WO1997039352A1

WO1997039352A1 - Assays for detection of purine metabolites

Info

Publication number: WO1997039352A1
Application number: PCT/US1997/002839
Authority: WO
Inventors: James L. Sherley; Stephen S. Ortiz
Original assignee: Fox Chase Cancer Center
Priority date: 1996-04-15
Filing date: 1997-02-25
Publication date: 1997-10-23
Also published as: AU1970897A

Abstract

A simple assay for purine bases or nucleosides in a liquid sample is provided wherein the purine base or nucleoside is converted to uric acid by the action of xanthine oxidase and purine nucleoside phosphorylase. In an exemplary embodiment, a commercially available uric acid reagent in combination with standards of known concentrations are used to determine the level of purine base or nucleoside in a sample. According to another aspect of the invention, test kits are provided for performing the above-described methods.

Description

ASSAYS FOR DETECTION OF PURINE METABOLITES

Pursuant to 35 U.S.C. §202(c) , it is hereby acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health.

FIELD OF THE INVENTION

This invention generally relates to detection and quantitation of selected analytes within a mixed population of analytes. Specifically, simple assays are provided to detect and measure purine bases and/or purine nucleotides in a biological sample.

BACKGROUND OF THE INVENTION

The concentration of a light-absorbing (i.e., colored) substance in solution can be estimated by comparing the intensity of its color with that of several standard solutions of known^' concentration. Such methods, using a spectrophotometer as a detector, are known as colorimetric methods; in various forms, they have played a major role historically in analytical chemistry.

Colorimetric assays have been developed to analyze levels of various components in biological or chemical solutions. Similarly, light-emitting (i.e., fluorescent) substances can be detected and quantified using appropriate detection devices, e.g., a fluorometer.

Antimetabolites that interfere with de novo purine synthesis constitute an important part of the arsenal of drugs available for the treatment of leukemia and other cancers. However, not all patients respond equally well to these drugs, and some eventually become refractory to therapy. Resistance to purine analogs has been attributed to the deficiency of enzymes necessary to convert them to their nucleotide forms. Other mechanisms have been observed in experimental tumors, such as increased degradation of drugs and inability of resistant cells to convert the ribonucleotide analogues to the deoxyribonucleotide analogue. Increased plasma concentrations of hypoxanthine during treatment with these drugs may also be a mechanism of resistance to antimetabolites acting in the purine biosynthetic pathways. Extracellular hypoxanthine is important for the activity of purine antimetabolites in two aspects: it can be utilized to restore purine nucleotide pools in cells, the de novo synthesis of which has been blocked, and it potentially may compete with some of these antimetabolites for transport into the cell or for the enzymes necessary to convert the drugs to their active nucleotides.

Thus, the levels of purine metabolites (inosine, hypoxanthine, xanthine, xanthosine, adenine and adenosine) have been extensively investigated in biological fluids in many diseases, including cancer, gout and Lesch-Nyhan syndrome. However, their determination, until a few years ago, presented several problems, as the analytical methods used were of dubious sensitivity and specificity. Methods exist for the detection of these compounds by HPLC, but these methods are expensive, requiring sophisticated instrumentation and technicians with specialized training. Because they require separation of purines from other plasma constituents, they are labor-intensive and poorly quantitative due to difficulties in ensuring uniform sample recovery.

It would therefore be beneficial to have available a rapid, inexpensive, and sensitive method for the determination of purine metabolites which can be performed by unskilled technicians. It is the object of the present invention to provide such a method. It is a further object of the invention to provide a kit for the determination of purine metabolite levels in a given biological sample i.e., blood, urine, seminal fluid, plasma or tissue extracts. SUMMARY OF THE INVENTION

The present invention provides simple assays for determining the presence or quantity of selected purine metabolites in a liquid sample. According to one aspect of the invention, a method is provided for determining the presence or quantity of a selected subpopulation of analytes in a mixed population of analytes, wherein the analyte subpopulation comprises purine metabolites capable of being further metabolized to uric acid. The method comprises providing a liquid sample of the mixed analyte population, adding to that sample at least one reagent that catalyzes the conversion of the purine metabolites to uric acid (under conditions whereby the purine metabolites in the subpopulation are converted to uric acid) , adding to the sample at least one reagent that reacts with uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide (and providing conditions whereby the hydrogen peroxide is produced in the sample) and quantitatively detecting the hydrogen peroxide in the sample. The quantity of hydrogen peroxide produced by the method is proportional to the quantity of purine metabolite analyte subpopulation in the sample.

In preferred embodiments of the aforementioned assay, the mixed analyte population is disposed within a biological fluid or tissue and a liquid sample is prepared from that biological fluid or tissue. The analyte subpopulation preferably comprises purine bases (such as adenine, xanthine and hypoxanthine) or purine nucleosides (such as inosine, xanthosine and adenosine) . Reagents that catalyze the conversion of purine bases to uric acid preferably include the enzyme xanthine oxidase; reagents for catalyzing the conversion of purine nucleosides to uric acid preferably comprise a combination of the enzymes purine nucleoside phosphorylase and xanthine oxidase. In preferred embodiments of the aforementioned method, the uric acid in the sample is exposed to the enzyme, uricase, producing as a byproduct a stoichiometrically equivalent amount of a hydrogen peroxide. The hydrogen peroxide is thereafter detected, preferably by optical means. Detection is accomplished by exposing the hydrogen peroxide in the sample substrates and other reactant which, upon reaction with hydrogen peroxide form the optically detectable product is preferably detected spectrophotometrically or spectrofluorometrically. According to another aspect of the invention, a method is provided for determining the presence or quantity of purine bases in a biological fluid or tissue. The method comprises providing a liquid test sample of the biological fluid or tissue and a volumetrically equivalent liquid control sample. To the test sample is added an amount of xanthine oxidase effective to catalyze the conversion of the purine bases to uric acid. The sample is placed under conditions whereby the purine bases are converted to uric acid. To the control sample is added a volume of a non-reactive liquid medium (such as water or buffer) equivalent to the volume of xanthine oxidase added to the test sample. The control sample is subjected to the same conditions as the test sample. To each of the test sample and control sample is added at least one reagent that reacts with uric acid in the samples to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide. The sample is subjected to conditions whereby the reaction to form the hydrogen peroxide occurs. Thereafter, the hydrogen peroxide is quantitatively detected in each of the test sample and the control sample, the quantity of hydrogen peroxide is proportional to the quantity of the uric acid in each sample. By comparing the difference in quantity of uric acid in the control sample versus the test sample the quantity of purine bases in the biological fluid or the tissue may be determined. According to another aspect of the present invention, a method for determining the presence or quantity of purine nucleosides in a biological fluid or tissue. The method comprises providing volumetrically equivalent liquid test sample and control sample of the biological fluid or tissue. To the test sample is added an amount of nucleoside phosphorylase and xanthine oxidase effective to catalyze the conversion of the purine nucleosides to uric acid. To the control sample is added a volume of a non-reactive liquid medium equivalent to the volume of nucleoside phosphorylase added to the test sample. The control sample is also given an equivalent amount of the xanthine oxidase as added to the test sample. The respective samples are subjected to conditions whereby, in the test sample the purine nucleoside are converted to uric acid. To each of the test sample and the control sample is added at least one reagent that reacts with the uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide. The hydrogen peroxide in each sample is quantitatively detected, that quantity being proportional to the quantity of uric acid present in each sample. The difference in quantity of uric acid in the control sample versus the test sample is compared, that difference being proportional to the quantity of purine nucleosides in the biological fluid or tissue.

According to other aspects of the invention, a test kit for determining the presence or quantity of a subpopulation of analytes comprising purine metabolites capable of being further metabolized to uric acid, in a mixed population of analytes, is provided. The test kit comprises at least one reagent that catalyzes the conversion of the purine metabolites to uric acid, and at least one reagent that reacts with the uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide. In a preferred embodiment of the invention, the test kit further comprises at least one reagent for detecting the hydrogen peroxide.

The assays in the test kits of the present invention provide a rapid, inexpensive and sensitive system for determining the presence or amount of purine metabolite in a biological sample. These methods and test kit provide an inexpensive alternative to sophisticated, instrument-dependent assays presently available, and can be performed in the clinic or in the field by unskilled technicians.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a simple method for determining the presence or quantity of a selected subpopulation of purine metabolites in a liquid sample, preferably obtained from a biological fluid or tissue. The method is designed to measure purine bases and purine nucleosides, either together or separately. The assays of the invention are based on the known enzymatic activity of xanthine oxidase and purine nucleoside phosphorylase, and relies on converting the substrates of these enzymes (e.g., the purine nucleotides, inosine, xanthosine and adenosine, and purine bases xanthine, hypoxanthine and adenine) to uric acid. The uric acid may be quantitatively detected in a variety of ways. In a most preferred embodiments of the present invention, the uric acid is exposed to the enzyme, uricase, under oxidative conditions, which results in the formation of a stoichiometrically equivalent amount of hydrogen peroxide (H₂0₂) as a reaction byproduct.

The ultimate step in the method of the present invention is based upon the detection of the byproduct, hydrogen peroxide. Hydrogen peroxide levels may be quantitated using several different procedures. In a preferred embodiment of the invention, a commercially available uric acid reagent kit (Sigma Chemical Co., St. Louis, MO)may be utilized. Purine metabolites initially present in a liquid sample are acted on by xanthine oxidase or purine phosphorylase. The resulting reaction product is uric acid which is then exposed to enzymes (uricase peroxidase) and other reagents present in the uric acid kit, which results in the generation of hydrogen peroxide. Hydrogen peroxide can be detected and quantitated many ways, as described in greater detail below. In the uric acid reagent kit, H₂0₂ is detected via its reaction with two substrates to form a quinoneimine dye that absorbs light at 520 nm (see Green and Hill (1984) Meth. Enz. 105: 3-22).

The method of this invention, exemplified by the determination of hypoxanthine and xanthosine, comprises in its most preferred form: incubating a liquid sample, i.e., plasma, tissue extract or urine, with enzymes selected from the group of xanthine oxidase and purine nucleoside phosphorylase or a combination thereof; adding the above components to a tube containing uric acid reagent (Sigma) ; and reading the absorbance values of the resulting reaction product in a spectrophotometer set at 520 nm. The presence of xanthine, xanthosine, inosine, hypoxanthine, adenine or adenosine are then determined. Another method for detecting H₂θ₂ is based upon the hydrolysis of a stable reagent, diacetyl- dichlorofluoroscin (LDADCF) , by sodium hydroxide to a less stable nonfluorescent compound dichlorofluoroscin (LDCF) , which is oxidized by hydrogen peroxide to the fluorescent dichlorofluorescin (DCF) . This fluorometric analysis is used successfully to detect ultramicroquantities of hydrogen peroxide (Sanchez-Ferrer et al., (1990) Analytical Biochem. 187:129-132).

Scopoletin may also be used to detect H₂0₂. The method is based on quantitation of decreases in scopoletin fluorescence following its oxidation by H₂0₂ and horseradish peroxidase (Clifford et al., (1984), Meth. Enz. JL0_5:393-398) . More specifically, when activated by light at 350 nm, scopoletin fluoresces with a peak at 460 nm. When oxidized by H₂0₂ and horseradish peroxidase, scopoletin loses its fluorescence in direct proportion to H₂0₂ concentration.

Another method for assessing hydrogen peroxide levels in a sample described by Pick and Mizel, (1981) J. Immunol. Meth. 4.6211-266. This is based on the horseradish peroxidase dependent conversion of phenol red by H₂0₂ into a compound with increased absorbance at 600- 610 nm. The spectrophotometric measurement is made after adjusting the pH of the reaction mixture to 12.5, in order to eliminate changes in the absorbance of phenol red due to its behavior as a pH indicator. Hydrogen peroxide levels may also be quantitated with dichlorophenol-indophenol. In this procedure, a sample is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate and/or other reductants present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the amount of H₂0₂ in the sample. Due to the observation that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min^'1 μM^"1 with generation of H₂0₂, this is not a particularly preferred method as it has the potential to overestimate the H₂0₂ content in the sample (Garcia-Castineiras et al., Exp. Eye Res. 55:9-

19).

It is apparent from the methods described above (which are exemplary, rather than all-inclusive) that various procedures may be utilized to detect the final reaction end product, H₂0₂, in the present assays. These methods are also contemplated for use in the instant invention. In a further aspect, the invention provides a kit of reagents for carrying out the assays of the invention. The kit contains reagents for converting prime nucleosides and/or bases to uric acid, as well as reagents for producing H₂0₂ from uric acid and, optionally, reagents for detecting the hydrogen peroxide. Thus for example, a kit according to the present invention may comprise reaction vials, preferably cuvettes, lyophilized enzymes (e.g., xanthine oxidase, nucleoside phosphorylase) , calibrated reference solutions, an appropriate diluent, and the uric acid reagent (Sigma) or an appropriate substitute (e.g., one or more of the individual components of the reagent obtained from Sigma or other suppliers, or modifications of the reagent, such as described by James and Price (1984) Ann. Clin. Biochem. 21' 405-410.

The examples set forth below are provided to describe the invention in further detail. They are not intended to limit the invention. Examples 1 and 2 describe assays for detecting purine bases and nucleosides. Example 3 shows typical results of the assays. Example 4 describes use of the assays to detect altered plasma hypoxanthine levels in cancer patients.

Example 1

Method for Detection of Purine Bases (e.g., adenine, xanthine, hypoxanthine)

The degradation of purines to the end product uric acid occurs via the action of deaminase enzymes that first convert adenine and guanine to hypoxanthine and xanthine, respectively. Deamination may also occur non- enzymatically. This is a more frequent occurrence for adenine because, unlike guanine, the amino group in adenine is attached to a 6-membered aromatic ring. Hypoxanthine and xanthine are then oxidized by xanthine oxidase to uric acid. To detect the levels of purine bases (xanthine, hypoxanthine, or adenine) in a liquid sample, duplicate tubes are prepared that contain 125 μl of the sample to be assayed. Xanthine oxidase (0.643 units, Sigma) in approximately 40 μl is added to the first tube. A comparable volume of distilled water is added to the second tube, which serves as a uric acid control. This sample is set up to determine the pre¬ existing uric acid level in the sample. Reaction mixtures are then incubated for 30 minutes at room temperature. After 30 minutes, 150 μl of the sample from each tube, (the control sample and the experimental sample) are added to two separate tubes containing 850 μl of Uric Acid Assay reagent prepared at 1.176 times the concentration specified by the supplier (Sigma Chemical Co. , St. Louis, MO) . Uric acid reagent is comprised of Uricase (Candida, 125 U/L) ; Peroxidase (horseradish, 5000 U/L); 4-Aminoantipyrine (4-AAP) (0.3 mmol/L); 3,5Dichloro-2-hydroxybenzensulfonate (DHBS) (2 mmol/L) in a physiological buffer (pH 7.2). Reaction mixtures are incubated for 10-30 minutes. The samples are then transferred to cuvettes and the absorbance measured by a spectrophotometer set at 520 nm. The enzymatic reactions involved in the assay are as follows: deamination xanthine oxidase 1. adenine > hypoxanthine >xanthine

+ H₂0 + 0₂ +H₂0₂

xanthine oxidase

2. xanthine + H₂0 + 0₂ > uric acid + H₂0₂

Uricase

3 . uric acid + 0₂ + H₂0 >allantoin + C0₂ +H₂0₂ Peroxidase

4. H₂0₂ + 4-AAP + DHBS > quinoneimine dye + 3H₂0

Uricase catalyzes the oxidation of uric acid to allantoin, carbon dioxide and hydrogen peroxide. The hydrogen peroxide formed at each step reacts in the presence of peroxidase with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS) to form a quinoneimine dye with an absorbance maximum at 520 nm. To determine the presence of a purine base, the absorbance value of the uric acid control sample is subtracted from the absorbance value of the sample exposed to xanthine oxidase. The resulting value is absorbance due to the presence of xanthine, hypoxanthine or adenine or a combination thereof.

Example 2 Method for Detection of Purine Nucleosides (e.g., inosine, xanthosine, adenosine) Interconversion between purine bases and nucleosides are catalyzed by purine nucleoside phosphorylase. The reaction is as follows:

phosphorylase

5. purine nucleoside + Pi +* purine + ribose-1-phosphate

The procedure for the detection of purine nucleosides is the same as above with the following modifications. The experimental tube contains the sample to be assayed, 0.643 units of xanthine oxidase and 2.5 units of nucleoside phosphorylase (Sigma Chemical

Co., St. Louis, MO). As a control for pre-existing uric acid and purine base levels, a duplicate tube is set up that contains the sample to be assayed, 0.643 units of xanthine oxidase and a comparable volume of distilled water instead of purine nucleoside phosphorylase. After a 30 minute incubation at room temperature, 150 μl of each sample is added to 850 μl of uric acid reagent and the samples are processed as in Example l. In this Example, the concentration of the purine nucleoside is calculated by subtracting the absorbance value of the xanthine oxidase sample from the absorbance value from the sample containing both xanthine oxidase and nucleoside phosphorylase. The remaining absorbance value is due to the presence of inosine, xanthosine or adenosine, or a combination thereof.

Example 3

Absorbances Observed for Purine Compounds Measured According to the Assays of Examples 1 and 2

While the purine base assay presently detects three different bases, we have found by HPLC analysis that hypoxanthine is by far the predominant purine base in human plasma samples. It is about 100-fold more abundant than xanthine and 5-10-fold more abundant than adenine. The absorbance values of 1 mM solutions of the purine bases and nucleosides is shown in Table 1 below:

Absorbance at 520 nm

Purine Purine Purine comnound Base Assay Nucleoside Assay

Hypoxanthine 1.18 ± 0.02 0.0

Xanthine 0.84 ± 0.01 0.0

Adenine 0.12 ± 0.01 0.0

Guanine 0.00 ± 0.0 0.0

Uric Acid 0.00 ± 0.0 0.0

Inosine (Not Done) 0.60

Xanthosine 0.0 ± 0.0 0.61

Adenosine (Not Done) 0.68

Guanosine 0.0 ± 0.0 0.00

Deoxyguanosine 0.0 ± 0.0 0.00

The detection of adenine and adenosine by the assay is thought to be due to contaminating deaminases in the xanthine oxidase preparation and/or spontaneous deamination. These reactions can convert adenosine to inosine and adenine to hypoxanthine, a substrate for xanthine oxidase. In another embodiment of the invention, 2'-deoxycorformycin can be included in the reaction mixture to inhibit such activities and provide a more specific assay for xanthine and hypoxanthine. The use of enzyme preparations with greater purity would also solve this problem. If contaminating deaminases are not responsible, it should be possible to alter the reaction conditions to eliminate spontaneous chemical deamination.

Example 4 Detection of Altered Plasma Hypoxanthine levels in Cancer Patients

It has been shown in earlier work that p53 is one of the most frequently mutated genes in human cancer. It is believed that p53 mutations result in altered guanine nucleotide metabolism (Sherley, J. (1991) J.

Biol. Chem. 266:24815-24828; Sherley et al., (1995) Proc. Natl. Acad. Sci. USA 92_.:136-140) . This alteration might be an increase in plasma hypoxanthine due to increased production of guanine nucleotides. Alternatively, decreases may be due to increased tissue utilization of hypoxanthine that results in carcinogenic levels of postulated critical cellular guanine ribonucleotides. Quantitative assays for plasma hypoxanthine will provide insight into the relationship between p53 mutation and altered guanine metabolism. Hypoxanthine is the body's primary currency for intercellular purine transport (Murray, A.W. (1971) Ann. Rev. Biochem. 4.0:811-826) . Hypoxanthine is produced by de novo biosynthesis in the liver, its primary site of production, and then transported to other tissues for utilization in the synthesis of adenine and guanine nucleotides via salvage pathways. The amount of dietary contribution to this primary de novo store is uncertain. However it is likely to be a minor contribution that depends on gut flora enzymes; as purines are not an essential dietary component and nucleic acids, and nucleotides do not freely cross the plasma membrane of cells (Murray, A.W. (1971) , supra ) . Hypoxanthine is transported from the liver primarily in "loaded" erythrocytes to other tissues, some of which do not engage in de novo purine biosynthesis at all. There is limited information available on the relationship between plasma and tissue cell levels of hypoxanthine. However, it is clear that hypoxanthine diffuses freely across the erythrocyte plasma membrane, and as a result, erythrocyte hypoxanthine content is determined by tissue-dependent metabolic gradients. Erythrocytes load with hypoxanthine in the high-hypoxanthine-content capillary beds of the liver and release hypoxanthine in its nucleoside form, inosine, in low-hypoxanthine-content tissue capillary beds (Murray, A.W. , (1971) Ann. Rev. Biochem. 40:811- 826) .

Hypoxanthine is catabolized sequentially by the enzyme xanthine oxidase/dehydrogenase, first to xanthine and then to uric acid. In humans, these two catabolites are excreted in the urine without further utilization (Murray, A.W. , (1971), supra ; Murray et al. , (1970)

Progr. Nucl. Acid Res. Mol. Biol. 10:87-119) . Unlike hypoxanthine, for which there are no clinical tests in common practice, the measurement of serum uric acid concentration is a routine clinical laboratory test. It provides diagnostic information for an array of pathological conditions including renal failure, gout, in-born errors of purine metabolism (e.g., Lesch-Nyhan's Disease), and hyper-catabolic states (e.g., cancer) Serum uric acid has been evaluated as a potential cancer risk marker. However, no significant association has been found (Hiatt et al., (1988) Cancer Research 48:2916- 2918; Kolonel, et al., (1994) Can. Epidem. Biomarkers Prev. 3_:225-228) . Hypoxanthine is arguably a much better candidate guanine nucleotide-related cancer risk factor because of its essential role in guanine nucleotide metabolism. Hypoxanthine has not been previously assessed for this purpose. To assess hypoxanthine levels in cancer patients, plasma samples from 677 individuals were collected from patients enrolled in the Family Risk Assessment Program (FRAP) at the Fox Chase Cancer Center (Philadelphia, PA) . In a number of cases, multiple samples were collected during different stages of disease and/or treatment for the same individual. The subject population was composed primarily of women (<50 male samples) , who had either sporadic or inherited breast and/or ovarian cancer, and their unaffected family members (who were for the most part, sisters and mothers) . The plasma collection included the following numbers of cancer types: breast, 107; ovary, 112; breast and ovary, l; endometrium, 27; cervix, 18; miscellaneous cancers, 34; no known cancer but with a family history of cancer, 318; no known cancer and no family history of cancer, 33. The two most represented cancers in the collection, breast and ovarian, are known to be associated with very frequent p53 mutations (about 50% and 85% respectively) . As noted earlier, this feature is deemed an essential prerequisite to increase the likelihood for success in establishing a significant association with cancer risk.

The colorimetric method of the instant invention, as set forth in Examples 1 and 2, was used to quantify hypoxanthine levels in human plasma. The assay was used to analyze plasma samples from 118 women with no known cancer and 49 ovarian cancer patients. The results obtained demonstrate that the cancer patients had a mean plasma hypoxanthine level that was 33% lower than that of the non-cancer group (p<0.01). This difference was not explained by differences in the age distributions of the two groups. While there are additional factors that must be analyzed in relation to this result, the data are consistent with the hypothesis of increased guanine nucleotide utilization in the tissues of cancer patients. Certain preferred embodiments of the invention have been described and exemplified herein. However, other embodiments will be apparent to persons skilled in the art. For example, the incorporation of other enzymes or particular enzyme inhibitors may allow variation in the purine specificity of the assay. Thus, the invention is not limited to the embodiments specifically described, but may be varied and modified within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for determining the presence or quantity of a selected subpopulation of analytes in a mixed population of analytes, the analyte subpopulation comprising purine metabolites capable of being further metabolized to uric acid, the method comprising: a) providing a liquid sample of the mixed analyte population; b) adding to the sample at least one reagent that catalyzes the conversion of the purine metabolites to uric acid, under conditions whereby the purine metabolites in the subpopulation are converted to uric acid; c) adding to the sample at least one reagent that reacts with uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide, under conditions whereby the stoichiometrically equivalent amount of hydrogen peroxide is produced in the sample; and d) quantitatively detecting the hydrogen peroxide in the sample, the quantity of hydrogen peroxide being proportional to the quantity of the analyte subpopulation in the sample.

2. The method of claim 1, wherein the mixed analyte population is disposed within a biological fluid or tissue and the liquid sample is prepared from the biological fluid or tissue.

3. The method of claim 2, wherein the biological fluid is selected from the group consisting of plasma, urine, seminal fluid, saliva, tissue extract, cell extract and conditioned medium of cultured cells.

4. The method of claim 3, wherein the biological fluid or tissue is plasma.

5. The method of claim 1, wherein the analyte subpopulation comprises purine bases.

6. The method of claim 5, wherein the purine bases are selected from the group consisting of adenine, xanthine and hypoxanthine.

7. The method of claim 5, wherein said at least one reagent that catalyzes the conversion to uric acid includes xanthine oxidase.

8. The method of claim 1, wherein the analyte subpopulation comprises purine nucleosides.

9. The method of claim 8, wherein the purine nucleosides are selected from the group consisting of inosine, xanthosine and adenosine.

10. The method of claim 8, wherein said at least one reagent that catalyzes the conversion to uric acid includes phosphorylase and xanthine oxidase.

11. The method of claim 1, wherein the reagent that reacts with the uric acid is uricase.

12. The method of claim 1, wherein the hydrogen peroxide is detected by adding to the liquid sample at least one reagent that reacts with the hydrogen peroxide to form a proportional amount of an optically detectable product.

13. The method of claim 12, wherein the optically detectable product is a spectrophotometrically detectable product.

14. The method of claim 13, wherein said at least one reagent that reacts with hydrogen peroxide includes a combination of peroxidase, 4-aminoantipyrine and 3, 5- dichloro-2-hydoxybenzenesulfonate, and the spectrophotometrically detectable product is a quinoneimine dye.

15. The method of claim 12, wherein the optically detectable product is a fluorescent product.

16. A method for determining the presence or quantity of purine bases in a biological fluid or tissue, the method comprising: a) providing a liquid test sample of the biological fluid or tissue; b) providing a liquid control sample of the biological fluid or tissue that is volumetrically equivalent to the test sample; c) adding to the test sample an amount of xanthine oxidase effective to catalyze the conversion of the purine bases to uric acid, under conditions whereby the purine bases in the sample are converted to uric acid; d) adding to the control sample a volume of a non-reactive liquid medium equivalent to the volume of xanthine oxidase added to the test sample; e) adding to each of the test sample and the control sample at least one reagent that reacts with uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide, under conditions whereby the stoichiometrically equivalent amount of hydrogen peroxide is produced in each sample; f) quantitatively detecting the hydrogen peroxide in each of the test sample and the control sample, the quantity of hydrogen peroxide being proportional to the quantity of the uric acid in each sample; and g) comparing the difference in quantity of uric acid in the control sample and in the test sample, the difference being proportional to the quantity of purine bases in the biological fluid or tissue.

17. The method of claim 6, wherein the biological fluid is selected from the group consisting of plasma, urine, seminal fluid, saliva, tissue extract, cell extract and conditioned medium of cultured cells.

18. The method of claim 17, wherein the biological fluid or tissue is plasma.

19. The method of claim 16, wherein the purine bases are selected from the group consisting of adenine, xanthine and hypoxanthine.

20. The method of claim 16, wherein the reagent that reacts with the uric acid is uricase.

21. The method of claim 16, wherein the hydrogen peroxide is detected by adding to each sample at least one reagent that reacts with the hydrogen peroxide to form a proportional amount of an optically detectable product.

22. The method of claim 21, wherein the optically detectable product is a spectrophotometrically detectable product.

23. The method of claim 22, wherein said at least one reagent that reacts with hydrogen peroxide includes a combination of peroxidase, 4-aminoantipyrine and 3, 5- dichloro-2-hydoxybenzenesulfonate, and the spectrophotometrically detectable product is a quinoneimine dye.

24. The method of claim 21, wherein the optically detectable product is a fluorescent product.

25. A method for determining the presence or quantity of purine nucleosides in a biological fluid or tissue, the method comprising: a) providing a liquid test sample of the biological fluid or tissue; b) providing a liquid control sample of the biological fluid or tissue that is volumetrically equivalent to the test sample; c) adding to the test sample an amount of a combination of nucleoside phosphorylase and xanthine oxidase effective to catalyze the conversion of the purine nucleosides to uric acid, under conditions whereby the purine nucleosides in the sample are converted to uric acid; d) adding to the control sample a volume of a non-reactive liquid medium equivalent to the volume of nucleoside phosphorylase added to the test sample and an equivalent amount of the xanthine oxidase as added to the test sample; e) adding to each of the test sample and the control sample at least one reagent that reacts with uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide, under conditions whereby the stoichiometrically equivalent amount of hydrogen peroxide is produced in each sample; f) quantitatively detecting the hydrogen peroxide in each of the test sample and the control sample, the quantity of hydrogen peroxide being proportional to the quantity of the uric acid in each sample; and g) comparing the difference in quantity of uric acid in the control sample and in the test sample, the difference being proportional to the quantity of purine nucleosides in the biological fluid or tissue.

26. The method of claim 25, wherein the biological fluid is selected from the group consisting of plasma, urine, seminal fluid, saliva, tissue extract, cell extract and conditioned medium of cultured cells.

27. The method of claim 26, wherein the biological fluid or tissue is plasma.

28. The method of claim 25, wherein the purine nucleosides are selected from the group consisting of inosine, xanthosine and adenosine.

29. The method of claim 25, wherein the reagent that reacts with the uric acid is uricase.

30. The method of claim 25, wherein the hydrogen peroxide is detected by adding to each sample at least one reagent that reacts with the hydrogen peroxide to form a proportional amount of an optically detectable product.

31. The method of claim 30, wherein the optically detectable product is a spectrophotometrically detectable product.

32. The method of claim 31, wherein said at least one reagent that reacts with hydrogen peroxide includes a combination of peroxidase, 4-aminoantipyrine and 3, 5- dichloro-2-hydoxybenzenesulfonate, and the spectrophotometrically detectable product is a quinoneimine dye.

33. The method of claim 29, wherein the optically detectable product is a fluorescent product.

34. A test kit for determining the presence or quantity of a selected subpopulation of analytes in a mixed population of analytes, the analyte subpopulation comprising purine metabolites capable of being further metabolized to uric acid, the kit comprising: a) at least one reagent that catalyzes the conversion of the purine metabolites to uric acid; and b) at least one reagent that reacts with uric acid to produce, as a reaction byproduct, a stoichiometrically equivalent amount of hydrogen peroxide.

35. The test kit of claim 34, which further comprises at least one reagent for detecting the hydrogen peroxide.