to allantoin with the formation of hydrogen peroxide.
The peroxide, the concentration of
which is directly proportional to the concentration of uric acid, could then be determined
by a number of methods.
Owing to their specificity, enzymatic methods have found widespread use. The reagents
mixture used in this method contains the enzymes: uricase, peroxidase, hydrogen – donor re
agent and coupling reagent. As hydrogen – donor reagent various derivatives of phenol or
aniline have been used.
Formerly, as the coupling reagent, 4-AA was used. In the present
work the use of the NCP reagent instead of 4-AA is proposed. This reagent forms a colored
compound (chromogen) with TOOS reagent in the presence of H
The concentration of the chromogen is directly proportional to the concentration of uric acid.
The chromogen shows an absorption maximum at 750 nm. The chromogen formed with
4-AA possesses an absorption maximum at 556 nm. This batochromic shift of 194 nm is es
pecially convenient in the analysis of hemolyzed or lipemic serum where undesirable prod
ucts are colored with an absorption near 500 nm and, thus, may strongly interfere with the
uric acid determination. The sensitivity of the uric acid determination with the NCPreagent is
considerably higher than that with 4-AA, as well.
The absorbance measurements were made with a double beam UV-VIS spectrophotometer Cintra
model 40 (GBC, Australia). The cells were thermostated with a temperature precision to ± 0.01 ºC. The
absorbance – concentration curves were obtained by using a single beam UV spectrophotometer Ultrospec
model 2000 (LKB-Pharmacia, UK). The pH measurements were made with a Corning model 250 pH-meter
equipped with an Orion sure – flow combined electrode (USA).
Reagents and solutions
Bidistilled water and analytical grade reagents were used for the preparation of the solutions. Uric acid
standard, uricase, peroxidase (POD) and ascorbate oxidase were the products of SERVA (FRG). The NCP and
TOOS reagents were the products of Dojindo Lab (Japan). The phosphate buffer and 4-aminoantipyrine (4-AA)
were the products of Merck (FRG). Sodium tetraborate, EDTA and Triton X-100 detergent were the products of
Serva (FRG); hydrogen peroxide (30 %) was from Zorka – [abac (Serbia nad Montenegro). The control serum
with a declared amount of uric acid was obtained from Boehringer Mannheim – Precinorm (Austria).
Reagent for the determination of the uricase activity
Prior to the uric acid determination, the optimal uricase activity needed for the determination had to be es
tablished. The composition of the solution used for uricase activity determination was: phosphate buffer (pH
7.40), 0.1 mol/L; sodium tetraborate, 20 mmol/L; EDTA, 1 mmol/L; Triton X-1000, 0.01 %. The activity of
uricase were 100, 200, 300 and 400 U/L. The primary standard solution of uric acid had a concentration of 1.428
mmol/L and was used as the analyt. An amount of 25 mL of the sample was added into 1.0 mLof the reagent and
the absorbance was measured at 750 nm using the reagent solution as the blank at a temperature of 37 ºC.
Reagent for the determination of uric acid
The composition of the reagent solution used for uric acid determination in serum was: phosphate
buffer (pH 7.40), 0.1 mol/L; NCP reagent, 0.075 mmol/L; TOOS reagent, 0.2 mmol/L; sodium tetraborate, 20
mmol/L; EDTA, 1 mmol/L; peroxidase (POD), 1000 U/L; ascorbate oxidase, 1000 U/L; uricase, 200 U/Land
Triton X-100, 0.01 %. Ascorbate oxidase was added in order to prevent the reaction between hydrogen perox
ide and vitamin C present in serum.
692 JELIKI]–STANKOV, DJURDJEVI] and STANKOV