine method; the capital
and running costs; the toxicity of the reagents; the
ability to produce results quickly; suitability for the
analysis of urine as well as of serum; and the
accuracy (or specificity) and precision.
Differential spectrophotometry offers an excellent
reference procedure. If the cost of purified uricase
and of ultra-violet spectrophotometers could be
reduced the technique would be excellent for routine
use also. Similar considerations apply to the auto-
mated kinetic assay using uricase described by Lum
and Gambino (1973). Methods which depend upon
measuring hydrogen peroxide, notably the manual
method of Kageyama (1971) and the automated
procedure of Gochman and Schmitz (1971), appear
to be as reliable as differential ultra-violet spectro-
photometry. These use colorimeters which operate in
the visible wave length range and relatively simple
standard equipment. Reports of the full evaluation
of these methods for the analysis of urine and of
blood in a wide range of clinical contexts are
awaited.
Manual methods based upon the reduction of
phosphotungstate or arsenophosphotungstate are
not recommended either for routine use or as
reference methods. The automated variants of these
procedures have reasonable precision, but are
susceptible to interference by other reducing agents,
especially in urine samples. The metal-complex
reactions have a high degree of precision, but can
also be influenced by reducing substances.
An automated technique based on measuring
hydrogen peroxide, such as that described by
Gochman and Schmitz (1971) may well prove to be
the most generally useful. Such a method has the
attraction that it utilises the high specificity of
uricase and measures a reaction product directly.
 109

Table 2. Wellcome Group Quality Control Programme: data showing the average precision of different methods of
serum acid determination for the period April-September, 1973.

Method code and method, as defined by the Programme Number of Average

participating precision
laboratories (mg/l00 ml)

1000 AutoAnalyzer Mk I. 134 0.35

(Phosphotungstate or arsenophosphotungstate reduction)
1020 AutoAnalyzer Mk II. 19 0.53
(Phosphotungstate or arsenophosphotungstate reduction)
1059 Any discrete analytical system
(Phosphotungstate or arsenophosphotungstate reduction) 2 0.21
1110 AutoAnalyzer Mk I or II. 12 0.28
(Cupric phenthroline, neocuproine or bathocuproine)
1120 SMA 12/60
(Cuproic phenanthroline, neocuproine or bathocuproine)
1139 Other automated methods employing metal complex 2 0.40
reduction
1200 SMA 12/60 45 0.21
(Phosphotungstate or arsenophosphotungstate reduction)
1509 All automated uricase methods 2 0.39
1999 Any automated method not specified above I
2000 Any manual or partly automated phosphotungstate or 95 0.60
arsenophosphotungstate method
2100 Any manual or partly automated spectrophotometric uricase 12 0.91
method
2110 Any manual or partly automated uricase-phosphotungstate
method

The prospects for the development of microbiological
sources of uricase appear good, and this will reduce
the running costs of enzymatic methods.
Data from the National Quality Control Scheme and
the Wellcome Group Quality Control Programme were
kindly provided by Professor T. P. Whitehead and
Dr. B. A. L. Hum respectively.
REFERENCES
Allan, R. D. An automated modification of Eichhorn's
uric acid method. J. med. Lab. Tech. 21 (1964) 240.
Alper, c., Seitchik, J. Comparison of the Archibald-Kern
and Stransky colorimetric procedure and the Praeto-
rius enzymic procedure for the determination of uric
acid. Clin. Chern. 3 (1957) 95.
Archibald, R. M. Colorimetric measurement of uric acid.
Clin. Chern. 3 (1957) 102.
Barron, E. J., Bouley, A. Automation of the enzymatic
method for the determination of uric acid, in Technicon
Symposia 1965. ed. L. T. Skeggs jun. Automation in
Analytical Chemistry, New York, Mediad, 1966, p.
395.
Benedict, S. R. The determination of uric acid. J. bioi.
Chern. 54 (I922a) 233.
Benedict, S. R. The determination of uric acid in blood.
J. bioi. Chern. 51 (1922b) 187.
Benedict, S. R. Hitchcock, E. H. On the colorimetric
estimation of uric acid in urine. J. bioi. Chern. 20
(1915) 619.
Blauch, M. B., Koch, F. C. A new method for the
determination of uric acid in blood, with uricase.
J. bioi. Chern. 130 (1939) 443.
Bloch, P. L., Lata, G. F. Fluorescence assay for picomole
quantities of uric acid: a new enzyme-coupled approach.
Analyt, Biochem. 38 (1970) I.
Brown, H. The determination of uric acid in human
blood. J. bioi. Chern. 158 (1945) 601.
Buchanan, O. H., Christman, A. A., Block, W. D. The
metabolism of the methylated purines. II. Uric acid
excretion following the ingestion of caffeine, theophyl-
line and theobromine. J. bioi. Chern. 157 (1945) 189.
Bulger, H. A., Johns, H. E. The determination of plasma
uric acid. J. bioi. Chern. 140 (1941) 427.
Von Bunemann, c., Kruse-Jarres, J. D. Vergleichende
Untersuchungen reduktometrischer und enzymatischer
Harnsaurebestimmungen. Z. klin. Chern. klin. Biochem,
11 (1973) 403.
Caraway, W. T. Determination of uric acid in serum by
a carbonate method. Amer. J. elin. Path. 25 (1955) 840.
Caraway, W. T., Marable, H. Comparison of the
carbonate and uricase-carbonate methods for the
determination of uric acid in serum. Clin. Chern. 12
(1966) 18.
110
Chalmers, R. A., Watts, R. W. E. An enzymatic spectro-
photometric method for the determination of oxy-
purines (hypoxanthine plus xanthine) in urine and
blood plasma. Analyst 93 (1968) 354.
Chalmers, R. A., Watts, R. W. E. The separate deter-
mination of xanthine and hypoxanthine in urine and
blood plasma by an enzymatic differential spectro-
photometric method. Analyst 94 (1969) 226.
Clarke, A. D. A modified glycerol-silicate technique for
the determination of serum uric acid. J. med. Lab. Tech.
20 (1963) 107.
Crowley, L. V. Determination of uric acid. An automated
analysis based on a carbonate method. Clin. Chern. 10
(1964) 838.
Crowley, L. V., Alton, F. I. Automated analysis of uric
acid. Amer. J. din. Path. 49 (1968) 285.
Dornagk, G. F., Schlicke, H. H. A colorimetric method
using uricase and peroxidase for the determination of
uric acid. Analyt, Biochem. 22 (1968) 219.
Eichhorn, F., Zelmanowski, S., Lew, E., Rutenberg, A.,
Fanias, B. Improvement of the uric acid determination
by the carbonate method for serum and urine. J. din.
Path. 14 (1961) 450.
Feichtmeir, T. V., Wrenn, H. T. Direct determination of
uric acid with uricase. Arner. J. din. Path. 25 (1955)
833.
Folin, D. A system of blood analysis. Supplement IV.
A revision of the method for determining uric acid.
J. bioi. Chern. 54 (1922) 153.
Folin, D. An improved method for the determination of
uric acid in blood. J. bioi. Chern. 86 (1930) 179.
Folin, D. The preparation of sodium tungstate free from
molybdate together with a simplified process for the
preparation of a correct uric acid reagent (and some
comments). J. bioi. Chern. 106 (1934) 311.
Folin, D., Denis, W A new (colorimetric) method for
the determination of uric acid in blood J. bioi. Chern.
13 (1912) 469.
Folin, D., Macallum, A. B. On the blue color reaction of
phosphotungstic acid (?) with uric acid and other
substances. J. bioi. Chern. 11 (1912) 265.
Folin, D., Wu, H. A system of blood analysis. J. bioi.
Chern. 38 (1919) 81.
Frabot, C. Colour reaction for tungsten. J. chem. Soc.
86ii (1904) 844. Ann. Chem. Anal. 9 (1905) 371.
Gochman, N., Schmitz, J. M. Automated determination
of uric acid with use of a uricase-peroxide system. Clin.
Chem. 17 (1961) 1154.
Godicke, W., Gddicke, I. The fiuorimetric determination
of uric acid by use of the uricase-peroxidase system and
3,5-diacetyl-I,4-dihydrolutidine as secondary substrate.
Clin. chim. Acta. 44 (1973) 159.
Jung, D. H., Parekh, A. C. An improved reagent system
for the measurement of serum uric acid. Clin. Chern.
16 (1970) 247.
Kabasakalian, P., KaIIiney, S., Westcott, A. Determina-
tion of uric acid in serum, with use of uricase and a
tribromophenol-aminoantipyrine chromogen. Clin.
Chern. 19 (1973) 522.
Kageyama, N. A direct colorimetric determination of
uric acid in serum and urine with uricase-catalase
system. Clin. chim. Acta. 31 (1971) 421.
Kalckar, H. M., Shafran, M. Differential spectrophoto-
metry of purine compounds by means of specific
enzymes. I. Determination of hydroxypurine com-
pounds. J. bioI. Chern. 169 (1947) 429.
Kanabrocki, E. L., Greco, J., Wilkoff, L., Veach, R.
Comparison of plasma uric levels obtained with five
different methods. Clin. Chern. 3 (1957) 156.
Kern, A., Stransky, E. Beitrag zur kolorimetrischen
Bestimmung der Harnsaure. Biochem. Z. 290 (1937)
419.
Lavery, T. D. A simplified method for estimating plasma
uric acid. Clin. chim. Acta. 21 (1968) 415.
Liddle, L., Seegmiller, J. E., Laster, L. The enzymatic
spectrophotometric method for determination of uric
acid. J. Lab. din. Med. 54 (1959) 903.
Lorentz, K., Berndt, W. Enzymic determination of uric
acid by a colorimetric method. Analyt, Biochem. 18
(1967) 58.
Lum, G., Gambino, S. R. Comparison of four methods
for measuring uric acid: copper chelate, phospho-
tungstate, manual uricase and automated kinetic
uricase. Clin. Chern. 19 (1973) 1184.
Mahler, J. L. A new bacterial uricase for uric acid
determination. Analyt, Biochem. 38 (1970) 65.
Morgenstern, S., Flor, R. V., Kaufman, J. H., Klein,
B. The automated determination of serum uric acid.
Clin. Chern. 12 (1966) 748.
Musser, A. W., Ortigoza, C. Automated determination
of uric acid by the hydroxylamine method. Techn. BI/II.
Regist. med. Techn. 36 (1966) 21.
Natelson, S. Uric acid. Stan. Meth. din. Chern. 1 (1953)
123.
Newton, E. B. A chromogenic tungstate and its use in
the determination of the uric acid of blood. J. bioI.
Chern. 120 (1937) 315.
Nishi, H. H. Determination of uric acid: An adaptation
of the Archibald method on the AutoAnalyzer. Clin.
Chern. 13 (1967) 12.
Orsulak, P. J., Haab, W., Appleton, M. D. Quantitative
estimation of uric acid, xanthine and hypoxanthine in
plasma using thin-layer chromatography. Analyt,
Biochem, 23 (1968) 156.
Patel, C. P. Semimicro method for determination of
serum uric acid using EDTA-hydrazine. Clin. Chern.
14 (1968) 764.
Peters, J. P., Van Slyke, D. D. Quantitative clinical
chemistry. (1932) Volume II. Methods. Williams and
Wilkins Company, Baltimore.
Pileggi, V. J., Di Giorgio, J., Wybenga, D. R. A one-tube
serum uric acid method using phosphotungstic acid as
protein precipitant and colour reagent. Clin. chim.
Acta. 37 (1972) 141.
Praetorius, E. An enzymatic method for the determination
of uric acid by ultraviolet spectrophotometry. Scand.
J. din. Lab. Invest. 1 (1949) 222.
Praetorius, E., Poulsen, H. Enzymatic determination of
uric acid with detailed instructions. Scand. J. din. Lab.
Invest. 5 (1953) 273.
Schaffer, N. K. Determination of uric acid in urine with
crude uricase. J. bioi. Chern. 153 (1944) 163.

Simmonds, H. A. A method of estimation of uric acid
in urine and other body fluids. Clin. chim. Acta. IS
(1967) 375.
Steele, T. H., Mansdorfer, M. C. An automated enzymic
spectrophotometric method for the determinaton of
uric acid. Tech. Bull. Regis. med. Tech. 39 (1970)
270.
Troy, R. J., Purdy, W. C. The coulometric determination
of uric acid in serum and urine. Clin. chim. Acta. 27
(1970) 401.
111
Wells, M. G. Improved method for the determination of
uric acid in blood and urine. Clin. chim. Acta. 22
(1968) 379.
Wells, M. G. Evaluation of an automated method for
the estimation of uric acid concentrations in blood and
urine. J. med. Lab. Tech. 27 (1970) 485.
Whitehead, T. P., Browning, D. M., Gregory, A. A
comparative survey of the result of analysis of blood
serum in clinical chemistry laboratories in the United
Kingdom. J. din. Path. 26 (1973) 435.