analyzer. (Union. Carbide. Corp., Tarrytown,. N. Y. 10591). All pipetting was performed automatically. Reagents: Reagents were obtained from Union Carbide.
CLIN. CHEM. 20/9, 123 1-1233 (1974)
Automated Enzymatic Micromethod for Determination of Uric Acid in Serum and Urine with a Centrifugal Analyzer Michael A. Pesce, Selma H. Bodourian, and John F. Nicholson
Uric acid is determined
in serum and urine by an enzy-
matic methodon a centrifugalanalyzer. The sample (8 tl) is mixed with the enzyme uricase in a sodium borate buffer. The resulting decrease in absorbance at 292 nm is linearly proportional to the concentration of uric acid from 1 to 75 mg of uric acid per deciliter. Use of a centrifugal analyzer eliminates measurement of a separate
serum blank. Recovery of uric acid added to serum and urine averaged 100% (range: 97-110%). Within-run precision (CV) on a serum pool for which the mean value was 5.96 mg/dl was 2.52%; day-to-day precision, measured on the same serum pool over a seven-day period, was 2.81% (mean: 6.05 mg/dI). Results obtained for serum with the uricase method on a centrif ugal analyzer and with the uricase method on the Du Pont aca yielded a correlation coefficient of 0.99.
Recently,
a centrifugal
analyzer
with
a 292-nm
filter
has
become available. With this analyzer, an initial absorbance reading may be obtained 3.0 s after mixing the samples and uric acid reagent. At this time uricase has not had a measurable effect, and thus the absorbance reading obtained is the same as if only sample and reagent were present. After 5.0 mm the reaction is complete, and the change in absorbance
is proportional
to the
amount
of uric
acid
present
in
the sample. With this method, blanking of the sample and reaction with uricase are accomplished in the same cuvette, thereby decreasing the amount of sample and time required for the test. We have evaluated this automated micromethod for the determination of uric acid in serum and urine and compared it with another automated method (the Du Pont aca).
Materials and Methods AdditIonal Keyphrases: #{149} uricase Uric acid concentration
Du
Pont
aca
can be specifically
serum and urine by measuring the decrease at 292 nm after incubation with the enzyme converts uric acid to allantoin (1-5). The
lowed
to go to completion
and
the
change
CentrifiChem
#{149}
determined
in
in absorbance uricase, which
reaction is alin absorbance
after the reagents and samples are mixed together is proportional to the concentration of uric acid. Most methods in which differential spectrophotometry is used for measuring uric acid are performed manually and require that a
separate sample blank be measured. These procedures usually require a large amount of sample and are time consuming.
Materials Instrumentation: We used a CentrifiChem centrifugal analyzer (Union Carbide Corp., Tarrytown, N. Y. 10591). All pipetting was performed automatically. Reagents: Reagents were obtained from Union Carbide Corp. and were reconstituted
0.10 U of uricase Stock
uric
acid
Babies Hospital,
The Children’s
Medical and Surgical Center of New York at Columbia-Presbyterian Medical Center; and The Department of Pediatrics, College of Physicians and Surgeons of Columbia University, 3975 Broadway, New York, N. Y. 10032. Received May 10, 1974; accepted June 13, 1974.
to the manufacturof the working rebuffer,
pH
9.3,
and
per milliliter. standard,
100
mg/dl.
uric acid (Eastman
Kodak Co., Rochester,
60 mg of lithium
carbonate
Dissolve
100
mg of
N. Y. 14650) and
in 15 ml of warm
distilled
water. Cool and dilute to 100 ml with distilled water. Working uric acid standards. Dilute the stock standard to 2, 5, and 10 mg/dl
Division of Clinical Chemistry,
according
The final concentration mol/liter sodium borate
er’s instructions. agent was: 0.2
with distilled
water.
Procedure Pipet an 8-pi volume of serum, dards
and
50 zl of diluent
and 350 zl of working
(water)
reagent
into
urine, into
or working the
sample
the reagent
stancavities
cavities
CLINICALCHEMISTRY,Vol. 20, No. 9, 1974
of
1231
the transfer disc. Use a temperature Record
the
The reference position contains water. of 30 #{176}C and a wavelength of 292 nm.
absorbance
3.0
s after
again 5 mm later. The change and 5 mm is used to determine either by using simultaneously from the following formula: Uric
acid,
mg/dl
where
LxA = change
=
the
rotor
Table 1. Decrease in Absorbance for Uric Acid, as Measured with the CentrifiChem, after Uricase Treatment
and
in absorbance between 3.0 s the uric acid concentration, determined standards or
x mol
E
