19
Clinica Ch~mica Actu, 64 (19’75) 19-25 0 Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CGA 7237
AN ALTERNATIVE METHOD FOR THE DETERMINATION OF URIC ACID IN SERUM JUI-CHANG
W. KUAN,
Department of Chemistry, Louisiana 70122 (U.S.A.) (Received
S.S. KUAN, University
and G.G. GUILBAULT of New Orleans, New Orleans,
April 4, 19’75)
Summa~ A novel fluorometric method for the determination of uric acid based on the coupled reactions of uric acid with uricase and peroxidase to form highly fluorescent 2,2’-dihydroxy-3,3’-dimethoxy-biphenyl-5,5’-diacetic acid is described. The calibration curve was constructed from a series of standard uric acid solutions vs. the corresponding relative fluorescence. It was linear up to 15 mgf dl (0.9 mmol/l). The serum or plasma samples must be deproteinated with (absolute) ethanol before assay and its uric acid content can be obtained from the calibration curve. This method is rather simple, having good precision and accuracy, High level ascorbic acid in the sample falsely elevates uric acid concentration. Comparison of the results obtained on the patient sera with a calorimetric phosphotungstate method and a standard enzymatic spectrophotometric method gave coefficients of correlation of 0.908 and 0.986, respectively.
Introduction The most commonly used methods for serum uric acid determination can be classified into two categories: (1) the non-enzymatic alkaline phosphotungstate method of Folin-Denis [l] and its modifications [2,3] ; (2) enzymatic methods based on the oxidation reaction of uric acid by uriease. The alkaline phosphotungstate methods which measure the blue color formation are restricted by interfering turbidity, lack of specificity or poor recovery. In the enzymatic method, the uric acid concentration is monitored either spectrophotometrically at around 292 nm before and after the uricase action [4-71, or calorimetrically by the chromogenic response of the coupling reactions of uric acid, uricase and a suitable chromogen in the presence of an oxidase [S--12]. The enzymatic methods are generally very specific with only minor or no interference and a relatively long incubation time.
20
With an approach similar to the calorimetric enzymatic, Inc~thotl. t tit> hydrogen peroxide evolved from the uric acid-uricase reaction c’an I)c used 1.0 oxidize a non-fluorescent compound to a highly fluorescent form by means of peroxidase, and the fluorescence produced can be related to uric* a(%1 (‘oncentration. Because of the inherent sensitivity of fluorometric methods and the specificity of enzymes, an attempt to develop a fluorometric clnzymatic, method for uric acid utilizing homovanillic acid as a fluorophor was thus initiated. Details of this new methodology are described in this paper.
Materials and methods Reagents Borate buffer, 0.02 M, pH 9.0. Dissolve 1.24 g of boric acid and 1.49 g of potassium chloride in about 800 ml of water. Adjust pH to 9.0 with 1 M NaOH. Make up to 1 liter with water. Homouanillic acid, 4 mg/ml. Dissolve 400 mg of homovanillic acid (98% pure, Aldrich Chem. Co., Inc., Milwaukee, Wise. 53233) in 100 ml of doubly distilled water. Keep refrigerated. This is stable for at least 2 months at 4°C. Uricase, 1 unit/ml. Dissolve 1 vial of 5 units uricase (Type IV, 1.5 unit/mg, purified from Candida utilis, Sigma Chem. Co., St. Louis, MO. 63178) in 5 ml of 0.02 M, pH 9.0 borate buffer. Keep refrigerated, where it remains stable for 2 weeks at 4°C. One unit of uricase will convert 1.0 pmole of uric acid to allantoin per min at pH 8.5 at 25°C. Stock peroxidase, 200 unit/ml. Dissolve 10 mg of peroxidase (Type II, from horse radish, 200 unit/mg solid, Sigma Chem. Co.) in 10 ml of doubly distilled water. Working peroxidase solutions are diluted from this stock solution. One unit of peroxidase will form 1 mg purpurogallin in 20 seconds at pH 6.0 at 20°C. Standard uric acid, 1 mg/ml. This can be obtained commercially from Harleco (Cat. No. 3054, Harleco, 60th and Woodland Ave., Philadelphia, Pa. 19143). Working standards are prepared from appropriate dilution of stock solution with doubly distilled water. Absolute ethanol, reagent grade, from Industrial Chemical Co., New York. Apparatus Fluorescence measurements were done with an Aminco Fluoro-microphotometer (American Instrument Co., Inc., Silver Spring, Md. 20910), with a Corning 7-60 primary filter and a Kodak Wratten 47B/2A combination secondary filter. A circulating water bath was employed to maintain the temperature at 25”C, and a Beckman linear recorder (Beckman Instruments, Inc., Fullerton, Calif. 92634) was coupled to the fluorometer. The fluorometer was standardized daily with polymer fluorescent standard No. 3 (p-Terphenyl, 3 X lO* M) from Perkin-Elmer (Perkin-Elmer Corp., Norwalk, Conn. 06856). An Adams Dynac Centrifuge (Cat. No. 0101, ClayAdams, Inc., Parsippany, N.J. 07054) was used in the deproteination process and a Vortex Genie Mixer (Scientific Industries, Inc., Springfield, Mass. 01103) was used for sample mixing.
21
10
Fig.
1. Calibration
curve
for uric acid
in borate
buffer
(a) PH 8.5,
(b)
PH 9.0.
Assay procedure
The serum sample, 0.2 ml, is deproteinated by the addition of 0.6 ml absolute ethanol, the mixture is allowed to stand at least 5 min and centrifuged at a high speed for about 10 min or until a clear supernatant fluid is obtained. Place, in order, into a test tube (1.1 cm X 10 cm) 0.16 ml of the deproteinated supernatant in 0.04 ml doubly distilled water, 2.5 ml, 0.02 M, pH 9.0 borate buffer, and 0.05 ml, 1 unit/ml uricase. Incubate for 10 min at 25°C. Then it must be treated with 0.2 ml, 4 mg/ml homovanillic acid, immediately followed by 0.05 ml, 32 units/ml peroxidase. Shake for 15 s (speed 3) with a Vortex Genie Mixer. After standing at 25°C for 10 min, the relative fluorescence at 425 nm (A,,,. = 315 nm) is recorded. Aqueous standard uric acid, 0.04 ml, in 0.16 ml absolute ethanol, is used and carried through the entire sequence as outlined above for plotting the calibration curve. The curve is linear up to 15 mg/dl uric acid (Fig. 1). The uric acid concentration of an unknown serum is obtained from the calibration curve. Results and discussion Order of reagent addition
In borate buffer at pH 9.0, uric acid reacts with uricase to produce hydrogen peroxide, allantoin . H202 and other degraded products [13-141. Homovanillic acid (HVA), in the presence of peroxidase, can be easily oxidized by hydrogen peroxide to form highly fluorescent compounds. However, it was found that uric acid could also be oxidized by peroxidase [ 151. When uricase and peroxidase were used in the same system at the same time, due to the competition between uric acid and homovanillic acid towards hydrogen perox-
ide formed, the rate of fluorescence and its intensity varied with thr ordrr 01’ addition of these reagents. This phenomenon has been rerbordetl I)y othc>~ investigators in a similar enzyme system with different chromogens for their photometric assays [9,10]. Peroxidase, if added to the system last, usually gave a higher reaction rate than if it was added sooner. In the sequence of uric, acid, buffer, uricase, homovanillic acid, and peroxidase without any incubation, the fluorescent change was proportional to uric acid concentration. IIowever, the rate of fluorescent change with regard to uric acid was small and the linear range was too narrow; hence, it did not always produce adequate accuracy for the kinetic determination of serum uric acid. With the same sequence, Ijut with uric acid incubated with uricase in borate buffer for a sufficient time to insure quantitative formation of H2 02, then followed by the addition of HVA and peroxidase, the relative fluorescence exhibited at a definite time was proportional to uric acid concentration. When mixing only HV:I and peroxidase together, a very slight fluorescent change is observed. This is probably due to the presence of some oxidant impurities in the peroxidase. Thus, a premixed solution of HVA and peroxidase is not recommended for use. Serum deproteination Serum must be deproteinated before assay since it contains catalase which could react with the H202 formed; almost all proteins act as H20, acceptors in the presence of peroxidase. The acid precipitants such as trichloroacetic and perchloric acid cannot be used because they would cause losses in uric acid by coprecipitation with the protein at low pH. Using ethanol or heat denaturation for deproteination were found to be quite satisfactory. Assay of dialysis patients ’ sera Blood samples were drawn from five kidney patients before, during and after they underwent renal dialysis and these samples were analyzed by the proposed method to correlate the uric acid level with renal function and the efficiency of dialysis. Serum uric acid levels are believed to elevate in renal malfunction. From the results of our assay (Table I) the uric acid values of some “before dialysis” sera were slightly above normal range, gradually decreased during dialysis and finally dropped within the normal range after the dialysis was accomplished. Judging from this observation, uric acid level can only be used as a supplementary indicator in diagnosis of renal impairment. TABLE
1
ASSAY
RESULTS
Patient
No.
OF
DIALYSIS
Uric
acid
Before
PATIENTS’SERA
concentration
dialysis
(mg/dl)* During
dialysis
After
dialysis
wok., ~)215 ~~~~~~~ -. ~.o~~~ ~~ 1
4.7
2
5.1
3.0
2.7
3
7.4
3.2
2.4
4
7.7
4.1
3.3
5
7.2
5.1
4.1
* Average
of 2 to 3 determinations.
23
Reproducibility and precision Two uric acid samples were analyzed with the same reagent concentrations for a period of six days. We found a withinday precision (CV) of 2.62%, mean of 3.98 mg/dl, standard deviation (SD) of 0.10; and CV = 0.49%, mean = 9.63 mg/dl, SD = 0.05. Day-today: CV = 1.18%, mean = 3.93, SD = 0.05;CV = 0.41%, mean = 9.65, SD = 0.04. The relative fluorescence reading contributed from any reaction mixture varies about +1.5 fluorescent unit which accounts for the relatively higher CV for low concentration uric acid samples. Effect of pH and incubation time The optimum for the catalytic reaction of uricase to uric acid in borate buffer is pH 9.0. The hydrogen peroxide-peroxidase reaction is favorable at pH 7.0, while the optimum for the oxidation of HVA to produce fluorescence is at pH 10-11. Although the fastest overall reaction rate was observed at the compromise pH of 8.5, when the reagents were mixed without incubation for the steady-state method, which has uric acid incubated with uricase, pH 9.0 provides better sensitivity (-10%) than pH 8.5 for uric acid determination (See Fig. 1 for comparison). A 12 mg/dl standard uric acid solution was used for incubation-time study and 10 min was found to be enough for complete conversion. Recovery study Standard uric acid solutions, give total uric acid concentration range of 95.5-104% (Table II).
added to the control serum Moni-Trol I to up to 11 mg/dl, showed a percent recovery
Ascorbic acid interference Using similar experimental conditions to those described previously, ascorbic acid interference was measured by adding it up to 100 mg/dl into deproteinated control serum Moni-Trol I. The solutions were mixed with uricase in borate buffer and incubated for 10 min at 25°C. Immediately after HVA and peroxidase were added, the reaction rate was recorded for at least 10 min. A similar reaction rate was observed irrespective of the ascorbic acid concentration. TABLE
II
RECOVERY
Uric
acid
OF
URIC
ACID
ADDED
TO
SERUM
Recovery
(mg/dl) _____
Added
Total
(cab)
Found
2.0
7.25
7.30
102.5
2.0
1.00
1.02
101.0
4.0
9.25
9.35
102.5
4.0
9.00
8.82
95.5
4.0
9.00
8.95
98.8
6.0
11.00
10.88
98.0
6.0
11.00
11.25
104.0 Meal-l
100.3
(%)
Fig. 2. Effect
of ascorbic acid (0, 20. 40, 60, 80, 100 mg/dl) on serum uric acid analvsis
But when ascorbic acid values were above 20 mgfdl, the conversion of HVA to the fluorescent compound was delayed. The length of this time lag increased as ascorbic acid co~~~~ntration increased (Fig. 2), but the lag decreased with the increase in uric acid content in serum. Ascorbic acid was found to react in the enzymatic system employed and produced fluorescence with a weaker intensity than uric acid. The fluorescence contributed by ascorbic acid was not significant if ascorbic acid level was below 6 mg/dl, this level being about 3 times higher than that of upper normal serum range. When it was below 20 mg/dl, an equivalent of about one-tenth the level that a similar concentration of uric acid would have contributed is observed.
Uric
Acid,
mpidl, Method
by
I;;ic
Present
Acid,
rily Mt?ih”d
Fig. 3. Comparison
uf the present method
with the ultravimet
Fig. 4. Comparison
of the present method
with the colori.,letric
method. method.
di.
by I’rrsrni
25
The time lag observed in this study indicates the inhibition of ascorbic acid, but this inhibition is reversible since ascorbic acid concentrations do not affect the rate of uric acid conversion as stated by Mahler et al. [13]. Comparison studies The correlation obtained on 30 routine clinical serum or plasma samples assayed by the present method and by the modified ultra-violet assay of Remp [16] is shown in Fig. 3. The figure shows the least-square regression equation and the correlation coefficient. Satisfactory correlation was obtained with this procedure. The correlation between results of this assay and those of the calorimetric method of Caraway, using phosphotungstic acid [17] was established on 26 sera. The correlation coefficient was 0.908 (Fig. 4). Acknowledgements The financial assistance of NIAMDD, NIH, USPHS (Contract No. 72-2216) and of NIH (Grant No. GM 18646) is gratefully acknowledged. Our thanks to the Methodist Hospital in New Orleans for the generous supply and assay of serum and plasma samples, and to Dr F.M. Gonzalez, Dr J. Barona, and Charity Hospital in New Orleans for their dialysis patients’ sera. References 1
0.
2
E.W.
Folin
and
3
J.J. Carroll,
4
E. Praetorius
Rice
5
C.A.
6
L. Liddle,
7
T.V.
8
J.H.
9
K.
W.
and
Denis.
B.S.
H. Cobum, and
Dubbs,
F.W.
J.E.
Feichtmeir
Davis and
Marymont. Lorentz
Jr and
and
Domagk
W.
G.F.
N. Kageyama.
and
12
N. Gochman
and J.M.
13
H.R.
H.M.
14
E.S.
Canellakis
15
E.S.
Canellakis.
16
D.G.
Remp,
York,
1970.
Mahler,
Caraway,
Am.
Anal.
Schlicke. Acta.
Schmitz,
.J. Biol.
J. Clin.
Tech. Anal.
31
G. Hubscher,
and
P.P.
Cohen,
J. Biol.
A.
Tuttle
in R.P. Vol.
and
MacDonald
P. Cohen, (ed.).
J. Clin.
Pathol..
25
(1956)
Regist.
Med. 58
158
903
833
Tech..
22 (1968)
(1971)
497
54 (1959)
25 (1955)
(1967)
17
(1971)
Science.
Chem., J. Biol. Standard
213
34
(1964)
630
219
(1955)
(1956)
(1955)
Methods 840
1154
124
Chem.,
6, p. 1
Am.
17 273
421
Chem.,
and
218
Med.,
Pathol.,
18
Chem., 5 (1953)
Chem..
Biochem.,
Baum
Clin.
Invest.,
Clin.
Bull.
(1971)
Clin.
Lab.
J. Lab.
Biochem.,
181
Babson,
J. Clin.
Adams.
Wrenn.
Chim.
A.L.
L. Laster,
M. London.
H.H.
Clin.
W.S.
469
8 (1962)
and
&and.
and
Berndt.
13 (1912)
Chem..
Douglass
and
H.T.
10
W.R.
R.
Seegmiller
Chem.,
Clin.
H. Paulsen.
11
17
J. Biol.
Grogan.
213
705
385 (1955) of Clinical
397 Chemistry.
Academic
Press.
New
Clinica Ch~mica Actu, 64 (19’75) 19-25 0 Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CGA 7237
AN ALTERNATIVE METHOD FOR THE DETERMINATION OF URIC ACID IN SERUM JUI-CHANG
W. KUAN,
Department of Chemistry, Louisiana 70122 (U.S.A.) (Received
S.S. KUAN, University
and G.G. GUILBAULT of New Orleans, New Orleans,
April 4, 19’75)
Summa~ A novel fluorometric method for the determination of uric acid based on the coupled reactions of uric acid with uricase and peroxidase to form highly fluorescent 2,2’-dihydroxy-3,3’-dimethoxy-biphenyl-5,5’-diacetic acid is described. The calibration curve was constructed from a series of standard uric acid solutions vs. the corresponding relative fluorescence. It was linear up to 15 mgf dl (0.9 mmol/l). The serum or plasma samples must be deproteinated with (absolute) ethanol before assay and its uric acid content can be obtained from the calibration curve. This method is rather simple, having good precision and accuracy, High level ascorbic acid in the sample falsely elevates uric acid concentration. Comparison of the results obtained on the patient sera with a calorimetric phosphotungstate method and a standard enzymatic spectrophotometric method gave coefficients of correlation of 0.908 and 0.986, respectively.
Introduction The most commonly used methods for serum uric acid determination can be classified into two categories: (1) the non-enzymatic alkaline phosphotungstate method of Folin-Denis [l] and its modifications [2,3] ; (2) enzymatic methods based on the oxidation reaction of uric acid by uriease. The alkaline phosphotungstate methods which measure the blue color formation are restricted by interfering turbidity, lack of specificity or poor recovery. In the enzymatic method, the uric acid concentration is monitored either spectrophotometrically at around 292 nm before and after the uricase action [4-71, or calorimetrically by the chromogenic response of the coupling reactions of uric acid, uricase and a suitable chromogen in the presence of an oxidase [S--12]. The enzymatic methods are generally very specific with only minor or no interference and a relatively long incubation time.
20
With an approach similar to the calorimetric enzymatic, Inc~thotl. t tit> hydrogen peroxide evolved from the uric acid-uricase reaction c’an I)c used 1.0 oxidize a non-fluorescent compound to a highly fluorescent form by means of peroxidase, and the fluorescence produced can be related to uric* a(%1 (‘oncentration. Because of the inherent sensitivity of fluorometric methods and the specificity of enzymes, an attempt to develop a fluorometric clnzymatic, method for uric acid utilizing homovanillic acid as a fluorophor was thus initiated. Details of this new methodology are described in this paper.
Materials and methods Reagents Borate buffer, 0.02 M, pH 9.0. Dissolve 1.24 g of boric acid and 1.49 g of potassium chloride in about 800 ml of water. Adjust pH to 9.0 with 1 M NaOH. Make up to 1 liter with water. Homouanillic acid, 4 mg/ml. Dissolve 400 mg of homovanillic acid (98% pure, Aldrich Chem. Co., Inc., Milwaukee, Wise. 53233) in 100 ml of doubly distilled water. Keep refrigerated. This is stable for at least 2 months at 4°C. Uricase, 1 unit/ml. Dissolve 1 vial of 5 units uricase (Type IV, 1.5 unit/mg, purified from Candida utilis, Sigma Chem. Co., St. Louis, MO. 63178) in 5 ml of 0.02 M, pH 9.0 borate buffer. Keep refrigerated, where it remains stable for 2 weeks at 4°C. One unit of uricase will convert 1.0 pmole of uric acid to allantoin per min at pH 8.5 at 25°C. Stock peroxidase, 200 unit/ml. Dissolve 10 mg of peroxidase (Type II, from horse radish, 200 unit/mg solid, Sigma Chem. Co.) in 10 ml of doubly distilled water. Working peroxidase solutions are diluted from this stock solution. One unit of peroxidase will form 1 mg purpurogallin in 20 seconds at pH 6.0 at 20°C. Standard uric acid, 1 mg/ml. This can be obtained commercially from Harleco (Cat. No. 3054, Harleco, 60th and Woodland Ave., Philadelphia, Pa. 19143). Working standards are prepared from appropriate dilution of stock solution with doubly distilled water. Absolute ethanol, reagent grade, from Industrial Chemical Co., New York. Apparatus Fluorescence measurements were done with an Aminco Fluoro-microphotometer (American Instrument Co., Inc., Silver Spring, Md. 20910), with a Corning 7-60 primary filter and a Kodak Wratten 47B/2A combination secondary filter. A circulating water bath was employed to maintain the temperature at 25”C, and a Beckman linear recorder (Beckman Instruments, Inc., Fullerton, Calif. 92634) was coupled to the fluorometer. The fluorometer was standardized daily with polymer fluorescent standard No. 3 (p-Terphenyl, 3 X lO* M) from Perkin-Elmer (Perkin-Elmer Corp., Norwalk, Conn. 06856). An Adams Dynac Centrifuge (Cat. No. 0101, ClayAdams, Inc., Parsippany, N.J. 07054) was used in the deproteination process and a Vortex Genie Mixer (Scientific Industries, Inc., Springfield, Mass. 01103) was used for sample mixing.
21
10
Fig.
1. Calibration
curve
for uric acid
in borate
buffer
(a) PH 8.5,
(b)
PH 9.0.
Assay procedure
The serum sample, 0.2 ml, is deproteinated by the addition of 0.6 ml absolute ethanol, the mixture is allowed to stand at least 5 min and centrifuged at a high speed for about 10 min or until a clear supernatant fluid is obtained. Place, in order, into a test tube (1.1 cm X 10 cm) 0.16 ml of the deproteinated supernatant in 0.04 ml doubly distilled water, 2.5 ml, 0.02 M, pH 9.0 borate buffer, and 0.05 ml, 1 unit/ml uricase. Incubate for 10 min at 25°C. Then it must be treated with 0.2 ml, 4 mg/ml homovanillic acid, immediately followed by 0.05 ml, 32 units/ml peroxidase. Shake for 15 s (speed 3) with a Vortex Genie Mixer. After standing at 25°C for 10 min, the relative fluorescence at 425 nm (A,,,. = 315 nm) is recorded. Aqueous standard uric acid, 0.04 ml, in 0.16 ml absolute ethanol, is used and carried through the entire sequence as outlined above for plotting the calibration curve. The curve is linear up to 15 mg/dl uric acid (Fig. 1). The uric acid concentration of an unknown serum is obtained from the calibration curve. Results and discussion Order of reagent addition
In borate buffer at pH 9.0, uric acid reacts with uricase to produce hydrogen peroxide, allantoin . H202 and other degraded products [13-141. Homovanillic acid (HVA), in the presence of peroxidase, can be easily oxidized by hydrogen peroxide to form highly fluorescent compounds. However, it was found that uric acid could also be oxidized by peroxidase [ 151. When uricase and peroxidase were used in the same system at the same time, due to the competition between uric acid and homovanillic acid towards hydrogen perox-
ide formed, the rate of fluorescence and its intensity varied with thr ordrr 01’ addition of these reagents. This phenomenon has been rerbordetl I)y othc>~ investigators in a similar enzyme system with different chromogens for their photometric assays [9,10]. Peroxidase, if added to the system last, usually gave a higher reaction rate than if it was added sooner. In the sequence of uric, acid, buffer, uricase, homovanillic acid, and peroxidase without any incubation, the fluorescent change was proportional to uric acid concentration. IIowever, the rate of fluorescent change with regard to uric acid was small and the linear range was too narrow; hence, it did not always produce adequate accuracy for the kinetic determination of serum uric acid. With the same sequence, Ijut with uric acid incubated with uricase in borate buffer for a sufficient time to insure quantitative formation of H2 02, then followed by the addition of HVA and peroxidase, the relative fluorescence exhibited at a definite time was proportional to uric acid concentration. When mixing only HV:I and peroxidase together, a very slight fluorescent change is observed. This is probably due to the presence of some oxidant impurities in the peroxidase. Thus, a premixed solution of HVA and peroxidase is not recommended for use. Serum deproteination Serum must be deproteinated before assay since it contains catalase which could react with the H202 formed; almost all proteins act as H20, acceptors in the presence of peroxidase. The acid precipitants such as trichloroacetic and perchloric acid cannot be used because they would cause losses in uric acid by coprecipitation with the protein at low pH. Using ethanol or heat denaturation for deproteination were found to be quite satisfactory. Assay of dialysis patients ’ sera Blood samples were drawn from five kidney patients before, during and after they underwent renal dialysis and these samples were analyzed by the proposed method to correlate the uric acid level with renal function and the efficiency of dialysis. Serum uric acid levels are believed to elevate in renal malfunction. From the results of our assay (Table I) the uric acid values of some “before dialysis” sera were slightly above normal range, gradually decreased during dialysis and finally dropped within the normal range after the dialysis was accomplished. Judging from this observation, uric acid level can only be used as a supplementary indicator in diagnosis of renal impairment. TABLE
1
ASSAY
RESULTS
Patient
No.
OF
DIALYSIS
Uric
acid
Before
PATIENTS’SERA
concentration
dialysis
(mg/dl)* During
dialysis
After
dialysis
wok., ~)215 ~~~~~~~ -. ~.o~~~ ~~ 1
4.7
2
5.1
3.0
2.7
3
7.4
3.2
2.4
4
7.7
4.1
3.3
5
7.2
5.1
4.1
* Average
of 2 to 3 determinations.
23
Reproducibility and precision Two uric acid samples were analyzed with the same reagent concentrations for a period of six days. We found a withinday precision (CV) of 2.62%, mean of 3.98 mg/dl, standard deviation (SD) of 0.10; and CV = 0.49%, mean = 9.63 mg/dl, SD = 0.05. Day-today: CV = 1.18%, mean = 3.93, SD = 0.05;CV = 0.41%, mean = 9.65, SD = 0.04. The relative fluorescence reading contributed from any reaction mixture varies about +1.5 fluorescent unit which accounts for the relatively higher CV for low concentration uric acid samples. Effect of pH and incubation time The optimum for the catalytic reaction of uricase to uric acid in borate buffer is pH 9.0. The hydrogen peroxide-peroxidase reaction is favorable at pH 7.0, while the optimum for the oxidation of HVA to produce fluorescence is at pH 10-11. Although the fastest overall reaction rate was observed at the compromise pH of 8.5, when the reagents were mixed without incubation for the steady-state method, which has uric acid incubated with uricase, pH 9.0 provides better sensitivity (-10%) than pH 8.5 for uric acid determination (See Fig. 1 for comparison). A 12 mg/dl standard uric acid solution was used for incubation-time study and 10 min was found to be enough for complete conversion. Recovery study Standard uric acid solutions, give total uric acid concentration range of 95.5-104% (Table II).
added to the control serum Moni-Trol I to up to 11 mg/dl, showed a percent recovery
Ascorbic acid interference Using similar experimental conditions to those described previously, ascorbic acid interference was measured by adding it up to 100 mg/dl into deproteinated control serum Moni-Trol I. The solutions were mixed with uricase in borate buffer and incubated for 10 min at 25°C. Immediately after HVA and peroxidase were added, the reaction rate was recorded for at least 10 min. A similar reaction rate was observed irrespective of the ascorbic acid concentration. TABLE
II
RECOVERY
Uric
acid
OF
URIC
ACID
ADDED
TO
SERUM
Recovery
(mg/dl) _____
Added
Total
(cab)
Found
2.0
7.25
7.30
102.5
2.0
1.00
1.02
101.0
4.0
9.25
9.35
102.5
4.0
9.00
8.82
95.5
4.0
9.00
8.95
98.8
6.0
11.00
10.88
98.0
6.0
11.00
11.25
104.0 Meal-l
100.3
(%)
Fig. 2. Effect
of ascorbic acid (0, 20. 40, 60, 80, 100 mg/dl) on serum uric acid analvsis
But when ascorbic acid values were above 20 mgfdl, the conversion of HVA to the fluorescent compound was delayed. The length of this time lag increased as ascorbic acid co~~~~ntration increased (Fig. 2), but the lag decreased with the increase in uric acid content in serum. Ascorbic acid was found to react in the enzymatic system employed and produced fluorescence with a weaker intensity than uric acid. The fluorescence contributed by ascorbic acid was not significant if ascorbic acid level was below 6 mg/dl, this level being about 3 times higher than that of upper normal serum range. When it was below 20 mg/dl, an equivalent of about one-tenth the level that a similar concentration of uric acid would have contributed is observed.
Uric
Acid,
mpidl, Method
by
I;;ic
Present
Acid,
rily Mt?ih”d
Fig. 3. Comparison
uf the present method
with the ultravimet
Fig. 4. Comparison
of the present method
with the colori.,letric
method. method.
di.
by I’rrsrni
25
The time lag observed in this study indicates the inhibition of ascorbic acid, but this inhibition is reversible since ascorbic acid concentrations do not affect the rate of uric acid conversion as stated by Mahler et al. [13]. Comparison studies The correlation obtained on 30 routine clinical serum or plasma samples assayed by the present method and by the modified ultra-violet assay of Remp [16] is shown in Fig. 3. The figure shows the least-square regression equation and the correlation coefficient. Satisfactory correlation was obtained with this procedure. The correlation between results of this assay and those of the calorimetric method of Caraway, using phosphotungstic acid [17] was established on 26 sera. The correlation coefficient was 0.908 (Fig. 4). Acknowledgements The financial assistance of NIAMDD, NIH, USPHS (Contract No. 72-2216) and of NIH (Grant No. GM 18646) is gratefully acknowledged. Our thanks to the Methodist Hospital in New Orleans for the generous supply and assay of serum and plasma samples, and to Dr F.M. Gonzalez, Dr J. Barona, and Charity Hospital in New Orleans for their dialysis patients’ sera. References 1
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