Determination of uric acid in human serum by an enzymatic method using N-methyl-N-(4-aminophenyl)-3-methoxyaniline reagent

ArticleinJournal of the Serbian Chemical Society 68(8-9) · January 2003with244 Reads
Impact Factor: 0.87 · DOI: 10.2298/JSC0309691J · Source: DOAJ
Abstract

In this work a new enzymatic method for the determination of uric acid in human serum has been developed. The method is based on the oxidative coupling reaction between the N-methyl-N-(4-aminophenyl)-3-methoxyaniline (NCP) reagent and the hydrogen – donor reagent, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), in the system involving three enzymes: uricase, peroxidase and ascorbate oxidase. Using this method uric acid could be determined in concentrations up to 1.428 mmol/L, with a relative standard deviation of up to 1.8 %. The effect of the medium pH and the NCP concentration on the linearity of the chromogen absorbance versus the uric acid concentration curve was investigated. The influence of the uricase activity on the maximum rate of uric acid oxidation was also examined. The use of the NCP reagent demonstrated a more precise and more sensitive determination of the uric acid compared to the determination with 4-aminoantipyrine (4-AA) as the coupling regent. The sensitivity of the method determined from the calibration curve was 0.71 absorbance units per mmol/L of uric acid; the limit of detection was LOD = 0.0035 mmol/L and the limit of quantification was LOQ = 0.015 mmol/L of uric acid.

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Available from: Predrag Djurdjevic, Jul 30, 2014
J.Serb.Chem.Soc. 68(8–9)691–698(2003) UDC 612.461.25+543-4:543.645:574.152
JSCS 3078 Original scientific paper
Determination of uric acid in human serum by an enzymatic
method using N-methyl-N-(4-aminophenyl)-3-methoxyaniline
reagent
MILENA JELIKI]-STANKOV
1, *
,
#
, PREDRAG DJURDJEVI]
2
and DEJAN STANKOV
3
1
Faculty of Pharmacy , Department of Analytical Chemistry, Vojvode Stepe 450, P. O. Box. 146, 11224
Belgrade,
2
Faculty of Science, P. O. Box 60, 34000 Kragujevac and
3
Hospital “Dr Dragi{a Mi{ovi}”,
Biochemistry Department, 11000 Belgrade, Serbia and Montenegr o
(Received 8 March 2003)
Abstract: In this work a new enzymatic method for the determination of uric acid in human
serum has been developed. The method is based on the oxidative coupling reaction between
the N-methyl-N-(4-aminophenyl)-3-methoxyaniline (NCP) reagent and the hydrogen – do-
nor reagent, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), in the system
involving three enzymes: uricase, peroxidase and ascorbate oxidase. Using this method uric
acid could be determined in concentrations up to 1.428 mmol/L, with a relative standard de-
viation of up to 1.8 %. The effect of the medium pH and the NCP concentration on the lin-
earity of the chromogen absorbance versus the uric acid concentration curve was investi-
gated. The influence of the uricase activity on the maximum rate of uric acid oxidation was
also examined. The use of the NCP reagent demonstrated a more precise and more sensitive
determination of the uric acid compared to the determination with 4-aminoantipyrine
(4-AA) as the coupling regent. The sensitivity of the method determined from the calibration
curve was 0.71 absorbance units per mmol/L of uric acid; the limit of detection was LOD =
0.0035 mmol/L and the limit of quantification was LOQ = 0.015 mmol/L of uric acid.
Keywor ds: uric acid, human serum, enzymatic determination, NCP reagent.
INTRODUCTION
Uric acid is a metabolic product of exogenous (brought in with food) or endogenous
purine bases. Since uric acid is found in human serum in relatively low concentrations (ref
-
erence range is 0.21 to 0.42 mmol/L in men and 0.16 to 0.36 mmol/L in women) it is nec
-
essary to use specific and sensitive methods for its determination. The existing methods
could be conveniently devided into two groups: reductive and enzymatic. The reductive
methods are non-specific and involve the oxidation of uric acid with phosphotungstate re
-
agent to allantoin with resultant blue coloring of tungstate solution. The enxymatic meth
-
ods are specific. They involve the catalytic oxidation of uric acid with the enzyme uricase
691
* Author for correspondence.
# Serbian Chemical Socitey active member.
Page 1
to allantoin with the formation of hydrogen peroxide.
1
The peroxide, the concentration of
which is directly proportional to the concentration of uric acid, could then be determined
by a number of methods.
2–10
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.
11
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
2
O
2
(oxidative coupling).
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.
EXPERIMENTAL
Apparatus
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
Page 2
Analytical pr ocedur e
A serum sample of 25 mL volume was added into 1.0 mL of the reagent solution (2.4). The absorbance
of the solution was measured at 750 nm after five minutes incubation at 37.0 ºC using the reagent solution as
the blank.
RESULTS AND DISCUSSION
The procedure of uric acid determination is based on the reactions:
Uric acid
uricase
¾®¾¾¾
allantoine + H
2
O
2
(1)
The concentration of chromogen (green colored dye stable fifteen minutes) which is
formed by oxidative coupling of NCP and TOOS reagents is directly proportional to the
concentration of the uric acid.
The spectra of the NCP reagent (Fig. 1, curve 1), TOOS reagent (Fig. 1, curve 2) and
the chromogen compound (Fig. 1, curve 3) were taken at pH 7.40. The absorption maxi
-
mum of the chromogen is at 750 nm. Thus, its absorption maximum is batochromically
shifted compared to its components.
The adventage of the application of NCP reagent lays in the fact that the formed
chromogen (Fig. 2, curve 2) has a batochromic shift of the absorption maximum at 750 nm
compared to the method that uses 4-AA instead of NCP, as a coupling reagent, when the
formed chromogen has an absorption maximum at 556 nm (Fig. 2, curve 1).
The effect of pH on the chromogen spectra was investigated in the pH interval from
6.5 to 7.5. The reason for choosing the pH interval was in the fact that all the enzymes used
show optimal activity under such conditions. The obtained spectra (Fig. 3) show that varia
-
tion of pH has no significant effect on the intensity and position of the adsorption maxi
-
mum of the chromogen. Thus, the working pH was set at 7.40.
URIC ACID DETERMINATION 693
Page 3
The effect of the NCP concentration on the absorbance of the chromogen (Fig. 4) was
investigated using NCP reagent in the concentration range from 0.025 to 0.075 mmol/L at
694 JELIKI]–STANKOV, DJURDJEVI] and STANKOV
Fig. 1. Absorption spectra of NCP (curve 1, 0.05 mmol/L; pH 7.40), TOOS (curve 2, 0.05 mmol/L; pH
7.40) and the chromogen (curve 3, NCP – 0.05 mmol/L, TOOS – 0.05 mmol/L; pH 7.40).
Fig. 2. Absorption spectra of the chromogen with 4-AA (curve 1, uric acid – 1.428 mmol/L, pH 7.40) and
the chromogen with NCP (curve 2, uric acid – 1.428 mmol/L, pH 7.40).
Page 4
constant concentration of TOOS, 0.2 mmol/L. The maximal absorbance of formed
chromogen, at 750 nm, was 3 units which is enough for the determination of uric acid up to
a concentration of 1.428 mmol/L.
During the investigations of the chromogen formation (Fig. 1, curve 3), the effect of
pH (Fig. 3) and NCP concentration on the absorbance of chromogene (Fig. 4), a hydrogen
peroxide concentration of 5.0 mmol/L was used.
Kinetic studies
The activity of uricase was followed kinetically as the rate of uric acid oxidation to
peroxide and allantoine (Eq. (1)). It was assumed that the reaction was finished when dA/dt
= 0. At a uricase activity of 200 U/L, complete oxidation of the uric acid (1.428 mmol/L),
was achieved in 5 min. Thus, the uricase was used at this level of activity in the determina
-
tion of uric acid (Fig. 5).
For the construction of a calibration curve for the determination of uric acid, standard
solutions of uric acid in the concentration range from 0 to 1.428 mmol/L were used. The
linear equation was y = 0.709 x + 0.00061, n = 5 with a correlation coefficient r = 0.9998,
where y denotes the absorbance and x is the concentration of the uric acid in mmol/L.
On the basis of the obtained calibration curve, uric acid was determined in samples
from 50 patients. Analysis was performed as described in the section Analytical Procedure.
In order to check the obtained results, uric acid was determined in the same samples of
human serum by the standard enzymatic method based on the use 4-AAas the coupling re
-
agent instead of NCP. The obtained results indicated a high level of correlation (a = 0.006,
b = 0.998 and r = 0.9993).
URIC ACID DETERMINATION 695
Fig. 3. Absorption spectra of the chromogen (0.1 M TOOS and 0.025 M NCP) in the pH region 6.50 – 7.50.
Page 5
Accuracy of the method was examined by the recovery test. To hyman pool serum
known amounts of uric acid were added and the concentration of the uric acid was deter-
mined from the calibration curve. The recovery was calculated from the expression:
696 JELIKI]–STANKOV, DJURDJEVI] and STANKOV
Fig. 5. The influence of the uricase activity on the rate of uric acid oxidation (uricase activity: curve1–100
U/L, curve 2 – 200 U/L, curve 3 – 300 U/L, curve 4 – 400 U/L: uricase activity shown as dA/dt vs. time
(curve 1’ – 4’).
Fig. 4. The effect of the NCP concentration on the formation of the chromogen (curve 1, NCP – 0.025
mmol/L, curve 2, NCP – 0.05 mmol/L, curve 3, NCP – 0.075 mmol/L; TOOS – 0.2 mmol/L, pH 7.40).
Page 6
% recovery = (FS US)/ADD ´ 100
where FS is concentration of the uric acid in the fortified serum sample. US is the concen
-
tration of uric acid in the unfortified (blank) serum and ADD is concentration of uric acid in
the added aliquot. The obtained results are given in Table I.
TABLE I. Application of the proposed method to the analysis of uric acid
Added/(mmol/L) Found
a
/(mmol/L)
Recovery ± RSD/%
0.286 0.282
98.6 ± 1.80
0.571 0.568
99.5 ± 1.52
0.857 0.863
100.7 ± 1.29
1.142 1.152
100.9 ± 1.35
1.428 1.417
99.2 ± 1.40
a
Mean value of ten determinations
The sensitivity of the method as determined from the calibration curve was 0.71
absorbance units per mmol/Lof uric acid. This is significantly higher than that obtained us-
ing 4-AA (0.35 absorbance units per mmol/L of uric acid). The detection limit (LOD) was
0.0035 mmol/L and the quantification limit (LOQ) for uric acid was 0.015 mmol/L.
CONCLUSIONS
Owing to the significantly higher absorbance of the chromogen and the increased sen-
sitivity of the method as a result of the batochromic shift of the absorption maximum of the
chromogen, the interference from the serum components on the determination of uric acid
was diminished. This enabled precise and accurate determination of uric acid in human se-
rum. The NCP reagent possesses better analytical properties than the so far used 4-AA re-
agent.
Acknowledgement: The authors are grateful to the Ministry of Science, Technology and Development
for financial support. (project 1941/2002).
IZVOD
ODRE\IVAWE MOKRA]NE KISELINE U SERUMU ENZIMSKOM METODOM
PRIMENOM N-METIL-N-(4-AMINOFENIL)-3-METOKSIANILIN REAGENSA
MILENA JELIKI]-STANKOV
1
, PREDRAG \UR\EVI]
2
I DEJAN STANKOV
3
1
Farmaceutski fakultet, Odsek za analiti~ku hemiju, Vojvode Stepe 450, p. pr. 146, 11224 Beograd,
2
Prirodno-matemati~ki fakultet, p. pr. 60, 34000 Kragujevac i
3
Bolnica Dr Dragi{a Mi{ovi}, Odsek za
biohemiju, Univerzitet u Beogradu, 11000 Beograd
U radu je predlo`ena nova enzimska metoda za odre|ivawe mokra}ne kiseline u huma
-
nom serumu. Metoda je zasnovana na kuplovawu N-metil-N-(4-aminofenil)-3-metoksianilin
(NCP), reagensa sa N-etil-N-(2-hidroksi-3-sulfopropil)-3-metilanilinom (TOOS), hidro
-
gendonor reagensom, u enzimskom sistemu tri enzima: urikaza, peroksidaza i askorbat
oksidaza. Mokra}na kiselina se ovom metodom mo`e odre|ivati u koncentracijama do 1.428
URIC ACID DETERMINATION 697
Page 7
mmol/L sa relativnom standardnom devijacijom do 1.8 %. Ispitan je uticaj pH medijuma i
koncentracije NCP reagensa na linearnost zavisnosti apsorbancije hromogena od koncen
-
tracije mokra}ne kiseline. Tako|e je ispitan uticaj aktivnosti urikaze na maksimalnu
brzinu oksidacije mokra}ne kiseline. Primena NCP reagensa omogu}ava preciznije i oset
-
qivije odre|ivawe mokra}ne kiseline u pore|ewu sa odre|ivawem uz 4-aminoantipirin
(4-AA) kao reagensa za kukplovawe. Osetqivost metode, odre|ena iz kalibracione krive,
iznosila je 0.71 apsorpcionu jedinicu po mmol/L mokra}ne kiseline; granica detekcije je
iznosila 0.0035 mmol/L a granica kvantifikacije 0.015 mmol/L.
(Primqeno 8. marta 2003)
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