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enzymatic analysis. One of the advantages of the present ... Common methods for clinical analysis of uric acid are based on the calorimetric reduction of alkaline ...
ANALYTICAL

BIOCHEMISTRY

(1978)

88,X%-.565

Determination of Uric Acid in Biological Fluids High-Pressure Liquid Chromatography J. A. MILNER Department

of Food

Science,

By

AND E. G. PERKINS

University

of Illinois,

Urbana,

Illinois

61801

Received September 30, 1977; accepted March 17, 1978 A high-pressure liquid chromatographic assay for uric acid in biological fluids has been developed. Blood uric acid can be analyzed in as little as 20 ~1 of plasma. The mean and range of plasma uric acid concentrations in healthy adults determined by high-pressure liquid chromatography were similar to those obtained by enzymatic analysis. One of the advantages of the present method is that naturally occurring metabolites in biological fluids or drugs do not interfere with the analysis. Data are presented for blood and urine specimens obtained from mice fed a known uricase inhibitor, potassium oxonate. Comparisons are made between the present method and methods previously employed for uric acid determination.

Common methods for clinical analysis of uric acid are based on the calorimetric reduction of alkaline phosphotungstate (l-3). These methods are satisfactory for routine analysis of uric acid concentrations in some patients. However, the usefulness of this method is c,‘ten limited by the presence of interfering substances such as drugs or their breakdown products and normal biological metabolites (4). Uricase has been incorporated into some assay methods for the purpose of increasing reaction specificity (5-7). These procedures are based either on determination of ultraviolet absorption at 292 nm before or after incubation with uricase (5) or on the determination of reducing substances before and after incubation with uricase (4). Methods employing uricase have increased sensitivity and reduced interferences from a number of The use of high-purity uricase of naturally occurring metabolites. microbiological origin has substantially decreased the cost of this method. These methods, however, are somewhat limited in applicability since they are extremely sensitive to uricase inhibitors. Several electrochemical methods are available for the analysis of uric acid (8). In addition chromatographic methods have been developed (9- 11). These methods, however, have not received wide attention. Liquid chromatography has met with increasing acceptance as a routine tool for clinical analysis in many laboratories. Although, less sensitive than electrochemical detection, ultraviolet detection offers the advantage of 0003-2697/78/0882-0560$02.00/0 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

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greater adaptability to analysis of other biological compounds and is more generally available in the laboratories. The determination of uric acid by high-pressure liquid chromatography (hplc) can provide for quantitative analysis and eliminate the interferences from drugs and other biological compounds as a result of selective wavelength detection and/or column separation. This report describes a simple, rapid, and inexpensive method for analysis or uric acid in biological fluids by high-pressure liquid chromatography. Comparisons are made between the described method and those employing alkaline phosphotungstate or uricase for analysis of uric acid in biological fluids. MATERIALS

AND METHODS

Apparatus. A Tracer, Inc., high-pressure

liquid chromatograph,’ based on a dual-piston reciprocating pump coupled with a variable-wavelength detector, was used for these determinations. Uric acid was separated on a laboratory packed stainless steel column (60 x 2 mm i.d.) containing a strong anion-exchange resin2 (37-50 pm). Operations were performed at room temperature. Samples were introduced with a 25-~1 syringe through a continuous-flow loop Rheodyne injector.3 The mobile phase was 0.01 M potassium chloride (pH 7.4) in glass-redistilled, degassed water at a flow rate of 1 ml min-‘. A variable-wavelength ultraviolet monitor’ set at 292 nm was used as the detector. Reagents. All reagents were of analytical reagent grade. Uric acid was obtained from Sigma Chemical Inc., St. Louis, MO. Uric acid standards (l-5 mg/lOO ml) were prepared in 0.1 N NaOH. Biological materials. Heparinized blood samples were collected from humans by venipuncture and from experimental mice by heart puncture. Mouse urine was also analyzed for uric acid content. Plasma (20 ~1) was treated with 0.1 M aqueous uranyl acetate (35 ~1) to precipitate proteins. After centrifugation the supernatant was used directly for analysis. Urine (1 ml) was extracted with basic methanol (4 ml, pH 8) for removal of organic components. Sodium hydroxide (1 N) was added to methanol to a final pH of 8 for preparations of the basic methanol extraction solution. After centrifugation, the supernatant was filtered through a Whatman No. 42 filter and a 0.45~pm Millipore filter, and then was evaporated to dryness under nitrogen in a 60°C water bath. Samples were then reconstituted in 0.2 ml of 0.1 N NaOH for analysis. ’ Included in the system was a Tracer Model 6970 liquid chromatograph, a Model 980 solvent programmer and a Model 970 variable-wavelength detector. Tracer Instruments, Inc., 6500 Tracer Lane, Austin, Texas 78721. * Waters Associates, Inc.. Maple Street, Milford, Massachusetts 01757. 3 High-pressure 907 variable-volume valve loop injector, Tracer Instruments. Austin, Texas 78721.

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MILNER

RESULTS

AND

PERKINS

AND DISCUSSION

The uric acid content of biological fluids can be readily detected by ultraviolet monitoring following elution by high-pressure liquid chromatography. A calibration curve of peak height versus concentration of uric acid in the range of O.l- 140 mg liters-1 is linear and passes through the origin (Fig. 1). The correlation coefficient of uric acid concentration against optical density was 0.997. Corroboration of the specificity for uric acid was performed by treatment of blood and urine specimens with uricase. No detectable response was observed when samples were pretreated with uricase (Fig. 2). The retention time of uric acid was 1.30 min under the present conditions. The precision obtained for repeated analysis of 20 ~1 of a 5.0 mg/lOOml standard was 0.3%. Precision for repeated analyses of plasma uric acid was similar. A comparison of the effects of various protein precipitating agents on uric acid recovery from plasma was performed. Perchloric acid, trichoroacetic acid, uranyl acetate, or a solution of sodium tungstate in dilute H,SO, was examined. Under our conditions uranyl acetate treatment yielded the highest recovery of uric acid. Analysis of uric acid from five human serum samples by hplc were compared with corresponding results obtained by analysis with uricase (Table 1). The correlation of the hplc method with the uricase methods was 0.97. Recovery of uric acid added to plasma samples was greater than 97%. When the supply of blood is limited, as occurs in infants and small experimental animals, the present method would be of considerable value. The presence of drugs, especially those that may act as metal-complexing agents, in blood specimens may interfere with methods employing uricase (4,12,13). Ionic selectivity for urine specimens is of considerable value be-

FIG.

I. Calibration

curve

for uric acid analysis

by high-pressure

liquid

chromatography.

URIC ACID IN BIOLOGICAL

563

FLUIDS

FIG. 2. Uric acid standards and sample: (A) 8 ~1 of 0.5 mg% uric acid standard; (B) 5 ~1 of mouse plasma extract, final concentration: 1.65 mg%; (C) standard as in a; and (D) standard after uricase treatment.

cause of the higher concentration of oxidizable metabolites. The presence of urinary compounds such as ascorbic acid, xanthine, cysteine, and glutathione normally affecting uric acid analysis by the phosphotungstic acid methods did not interfere significantly in the present method of analysis. Potassium oxonate has been shown to be a potent inhibitor of in vitro uricase activity (14). Urine obtained from mice fed 3% potassium oxonate had a virtually undetectable uric acid content when a method employing TABLE DETERMINATION

1

OF PLASMA URIC ACID BY hplc AND ENZYMATIC

VALUES IN NORMAL ANALYSIS

ADULTS

Method

Mean SEMd Range Number

hplc”

Enzymatic”

3.45’ 0.77

3.50 0.75 I .47-5.50 5

1.34-5.47

5

(1See Materials and Methods for analysis. b See Ref. (5). r Values are expressed as milligrams per 100 ml. P Standard error of the mean.

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MILNER

AND PERKINS TABLE

URINE

2

AND PLASMA URIC ACID FROM MICE FED A DIET WITH OR WITHOUT 3% POTASSIUM OXONATE~

Plasma (mgil00 ml) - Potassium oxonate + Potassium oxonate

1.8 2 0.1 (A) 4.7 k 1.4 (B)

Urine (pglpg of creatinine)” - Potassium oxonate + Potassium oxonate’

2.0 k 0.2 (A) 3.0 2 0.3 (B)

’ Values followed by unlike letters differ, P < 0.05. ’ Twenty-four-hour urine collection. r Undetectable by enzymatic analysis (7).

uricase was used for analysis. However, analysis of these samples by hplc revealed a mild uricosuria compared to urine obtained from mice fed a normal diet (Table 2). These data illustrate the potential problems of drugs or their breakdown products interfering with the quantitative determination of uric acid in clinical patients. Analysis of biological metabolites by hplc is not routinely used as a method for clinical analysis. More analysis by hplc is likely to be used in clinical situations with the advent of new and diverse procedures. The present analysis of uric acid in biological materials employs a laboratory packed column. The use of such a column substantially decreases the operating cost for this separation. Detection of uric acid by a multiwavelength monitor eliminates the need for a specific electrochemical detector, yet allows for maximum versatility for other analyses. The sensitivity of the present method as demonstrated is more than adequate for analysis of uric acid in most biological fluids. ACKNOWLEDGMENTS The authors wish to thank Ms. J. Gnaedinger for her excellent technical assistance and Ms. S. Mangoff for assistance in obtaining the biological samples used in this manuscript. Supported in part by Illinois Agriculture Experiment Station.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Archibald, R. M. (1970) Clin. Chem. 3, 102-105. Jung, D. H., and Parekh, A. C. (1970) C/in. Chem. 16, 247-250. Henry, R. J., Sobel, C., and Kim, J. (1957) Amer. J. Clin. Pathol. 28, 152- 160. Gochman, N., and Schmitz, J. M. (1971) Chin. Chem. 17, 1154-1159. Kalckar, H. M. (1947) J. Biul. Chem. 167, 429-443. Praetorius, E. (1949) Stand. J. Clin. Lab. Invest. 1, 222-230. Liddle, L.. Seegmiller, J. E.. and Laster, L. (1959) J. Lab. Clin. Med. 54, 903-913. Troy, R. J., and Prudy. W. C. (1970) Clin Chim. Acta. 27, 401-408.

URIC ACID IN BIOLOGICAL 9. 10. 11. 12.

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Pauhla, L. A., and Kissinger, P. T. (1975) Chin. Chim. Acta 59, 309-312. Simkin, P. A. (197O)J. Chromatogr. 47, 103-107. Kelley, W. N., and Wyngaarden, J. (1970) Clin. Chem. 16, 707-713. Baum, H., Hiibscher, G., and Mahler, H. R. (1956) Biochim. Biophys. Acta 22, 5 14-527. 13. Mahler, H. R., Hiibscher, G., and Baum, H. (1955)J. Biol. Chem. 216, 625-641. 14. Johnson, W. J., Stavric, B., and Chartrand, A. (1969) Proc. Sot. Exp. Biol. Med. 131, 8-12.