Uric acid, from xanthine

Xanthine oxidase, mol wt ca 275,000, present in milk, Hver, and intestinal mucosa (131), is required in the cataboHsm of nucleotides. The free bases guanine and hypoxanthine from the nucleotides are converted to uric acid and xanthine in the intermediate. Xanthine oxidase cataly2es oxidation of hypoxanthine to xanthine and xanthine to uric acid. In these processes and in the oxidations cataly2ed by aldehyde oxidase, molecular oxygen is reduced to H2O2 (133). Xanthine oxidase is also involved in iron metaboHsm. Release of iron from ferritin requires reduction of Fe " to Fe " and reduced xanthine oxidase participates in this conversion (133). 

Figure 34-8. Formation of uric acid from purine nucleosides byway of the purine bases hypoxanthine, xanthine, and guanine. Purine deoxyribonucleosides are degraded by the same catabolic pathwayand enzymes,all of which existin the mucosa of the mammalian gastrointestinal tract.

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

Xanthine oxidase catalyzes the oxidation of hypox-anthine and xanthine to uric acid. Xanthine oxidase is a complex metalloflavoprotein containing one molybdenum, one FAD and two iron-sulfur centers of the ferredoxine type in each of its two independent subunits. Usually, the enzyme is isolated from cow s milk. The enzyme is inhibited by allopurinol and related compounds. The production of uric acid from the substrate (xanthine) can be determined by measuring the change in optical density in the UV range. 

Figure 10-21 Oxidation of Hypoxanthine and Xanthine to Uric Acid by Xanthine Oxidase. Source From J.R. Whitaker, Principles ofEnzymology for the Food Sciences, 1972, Marcel Dekker, Inc.

RP-HPLC procedures for the determination of creatinine and purine metabolites, such as allantoin, uric acid, xanthine, and hypoxanthine in ruminant urine, were described. Chromatography was achieved with a Cig column under isocratic conditions, and detection at 218 nm without allantoin derivatization. The chromatographic conditions were a compromise between the sensitivity and specificity of the measurement of each analyte, analysis time, and resolution of all analyte peaks from interfering compounds.Uremic toxins creatine, creatinine, uric acid, and xanthine were simultaneously determined in human biofluids, simply after dilution, with UV detection at 200 nm. This method was compared, for creatinine and uric acid, with conventional routine methods and did not give significantly different results. 

Allopurinol is a uricosuric drug used in chronic gout that prevents formation of uric acid from purines by acting as a suicide substrate of xanthine oxidase. The drug is commonly used in patients undergoing treatment of cancer to slow down formation of uric acid derived from purines released by the cytotoxic action of drugs or radiation. The metabolism of 6-mercaptopurine (6-MP), a substrate for xanthine oxidase, is also inhibited by allopurinol, necessitating a major dose reduction to avoid its toxic effects. 

Purine is obtained from trichloropurine by treatment with hydriodic acid, which converts it into diiodopurine, and subsequent reduction of the latter by zinc-dust. Purine is of interest as it is the compound from which uric acid and xanthine may be considered as derived by substitution. For convenience of reference the atoms in purine are numbered in the following way —... 

The biosynthesis of uric acid from the immediate purine precursor xanthine that results from adenine, via the intermediate hypoxanthine, or from guanine is illustrated in Figure 36.32. The enzyme xanthine oxidase (a molybdenum hydroxylase enzyme) is involved in two steps, the conversion of hypoxanthine to xanthine, and the final step, the conversion of xanthine to uric acid. Allopurinol originally was designed... 

Adenosine formation is accelerated under hypoxic conditions (Rudolphi et al. 1992), but it is metabohsed only after reperfusion. Now superoxide dismutase and catalase are insufficient to serve detoxification of the reactive oxygen species resulting from the biotransformation of hypoxanthine to xanthine and of xanthine to uric acid. While xanthine oxidase inhibitors as oxypurinol do protect against ischaemic damage (Helfman and Phillis 1989, Lin and Phillis 1992), adenosine deaminase inhibitors as trazodone (Sheid 1985) are expected to reduce the formation of hypoxanthine and xanthine, the substrates for xanthine oxidase. 

Fig. 4. 27. Concentrations of ATP, adenosine (ADO), uric acid (DA), xanthine (X), and hypoxanthine (HX) in experimental rat DS sarcomas as a function of tumor volume. Values are means SEM (from Vaupel 1994a)...

Uric Add Formation. In vertebrates purines are oxidized to uric acid. This reaction is catalyzed by xanthine oxidase (or dehydrogenase), which attacks both hypoxanthine and xanthine.Since adenine and guanine nucleotides can give rise to the hydroxylated purines either as the nucleotide, nucleoside, or free base, all of the naturally occurring purines of animals can be converted to uric acid. Adenine may also be oxidized to 2,8-dihydroxy-4-aminopurine, which is excreted in the urine. The formation of uric acid from any of its precursors is followed conveniently spectrophotometrically (Fig. 29). ... 

Usually, in reversed-phase systems, ascorbic acid is eluted within 3 to 10 min. However, as extracting biological samples there may be compounds that elute far after ascorbic acid. Gradient elution may be needed to shorten the column purification time between injections. Tanishima and Kita (74) used a stepwise increasing methanol concentration to remove uric acid from the column after plasma samples. Lazzarino et al. (88) separated ascorbic acid, uric acid, xanthine, hypoxanthine, malondialdehyde, and several nucleotide derivatives with ion pairing and methanol gradient in a reversed phase system. The method was used for determining those compounds in ischemic-reperfused rat heart. 

An intermediate in the formation of uric acid from purines. It is excreted in the urine in large amounts in the inborn error of metabolism, xanthinuria, a condition in which xanthine stones can be found. It can be detected in urinary stones by its reaction with Ehrlich s diazo reagent to give a red colour. 

Amino[2- C]uracil (301. accessible from ethyl cyanoacetate and [2- C]urea as previously mentioned, is one of the key intermediates in the Traube-type synthesis of labeled uric acid and xanthine, whose methyl homologues are important biologically active natural products. Nitrosation at C5 with nitrous acid and reduction of the nitroso group with sodium dithionate installed the 5-amino group. Heating of the resulting 5,6-[2- C]diaminouracil (441 in formamide or formic acid resulted in the initial formation of the iV-formamide at C5, which cyclized with elimination of water to afford [2- C]xanthine (451 ". (V-methylation with dimethyl sulfate in basic medium under adequate reaction conditions enabled the separate preparation of [2- C]theobromine ... 

Figure 9.12 Synthesis of [2d C]xanthine and [2- C]uric acid from [2- " C]urea...

Ureides are a class of cyclic or acyclic nitrogenous organic compounds derived from or structurally related to urea. Representatives of this class which have been found in plants include allantoin, allantoic acid, citrulline, uric acid, hypoxanthine, xanthine, caffeine, hydroxycitruUine, and albizziine. The structures of allantoin (ALN), allantoic acid (ALA), and citrulline (CIT), ureides which occur throughout the plant kingdom, are presented in Fig. 1. These ureido compounds play an essential role in the assimilation, metabolism, transport, and storage of nitrogen in plants. In this chapter, attention will be focused on the occurrence, function, synthesis, and metabolism of these key metabolites. 

Anti-gout Drugs. Figure 1 Xanthine oxidase-catalyzed reactions. Xanthine oxidase converts hypoxanthine to xanthine and xanthine to uric acid, respectively. Hypoxanthine and xanthine are more soluble than uric acid. Xanthine oxidase also converts the uricostatic drug allopurinol to alloxanthine. Allopurinol and hypoxanthine are isomers that differ from each other in the substitution of positions 7 and 8 of the purine ring system. Although allopurinol is converted to alloxanthine by xanthine oxidase, allopurinol is also a xanthine oxidase inhibitor. Specifically, at low concentrations, allopurinol acts as a competitive inhibitor, and at high concentrations it acts as a noncompetitive inhibitor. Alloxanthine is a noncompetitive xanthine oxidase inhibitor. XOD xanthine oxidase. 

Xanthine dehydrogenase that mediates the conversion of hypoxanthine into xanthine and uric acid has been studied extensively since it is readily available from cow s milk. It has also been studied (Leimkiihler et al. 2004) in the anaerobic phototroph Rhodobacter capsulatus, and the crystal structures of both enzymes have been solved. Xanthine dehydrogenase is a complex flavoprotein containing Mo, FAD, and [2Fe-2S] redox centers, and the reactions may be rationalized (Hille and Sprecher 1987) ... 

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