CLIN. CHEM.

26/2, 227-231 (1980)

Use of 3, 5-Dichloro- 2-hydroxybenzenesulfonic Acid/4-Ami nophenazone

Chromogenic System in Direct Enzymic Assay of Uric Acid in Serum and
Urine
Piero Fossati,1 Lorenzo Prencipe,2 and Giovanni Berti
A new direct colorimetric procedure for uric acid assay in
serum or urine is described, utilizing a 3,5-dichloro-2hydroxybenzene
sulfonic acid/4-aminophenazone
chromogenic system in the presence of horseradish peroxidase
and uricase from Aspergillus
flavus. This chromogen
system has a high absorptivity, affording useful results with
sample/reagent
volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A
single working reagent is used; the reaction is complete
in less than 15 mm at room temperature.
The red dye
formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method
is linear for uric acid concentrations
up to 1500 jzmol/L.
Average analytical recovery of uric acid in human sera and
urine exceeded
99%; within-run and between-run
precision studies

showed

Cvs

of 1.2

and 2.2%,

tively. The new procedure correlated

case/catalase
and uricase/ultraviolet
method is suitable for automation.
AddItIonal Keyphrases:
Aspergillus

respec-

well with the unmethods.

The

enzymic methods

uricase from

flavus

A direct method,
which is at the same time reliable, convenient
(i.e., quick, simple, and not requiring
expensive
instruments),
and possibly suitable both as a manual or automated procedure
is not now available
for uric acid determination in serum or other biological
fluids, although
many
methods
have been published.
The chemical methods based
on reduction
of alkaline
phosphotungstate
by uric acid, in
addition
to requiring
deproteinization,
are subject to interference from reducing
compounds,
including
common
metabolites
and drugs or their breakdown
products
(1).
The enzymic methods
overcome
in part the interference
problem,
because
they are based on the use of uricase
(urate:oxygen
oxidoreductase,
EC 1.7.3.3), which is an enzyme
specific for uric acid, but still suffer some disadvantages.
The ultraviolet
procedure
at 293 nm (2) is often used as a
comparison
method, but the reading wavelength
and the small
absorbance
changes make it unsuitable
for routine work because of poor reproducibility
(3). In addition,
difficulties
are
encountered
in the application
of the procedure to automated
instruments.
In the Haeckel method (4), the hydrogen peroxide produced
by the uricase-catalyzed
oxidation
of uric acid is detected
through
the catalase-catalyzed
oxidation
of ethanol to acetaldehyde,
coupled to the oxidation
of the latter to acetate in
the presence
of NAD
(or NADP)
and aldehyde
dehydrogenase (aldehyde:NAD(P)
oxidoreductase,
EC 1.2.1.5), by
measuring
the change of absorbance
of NADH (or NADPH)

1 Ames Research
and Development
Laboratory, Miles Italians
S.p.A., Cavenago Brianza (Milan), Italy.
2Laboratorio di Biochimica, Ospedale Maggiore Ca Grands, Milan,

Italy.
Received

July

16, 1979; accepted

Oct. 5, 1979.

at 340 nm. An initial reading to be used as sample blank is

needed,
and this makes the method
quite laborious
as a
manual procedure.
Also, highly purified aldehyde
dehydrogenase is not always readily available.
In the Kageyama
method (5), the hydrogen peroxide in the
presence of catalase (hydrogen-peroxide:hydrogen-peroxide
oxidoreductase,
EC 1.11.1.6) converts
methanol
to formaldehyde; this last reacts with ammonium
ions and acetylacetone, giving the yellow dye 3,5-diacetyl-1,4-dihydrolutidine,
which is measured
at 410 nm. This method, although
highly
reliable, has certain disadvantages
in routine use: it requires
a sample blank for each specimen
and a long incubation
time.
In an attempt
to find an improved
detection
system to be
coupled to the enzymic oxidation
of uric acid, we took into
consideration
the chromogenic
system phenol/4-aminophenazone, which, in the presence of peroxidase
(hydrogen-peroxide oxidoreductase,
EC 1.11.1.7), is oxidized by hydrogen
peroxide to form a red quinoneimine
dye. This system has
become very popular in clinical chemistry after its application
by Trinder
(6) to the enzymic determination
of glucose.
However,
there are two big obstacles
to the use of this
chromogenic
system
in the determination
of serum uric
acid:
#{149}
low concentration
of the analyte in serum: the serum!
reagent volume ratio needed for adequate
sensitivity
is
so high as to invite interference
from other serum components and so make the method unreliable.
#{149}
incompatibility
between the working pH of horseradish
peroxidase
and that of uricase of animal origin (most
commonly
used): this makes it difficult
to oxidize uric
acid and develop the color in a single step.
The
method

following
in which

adjustments
provide
a simple,
reliable
the above difficulties
were overcome:

#{149}
by using a substituted
phenol, namely, 3,5-dichloro-2hydroxybenzenesulfonic
acid, which by oxidative
coupling with 4-aminophenazone
gives a quinoneimine
dye
with a molar absorptivity
about fourfold that of phenol
(7). This makes it possible to work with a serum/reagent
volume ratio low enough to eliminate
interferences
for
practical
purposes.
#{149}
by using a bacterial
uricase with a working pH range
materially lower than that of animal uricase, which works
without
major loss of activity at the pH of maximum
horseradish
peroxidase
activity.
Figure 1 shows the sequence of the reactions.
Interference
from bilirubin
up to about 170 iimol/L was
eliminated
by use of ferrocyanide,
which, through the peroxidase-hydrogen

peroxide

oxidation

to ferricyanide,

seems

to

increase the specificity

of the color reaction.
Interference
from ascorbic
acid, especially
important
in
urine, was eliminated
by using ascorbate
oxidase (L-ascorbate:oxygen
oxidoreductase,
EC 1.10.3.3).
In addition
to being highly reliable, this method
affords
several practical
advantages,
namely: (a) direct assay of uric
CLINICAL CHEMISTRY,

Vol. 26, No. 2, 1980

227

OH

H2N-C-N

II

OH

HO

2 H20

02

Uricase

-f CO2 + H2

OH
Uric

acid

Allantoin

OH

SO3H
SO3H

H2N

CH3
-{-2H202

0 CH3

CI

K4Fe (CN)6

Fig. 1. Reaction

monoimine

sequence

of uric acid enzymatic

assay

Apparatus
We used a Model 124 double-beam
Model
56 recorder
(Perkin-Elmer
06856).

spectrophotometer
Corp.,
Norwalk,

with
CT

Chemicals
Potassium
monohydrogen
phosphate,
potassium
dihydrogen phosphate,
4-aminophenazone,
Triton X-100, potassium ferrocyanide,
lithium carbonate,
bilirubin,
and ascorbic
acid were from E. Merck, F.R.G. (reagent-grade
materials).
Uric acid was SRM no. 913; National
Bureau of Standards,
Washington,
DC. 3,5-Dichloro-2-hydroxybenzene
sulfonic
acid (DHBS)
was from BDH Chemicals
Ltd., England.
Horseradish
peroxidase
(100 kU/g) was from Miles Laboratories Ltd., Research
Products
Division,
England.
Uricase
from
Aspergillus
flavus
(freeze-dried,
8 kU/g) was from
Sempa Chimie, France. Ascorbate
oxidase was obtained
from
green squash (8); the product had a specific activity of 200 U
per mg of protein,
according
to the spectrophotometric
described

by Oberbaker

Uricase
reagent:
Uricase, 6 kU/L, in doubly-distilled
water.
Stable for months
at room temperature,
barring
bacterial
contamination.
Working
reagent:
Mix the buffer/enzymes/4-aminophenazone, DHBS, and uricase reagents in the ratio 1.5/0.5/0.02;
store in a dark bottle. The mixture should be used during only
one working day. A moderate
increase in the reagents
pink
color does not influence
the results.
Stock uric acid standard
solution,
1 gIL (5.949 mmol/L):
Dissolve 100 mg of uric acid and 60 mg oflithium
carbonate
in 30 mL of doubly-distilled
water at 60 #{176}C
and dilute to 100
mL with doubly-distilled
water. Formaldehyde
must not be
added as a preservative
because it inhibits uricase from Aspergillus
flavus. This stock solution
is stable for about one
month if frozen and protected
from light.
Working
uric acid standard:
Dilute the stock uric acid
standard
solution
daily with doubly-distilled
water to make
a working uric acid standard
solution equivalent
to 60 mg/L
(357 tmol/L).

Procedure
(serum, plasma, or 10-fold diluted
standard
to 2 mL of working reagent. Mix,
and let stand
for 15 mm at room temperature.
Read the absorbance
at 520 nm vs the reagent blank.
Add

urine)

50 L

of sample

or working

(9).

and Standards3

Buffer/enzymes/4-aminophenazone
200

mmol/L,

pH

Phosphate

reagent:
7.0;

horseradish

peroxidase,

0.20

kU/L; ascorbate oxidase, 0.20 kU/L; 4-aminophenazone,

0.33
mmol/L; and potassium ferrocyanide,
40 tmol/L. This reagent
is stable for six weeks at 2-8 #{176}C.
DHBS
reagent:
3,5-Dichloro-2-hydroxybenzenesulfonic
acid, 8 mmol/L,
and Triton X-100, 5 g/L. Stable for months
at room

temperature

if protected

from

direct

light.

Table 1. Range of Absorbances Found for Sample

Blanks from 50 Abnormal Specimens at Different
Wavelengths
Wavelength,

500

Available as a kit from Miles Italiana,

228

Ames Division, Cavenago

Italy.
CLINICAL

CHEMISTRY,

Vol. 26, No. 2, 1980

nm

Absorbance

range

520

0.010-0. 125
0.007-0.095
0.006-0.065
0.004-0.045
0.002-0.025

525

0.002-0.025

530

0.002-0.025

505
510

515

Brianza,

CI

N-(4-antipyryl)-3-chloro-5sulfonate-p-benzoquinone-

Materials and Methods

buffer,

K3Fe (CN)6

CH3 + 4H20

4-am i nophen azone

acid in a small sample with a single reagent, (b) no need for

a serum blank, (c) short incubation
(15 mm at room temperature), (d) reading in the visible spectrum,
(e) linearity up to
1500 mol/L,
and (f) suitability
for automation.

Reagents

C6H5

3,5-dichloro-2hydroxybenzenesulfonic acid

method

Peroxidase
//___-S\:

Table 3. Results of Tests for Interference

Table 2. Precision Data

Within-run
Serum

Substances
SD

CV, %

361

3.93

20

571

6.96

1.08
1.20

20

1016

8.86

0.87

0.02
0.04

0.64
0.43

Mean

Normal

20

Abnormal
Abnormal

tmoI/L

Urine

mmol/L

Normal
Abnormal

20

3.90

20

9.39
jmol/L

Normal
Abnormal

20

313

20

492

6.96
5.41

2.22
1.10

Glucose
Reduced glutathione

330 tmol/L
360 mmol/L

Sodium fluoride
Sodium oxalate

30 mmol/L
40 mmol/L

Sodium citrate

4 000 mg/L
tetraacetate

L-DOpa

Comparison

with

an

Unicam

SP

1700

Spectrophotometer

(Philips).

Results
Analytical

Variables

Reading wavelength:
Maximum
absorbance
of the reaction
product was at 512 nm. We also ran 50 serum blanks, chosen
from among
the apparently
least-desirable
specimenssamples showing massive hemolysis,
high bilirubin
content,
and gross turbidity-and
we always read the minimum
absorbance
at 520 nm (Table 1). This wavelength
was accordingly adopted
in the final procedure,
instead of 512 nm; the
loss of sensitivity
was less than 3%.
Color development
rate: An overall control was conducted,
both with sera (three different concentrations
of uric acid: 245,
586, and 1478 tmol/L)
and with the working standard,
at 22,
30, and 37 #{176}C.
Color development
was complete within 15 mm
at each of these temperatures.
Color stability:
This was studied in four different
pools of
human sera (normal and abnormal
for uric acid), in two pools
of human urine (normal and abnormal),
and on the working
standard.
No important
changes of absorbance
were detected
during 30 mm of observation
after the recommended
incubation time.

7 mmol/L
6 mmol/L

3,4-Dihydroxyphenylacetic

acid

10 zmol/L

130 imol/L
3 mmol/L

Gentisicacid
Xanthine

Interferences:
The possibility
that interfering
substances
in the sample could produce false-negative
or false-positive
results was taken into account.
Serum samples
containing
normal concentrations
of uric acid and different
concentrations of presumed
interfering
substances
were analyzed,
as
were also uric acid solutions containing
known amounts of the
more commonly
used anticoagulants.
No interference
was
found up to the concentrations
shown in Table 3.
The effect of bilirubin
was assessed
by adding
known
amounts of the substance to portions of a serum pool of known
uric acid content. The test was repeated without ferrocyanide
in the working reagent, to assess the usefulness of this product.
The results were evaluated
in terms of uric acid recovery vs
the sample without added bilirubin
(Table 4). No significant
interference
was found from bilirubin
up to 170 gmol/L.
The effect of ascorbic acid was assessed by adding known
amounts
of this compound
to portions
of serum and pooled
specimens
of urine of known uric acid content; samples were
assayed promptly
after the addition of ascorbic acid. The results were evaluated
in terms of uric acid recovery
vs the
sample without
added ascorbic
acid. No interference
from
ascorbic acid was detected up to 570 fzmol/L in serum and 5.70

mmol/L

in urine.

We also tested ascorbic acid for stability in serum and urine,

adding it to serum and urine pools of known uric acid content

Table 4. Bilirubin Interference

Reliability
Linearity
was assessed

10 tmol/L
5 mmol/L

acid

Salicylicacid
Acetaminophen

Methods

Uricaselcatalase
method
(5): Reagents
were prepared
according
to Scheibe et al. (10); photometric
measurements
were taken with an LKB 7400 Calculating
Absorptiometer
(Bromna,
Sweden) and sample/reagent
dilutions
were made
with an automatic
pipette
(Micromedic
Systems,
Horsham,
PA 19044).
Uricase/ultraviolet
method:
Reagents
were prepared
according to Scheibe et al. (10); photometric
measurements
were

taken

10 mmol/L
1 500 /LmoI/L
20 4umol/L

Allopuninol
a-Methyldopa
Acetylsalicylic

up to:

13000 mg/L
1 800 zmol/L
110 mmol/L

Creatinine

Sodium heparinate
Disodium ethylenediamine

Between-run
Serum

No Interference

Hemoglobin

and sensitivity:
Linearity
by use of aqueous solutions

of reagent responses
of uric acid, 0 to 1500

izmol/L.
Net absorbance
change was plotted
vs uric acid
concentrations.
The response was linear up to 1500 izmol/L,
with a linear regression
equation
y = 0.000599x
- 0.00072,
correlation
coefficient
0.9999.
Analytical
recovery: The recovery of uric acid added to a
pool of human sera in concentrations
ranging from 90 to 1200
tmol/L
and to a pool of urine in concentrations
ranging from
1.2 to 12 mmol/L was 99.3 (CV 1.2)% and 99.0 (CV 1.0)%, respectively.
Precision:
Studies of within-run
precision
for serum and
urine and between-run
precision
for daily analysis of frozen
serum pools for 20 days gave the results shown in Table 2.

BilIrubin
In serum, mol/L

10

AnalytIcal recovery (%)

A

100.0

1.2

of urIc acid
B

100.0 1.2

23

100.0

97.0

37

99.7

94.0

62
112

99.4
99.0
99.0

83.6
74.0

147

65.7

174
210

98.9
107.0

60.1

305

118.0

38.5

55.5

A, by the present proposed method; B. with use of a working solution not

including ferrocyanide.

CLINICAL CHEMISTRY, Vol. 26, No. 2, 1980

229

900

method
and by the uricase/catalase
method.
The results,
elaborated
statistically
by a linear method (11), are shown in
Figures 2-4.

780

Discussion
We have incorporated
the specificity of uricase into a quick,
direct colorimetric
procedure
that is suitable
for routine
hospital laboratory
use. In the optimization
of the proposed
method, the reaction pH and the concentrations
or activities
of all components
were taken into account; optimal conditions
were taken to be those affording maximum absorbance
within
15 mm, greatest
possible color stability,
and maximum
lin-

540

oglx-297
=

120

C
a.
180

60

60

160

420

30
Urecase

catalase

melhod

660

780

uric acid concentration

540

pmol/iiterl

900

Fig. 2. Correlation between present method (y) and uricase/

catalase method (x)forthe determination of uricacid concen-

tration in serum

to give concentrations
of 285 tmol/L
and 2.85 mmol/L,
respectively,
and assaying the uric acid with a reagent not containing ascorbate
oxidase. Ascorbic acid no longer interfered
with the uric acid assay 90 mm after its addition to serum; in
contrast,
it still interfered
quite materially
in urine assays
made as late as 24 h after the addition.
Comparative
studies: The method was compared
with two
other enzymic methods
in a different
series of samples-all
of which, however, had been received with specific requests
for uric acid assay. The other methods were: uricase/catalase
assay (120 samples)
and uricase/ultraviolet
assay (120 samples).
As for urine, 50 hospital
specimens
were assayed by our

780

660

540

420-

180

601

60

180
urcase-

ultraviolet

300
n,ethod

420
(293

nnv(. uric

540

660

acid concentration

780

900

5n0I/lte,)

Fig. 3. Correlation between present method (y) and uricase/

ultraviolet (293 nm) method (x) for the determination of uric acid
concentration
230

in serum

CLINICAL CHEMISTRY,

Vol. 26, No. 2, 1980

earity.
The problems
encountered
in setting up the procedure
involved several aspects of the reaction,
namely, the simultaneous assaying of uricase and peroxidase,
test sensitivity,
and
competition
by some interfering
substances
with the chromogenic system, reflecting
the relative nonspecificity
of peroxidase.
The use of uricase from Aspergillus
flavus, which shows
maximum
activity at pH 8.5 and at 30 #{176}C
(12) as opposed to
pH 9.0 and 37 #{176}C
for uricase of animal origin, permitted
testing at a neutral pH, optimal for horseradish
peroxidase,
and at room temperature,
with a loss of uricase activity not
exceeding
20%.
The well-known
Trinder chromogenic
system (6) was used
in the present work in the modified version of Bahram,
utilizing DHBS (7). This substituted
phenol gives rise to a quinoneimine
dye with an absorptivity
about fourfold that obtained with phenol, affording sufficient
sensitivity
with a low
sample/reagent
volume ratio (0.025) and with little or no interference
from metabolites,
drugs, or anticoagulants.
Bilirubin is reported
(13) to interfere
with the quantitation
of hydrogen
peroxide
in systems including
peroxidase,
the
effect being roughly proportional
to its concentration
and also
dependent
on reagent composition.
Actually, the mechanism
by which bilirubin
acts is quite complex and not fully understood. According to Witte et al. (13), in addition to a positive
interference
from an overlap in the spectra for bilirubin
and
Trinders
chromophore,
there is also a chemical mechanism
that produces a negative interference.
They speculate that the
main cause of the chemical subtraction
of color could be the
consumption
of a possible electrophilic
4-aminophenazone
intermediate
by bilirubin.
In our method, the spectral interference
was substantially
overcome by reading at 520 nm, where absorbance
by bilirubin
and other pigments or serum turbidity
is least (Table 1). The
negative
chemical
interference
was eliminated
up to 170
fimol/L by use of potassium
ferrocyanide.
The following is a tentative
explanation
of how potassium
ferrocyanide
works: ferrocyanide
is oxidized to ferricyanide
by hydrogen peroxide in the presence of peroxidase
(14); this,
in turn, oxidizes the chromogen
(15). The latter
oxidation
perhaps involves a phenol (as opposed to 4-aminophenazone)
intermediate
(16), which does not react with bilirubin.
With
bilirubin
concentrations
exceeding
170 tmol/L,
however,
spectral interference
from bilirubin itself tends to give unduly
high values.
Ascorbic acid has a depressing
action on the Trinder
chromogen; this has been imputed both to the subtraction
of hydrogen peroxide and to actual chromophore
destruction
(17).
The substance,
however, can easily be oxidized by ascorbate
oxidase (18, 19) to dehydroascorbic
acid, which does not affect
the chromogen
system. Under our reaction conditions,
with
0.15 U of this enzyme per milliliter
of working reagent,
an
ascorbic acid concentration
of 570 mol/L
in serum had no
effect on uric acid determination.
Ascorbic acid, on the other
hand, is rapidly dissipated
from blood; after administration
of even large doses, reported
maximum
concentrations
in

References

6000

5400

4800
E
4200
Si

3600

0.940

7.14

Sy/x0=t7

3000

50

2400.
Si

a
0

1800.

0
0

1200.

600.

600

1200

1800

2400

3000

3600

4200

Uricase catalase method: uric acid concentration

4800

5400

6000

Ipmol/liter]

Fig. 4. -Correlation between present method (y) and uricase/

catalase method (x) for the determination of uric acid concentration in urine

serum do not exceed 190 omol/L

(20). In addition,
ascorbic
acid is quite unstable
in serum; 90 mm standing
at room
temperature
is enough to eliminate
interference
with the uric
acid assay by 285 tmol of ascorbic acid per liter. In urine,
considerably
higher amounts
of ascorbic acid can be found,
and the product is far more stable. Nevertheless,
by using the
amount
of ascorbate
oxidase stated above, urinary ascorbic
acid concentrations
as high as 5.70 mmol/L had no effect on
uric acid assay.
As for interference
from other substances,
including several
drugs in common use, we had no trouble with serum assays at
concentrations
vastly in excess of clinically realistic concentrations
in blood (Table 3), with the possible exceptions
of
a-methyldopa,
L-dopa, 3,4-dihydroxyphenylacetic
acid, and
gentisic acid, which gave borderline
interference
only at high
or very high doses.
The substances
just named, on the other hand, definitely
disturbed
the color reaction
in the urine-as
was to be expected, because the concentrations
in urine much exceed those
in serum. In all these cases the uric acid assays were on the low
side.
Clouding during the test was avoided by use of Triton X-100
surfactant,
which proved effective at the suggested
concentration.
Samples
with uncommonly
high bilirubin
or serum lipid
concentrations
may assay poorly by the proposed method. In
such exceptional
cases, a parallel blank run without uricase
might be useful.

1. Henry, R. J., Cannon, D. C., and Winkelman, J. W., Clinical

Chemistry: Principles
and Technics, 2nd ed., Harper & Row,
Hagerstown, MD, 1974, p 526.
2. Praetorius, E., and Poulsen, H., Enzymatic determination
of uric
acid, with detailed directions. Scand. J. Clin. Lab. Invest. 5, 273
(1953).
3. Bywaters, E. G., and Holloway, V. P., Measurement
of serum uric
acid in Great Britain in 1963. Ann. Rheum. Dis. 23, 236 (1964).
4 Haeckel, R., The use of aldehyde dehydrogenase
to determine
H202-producing
reactions: 1. The determination
of the uric acid
concentration.
J. Clin. Chem. Clin. Biochem. 14, 101 (1976).
5. Kageyama, N., A direct colorimetric determination
of uric acid in
serum and urine with uricase-catalase
system. Clin. Chim. Acta 31,
421 (1971).
6. Trinder, P., Determination
of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6, 24
(1969).
7. Barham, D., and Trinder, P., An improved colour reagent for the
determination
of glucose by the oxidase system. Analyst 97, 142
(1972).
8. Marchesini, A., Capelletti, P., Canonica, L., et al., Evidence about
catechol oxidase activity of the enzyme ascorbate oxidase extracted
from Cucurbita pepo medullosa. Biochim. Biophys. Acta 484, 290
(1977).
9. Oberbaker, M. F., and Vimes, H. N., Spectrophotometric
assay of
ascorbic acid oxidase. Nature 23, 1203 (1963).
10. Scheibe, P., Bernt, E., and Bergmeyer, H. U., Uric acid. In
Methods of Enzymatic
Analysis, Section D, 2nd EngI. ed., H. U.
Bergmeyer, Ed., Academic Press, New York, NY, 1974, p 1951.
11. Westgard, J. 0., and Hunt, M. R., Use and interpretation
of
common statistical tests in method-comparison
studies. Clin. Chem.
19,49 (1973).
12. Laboureur, P., and Langlois, C., Urate oxidase dAspergillus
flavus. Il-Metabolisme,
inhibition, specificit#{233}.
Bull. Soc. Chim. Biol.
50, 827 (1968).
13. Witte, D. L., Brown, L. F., and Feld, R. D., Effects of bilirubin on
detection of hydrogen peroxide by use of peroxidase. Gun. Chem. 24,
1778 (1978).

14. Critchlow, J. E., and Dunford, H. B., Studies on horseradish

peroxidase. X. The mechanism of the oxidation of p-cresol, ferrocyanide, and iodide by compound II. J. Biol. Chem. 247, 3714 (1972).
15. Emerson, E., The condensation of aminoantipyrine.
II. A new
color test for phenolic compounds. J. Org. Chem. 8, 417 (1943).
16. Jone, P. F., and Johnson, K. E., Estimation of phenols by the 4aminoantipyrine
method: Identification
of the colored reaction
products by proton magnetic resonance spectroscopy. Can. J. Chem.
51, 2860 (1973).
17. Fossati, P., and Prencipe, L., La reazione di Emerson-Trinder:
studio dei van cromogeni e analisi delle principali interference. Quad.
Sclavo Diagn. Clin. Lab. 14, 164 (1978).
18. Dawson, C. R., and Magee, R. J., Ascorbic acid oxidase. In
Methods in Enzymology
II,S. P. Colowick and N. 0. Kaplan, Eds.,
Academic Press, New York, NY, 1955, p 831.
19. Klose, S., Stoltz, M., Munz, E., and Portemhauser,
R., Determination of uric acid on continous-flow
(AutoAnalyzer II and SMA)
systems with a uricase/phenol/4-aminophenazone
color test. Clin.
Chem. 24, 250 (1978).
20. King, G. C., and Burns, J. J., In Second Conference on Vitamin
C. Ann. N.Y. Acad. Sci. 258 (1975).

CLINICAL

CHEMISTRY,

Vol. 26, No. 2, 1980

231