Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5'-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the 'de novo' pathway or (from unchanged hypoxanthine) by ther salvage pathway.     

Analysis of purines in urinary calculi by high-performance liquid chromatography

A high-pressure liquid chromatography method has been developed for the analysis in urinary calculi of six purines: uric acid, 2, 8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol, and oxypurinol. Separation was conducted isocratically on a reversed-phase column, using 50 mM phosphate buffer (pH 5.5) / methanol (97/3, v/v) as mobile phase. Limits of detection, depending on compound, ranged from 7 to 28 microg/g stone weight. Hitherto, no reports have appeared on other purines present with uric acid in stones, due to lack of a sensitive and specific analytical method. We have now found that all calculi with more than 4% uric acid also contained 1-methyluric and 7-methyluric acids and trace amounts of hypoxanthine, xanthine, and 2,8-dihydroxyadenine. Accurate identification and quantitation of purines in urinary calculi are important for the diagnosis of rare metabolic diseases leading to urolithiasis (xanthinuria, dihydroxyadeninuria), as well as for prevention of iatrogenic complications during treatment with allopurinol of uric acid urolithiasis. The method may be used for reference purposes in clinical laboratories and for research on the pathogenesis of urolithiasis in disorders of purine metabolism.     

Simultaneous determination of inosine, hypoxanthine, xanthine, and uric acid and the effect of metal chelators

We describe a sensitive, reproducible method for the simultaneous determination of the ATP catabolites inosine, hypoxanthine, xanthine, and uric acid in biological samples and organ perfusate using reverse-phase chromatography and multiwavelength detection at 254, 270, and 292 nm. Sample preparation includes precipitating proteins with perchloric acid, neutralizing the sample, passing the supernatant over a polyethyleneimine column, and analyzing the collected fractions by high-performance liquid chromatography. Addition of metal chelators to the perchloric acid resulted in increased values for xanthine, hypoxanthine, and uric acid. The method was sensitive (limit of detection, 0.08 nmol on column; S/N = 4) and linear over the range 0.5-30 microM. Precision and accuracy of the method were evaluated for lung tissue and lung perfusate. Coefficients of variation ranged from 2.8 to 6.1% for perfusate and from 1.7 to 12.6% for tissue. Recoveries for all compounds exceeded 90%. We applied this method to rat lung tissue, lung perfusate, and rat and human blood. Advantages of this method are simultaneous quantitation with excellent sensitivity of all compounds, simplified peak identification by using multiwavelength detection, and improved accuracy by preventing loss of compounds with metal chelators.     

Identification of a new point mutation in the human xanthine dehydrogenase gene responsible for a case of classical type I xanthinuria

A 60-year-old Japanese man was diagnosed as having hypouricemia at an annual health check-up. The routine laboratory data was not remarkable except that the patient's hypouricemia and plasma levels of xanthine and hypoxanthine were much higher than those of normal subjects. Furthermore, the patient's daily urinary excretion of xanthine and hypoxanthine was markedly increased compared with reference values. The xanthine dehyrogenase activity of the duodenal mucosa was below the limits of detection. Nevertheless, allopurinol was metabolized to oxypurinol in vivo. Based on these findings, a subtype of classical xanthinuria (type I) was diagnosed. The xanthine dehyrogenase protein was detected by Western blotting analysis. Sequencing of the cDNA of the xanthine dehyrogenase obtained from the duodenal mucosa revealed that a point mutation of C to T had occurred in nucleotide 445. This changed codon 149 from CGC (Arg) to TGC (Cys), a finding that has not been previously reported in patients with classical xanthinuria type I.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.     

High-performance liquid chromatographic determination of hypoxanthine and xanthine in biological fluids

A rapid and selective reversed-phase high-performance liquid chromatographic method for the simultaneous determination of hypoxanthine and xanthine in biological fluids was developed. The identification of hypoxanthine and xanthine was confirmed by xanthine oxidase reaction. This method was applied to the investigation of purine metabolism in subjects with xanthine oxidase deficiency or gout. Hypoxanthine concentrations three to ten times higher than those determined in plasma were found in erythrocyte samples from normal subjects and from patients with xanthine oxidase deficiency or hyperuricemia under allopurinol therapy.     

Effects of long-term beer ingestion on plasma concentrations and urinary excretion of purine bases.

To investigate the long-term effects of beer ingestion on plasma concentrations of purine bases (hypoxanthine, xanthine, and uric acid), ten healthy males ingested beer (15 ml/kg body weight) every evening for three months. Blood and 24-hour urine samples were collected in the morning on one day before and one, two, and three months after starting the experiment to determine the plasma concentrations and urinary excretion of uric acid, hypoxanthine, and xanthine. Plasma concentrations and urinary excretion of uric acid, hypoxanthine, and xanthine in five of the participants that did not regularly ingest beer at a quantity of more than 15 ml/kg body weight in a single day prior to the experiment were not increased during the experimental period. In contrast, plasma concentrations and urinary excretion of uric acid were increased in five participants who regularly ingested more than 15 ml/kg body weight of beer in a single day prior to the experiment, although hypoxanthine and xanthine levels were not significantly increased during the experimental period. In both groups, uric acid clearance and purine ingestion were not significantly different throughout the study. Our results suggest that the production of uric acid caused by ethanol ingestion from beer is a significant contributor to the increase in plasma uric acid concentration in patients that regularly consume more than 15 ml/kg body weight of beer each day. Therefore, patients with gout should be encouraged to refrain from drinking large amounts of beer on a daily basis.     

Effect of purine-free low-malt liquor (happo-shu) on the plasma concentrations and urinary excretion of purine bases and uridine--comparison between purine-free and regular happo-shu.

To determine whether purine-free and regular low-malt liquor beverages (happo-shu) increase the plasma concentration and urinary excretion of purine bases (hypoxanthine, xanthine, uric acid) and uridine, 6 healthy males were given regular (10 ml/kg of body weight) and purine-free happo-shu (10 ml/kg of body weight). Plasma concentration-time curves were plotted, and the areas under the curves for uric acid and total purine bases (the sum of hypoxanthine, xanthine, and uric acid) were greater in the regular than in the purine-free happo-shu ingestion experiment (both p < 0.05). In addition, the total urinary excretion of xanthine, total purine bases, and uridine was greater in the regular than in the purine-free happo-shu ingestion experiment (p < 0.05 in all cases), although the total urinary excretion of hypoxanthine and uric acid was no different between the regular and the purine-free happo-shu ingestion experiments. These results suggest that uridine contained in regular happo-shu might contribute to an increase in the urinary excretion of uridine along with ethanol, and that the purines contained in regular happo-shu may contribute to the increase in plasma concentration of uric acid due to purine degradation.     

Identification of xanthine and hypoxanthine as components of assembly pheromone in excreta of argasid ticks

In addition to guanine, xanthine and hypoxanthine were identified in white spherules in excreta of five species ofArgas andOrnithodoros ticks by a reverse-phase high-performance liquid chromatography (HPLC) and a gas chromatographic method with mass spectrometric detection, (GC/MS). The mutual relationships of these purines in excreta ofArgas (Persicargas) persicus were found to be less than 1.5% for hypoxanthine, less than 9.0% for xanthine and 89.8–98.6%, for guanine. In excreta of other species, the relationships of purines were similar, with the exception ofArgas (A.) reflexus andA. (A.) polonicus, where the amount of hypoxanthine was rather elevated. Uric acid was also identified in some cases. The assembly efficacy of xanthine and hypoxanthine is similar to that of guanine, but xanthine significantly enhances the assembly efficacy of commercial guanine when mixed in ratio of about 125. Thus, xanthine seems to be the second important component of assembly pheromone of argasid ticks.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Distinction between Hypoxanthine and Xanthine Transport in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these purines.     

Febuxostat (TMX-67), a novel, non-purine, selective inhibitor of xanthine oxidase, is safe and decreases serum urate in healthy volunteers

In order to evaluate the safety, pharmacological properties, and urate-lowering efficacy of febuxostat, a non-purine, selective inhibitor of xanthine oxidase, a Phase 1, 2-week, multiple-dose, placebo-controlled, dose-escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self-limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.     

Analysis of uric acid and oxypurines in normal subjects and in gout patients

The behavior of plasma and urine oxypurines (hypoxanthine and xanthine) and of uric acid has been studied in normal subjects and in gout patients. Oxypurines and uric acid were increased in the plasma of gout patients but only the urinary excretion of hypoxanthine was higher in this group. The interpretation of the observed variations is discussed.     

Isolation of hypoxanthine from calf brain

E xcept for the crystalline deposits of hypoxanthine found in skeletal muscle of patients with congenital xanthine oxidase deficiency (xanthinuria) there are few reports concerning oxypurines in the mammalian tissues (P arker , S nedden and W atts , 1969, 1970). Since it is difficult to separate hypoxanthine from xanthine in biological fluids (S immonds and W ilson , 1967), the distribution of hypoxanthine in mammalian tissues is not known in detail.
This paper shows that cubic or rod crystals of C₅H₄ON₄are easily isolated from calf brain by column chromatography with ion exchange resin Amberlite CG120 and their identity with hypoxanthine was shown by means of nuclear magnetic resonance and mass Spectrometry.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Febuxostat (TMX‐67), a Novel,Non‐Purine,Selective Inhibitor of Xanthine Oxidase,Is Safe and Decreases Serum Urate in Healthy Volunteers

In order to evaluate the safety, pharmacological properties, and urate‐lowering efficacy of febuxostat, a non‐purine, selective inhibitor of xanthine oxidase, a Phase 1, 2‐week, multiple‐dose, placebo‐controlled, dose‐escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self‐limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.     

Chemical diagnosis of Lesch-Nyhan syndrome using gas chromatography-mass spectrometry detection

Lesch-Nyhan syndrome (LNS) is caused by a severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and clinically characterized by self-injurious behavior and nephrolithiasis; the latter is treatable with allopurinol, an inhibitor of xanthine oxidase which converts xanthine and hypoxanthine into uric acid. In the HPRT gene, more than 200 different mutations are known, and de novo mutation occurs at a high rate. Thus, there is a great need to develop a highly specific method to detect patients with HPRT dysfunction by quantifying the metabolites related to this enzyme. A simplified urease pretreatment of urine, gas chromatography-mass spectrometry, and stable isotope dilution method, developed for cutting-edge metabonomics, was further applied to quantify hypoxanthine, xanthine, urate, guanine and adenine in 100 microl or less urine or eluate from filter-paper-urine strips by additional use of stable isotope labeled guanine and adenine as the internal standards. In this procedure, the recoveries were above 93% and linearities (r(2)=0.9947-1.000) and CV values (below 7%) of the indicators were satisfactory. In four patients with proven LNS, hypoxanthine was elevated to 8.4-9.0 SD above the normal mean, xanthine to 4-6 SD above the normal mean, guanine to 1.9-3.7 SD, and adenine was decreased. Because of the allopurinol treatment for all the four patients, their level of urate was not elevated, orotate increased, and uracil was unchanged as compared with the control value. It was concluded that even in the presence of treatment with allopurinol, patients with LNS can be chemically diagnosed by this procedure. Abnormality in the levels of hypoxanthine and xanthine was quite prominent and n, the number of standard deviations above the normal mean, combined for the two, was above 12.9.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Effects of allopurinol on beer-induced increases in plasma concentrations and urinary excretion of purine bases (uric acid, hypoxanthine, and xanthine).

To determine the effects of allopurinol on beer-induced increases in plasma and urinary excretion of purine bases (hypoxanthine, xanthine, and uric acid), we performed three experiments on five healthy study participants. In the first experiment (combination study), the participants ingested beer (10 ml/kg body weight) eleven hours after taking allopurinol (300 mg). In the second experiment (beer-only study), the same participants ingested beer (10 ml/kg body weight) alone, while in the third experiment (allopurinol-only study), they took allopurinol (300 mg) alone. There was a two-week interval between each of the studies. Beer-induced increases in plasma concentration and urinary excretion of hypoxanthine in the combination study were markedly higher than those in the beer-only study. On the other hand, the sum of increases in plasma concentrations of purine bases in the beer-only study was greater than in the combination study, whereas the increase in plasma uridine concentration in the combination study did not differ from the beer-only study. In addition, allopurinol administration inhibited the beer-induced increase in plasma concentration of uric acid. These results suggest that abrupt adenine nucleotide degradation may increase plasma concentration and urinary excretion of hypoxanthine under conditions of low xanthine dehydrogenase activity, which is mostly ascribable to allopurinol. Further, the difference in the sum of increases in plasma concentrations of purine bases between the combination study and beer-only study was largely ascribable to a greater increase in urinary excretion of hypoxanthine in the combination study. In addition, allopurinol intake seems to be effective in controlling the rapid increase in plasma uric acid caused by ingestion of alcoholic beverages.     

Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor

The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor. Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C. Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells. Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography. The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography. Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5. The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP). At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively. The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine.