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.     

Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C₁₈ columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at −10°C. The low values of the intra-assay coefficient of variations (2.5–4.6%) and the low values of the detection limits (0.187–0.004 nmol) point to the satisfactory precision and sensitivity of the method.     

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.     

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.     

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 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.     

Hypoxanthine Transport and Metabolism in the Central Nervous System

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypoxanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [14C]hypoxanthine; however, 14C was readily released from choroid plexus principally as [14C]-hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid-soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [14C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C-nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.     

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Purine catabolism in advanced carotid artery plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

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.     

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.     

Effect of allopurinol on the utilization of purine degradation pathway intermediates by tobacco cell cultures

Suspension cultured Nicotiana tabacum (tobacco) cells grow slowly on intermediates of the purine degradation pathway (hypoxanthine, xanthine, uric acid, allantoin, and urea) as their sole nitrogen source indicating that this degradation pathway is operative in these cells. The hypoxanthine analog, allopurinol inhibited tobacco cell growth on hypoxanthine but not uric acid. This helps confirm that the site of action of allopurinol is the conversion of hypoxanthine to uric acid by xanthine oxidase. Attempts to select cells which could grow in the presence of allopurinol with hypoxanthine as the nitrogen source were not successful.     

Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos.

The importance of de novo purine synthesis as opposed to the reutilisation of metabolites by salvage pathways, and the nature of the excretory product(s) of purine degradation, have been examined in cultured preimplantation mouse embryos. In the presence of azaserine and mycophenolic acid, which inhibit de novo purine synthesis, embryo cleavage was blocked prior to compaction, the precise stages at which this occurred depended on whether the cultures were established on day 1 or day 2 after fertilisation, and indicated that salvage pathways were insufficient to fulfil the demand for nucleotides during early preimplantation development. The end-product of purine degradation appeared to be xanthine, which was excreted in very small amounts on days 1, 2 and 3, with a pronounced rise from the early to late blastocyst. Uric acid formation or excretion could not be detected. Exogenous hypoxanthine and adenine, which partially inhibited development, were taken up by the embryos and converted to xanthine, most probably by salvage pathways, since the enzyme xanthine oxidase, which converts hypoxanthine directly to xanthine and then to uric acid, could not be detected. Exogenous guanine had little effect on development and was also converted to xanthine, but in this case, the conversion was probably in a single step, via the enzyme guanase.     

Absorption and metabolism of purines by the small intestine of the chicken.

1. Absorption of purines and their metabolism by the small intestine were estimated by using the everted gut sacs from the duodenum, jejunum and ileum of the chicken. 2. When no purine was added to the mucosal fluid, large amounts of uric acid, much less but appreciable adenine, hypoxanthine and xanthine and no detectable guanine were released from both sides of all segments of the small intestine, and these released amounts were largest in the duodenum. 3. Similar absorption rates of adenine from the jejunum and ileum were about 1.7-3.0 times as high as those of hypoxanthine and uric acid from these intestines and those of adenine and uric acid from the duodenum (P less than 0.05). 4. Guanine was not absorbed unchanged from any segments of the intestine and a little xanthine was absorbed only from the jejunum and ileum. 5. Guanine and xanthine seem to be absorbed in uric acid form, hypoxanthine in xanthine and uric acid forms and adenine in hypoxanthine form, from the small intestine especially from the jejunum. 6. Adenine, guanine, xanthine and hypoxanthine were greatly metabolized in the mucosa of the duodenum, and the conversions of hypoxanthine to xanthine and uric acid were most active.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Regulation of purine hydroxylase and xanthine dehydrogenase from Clostridium purinolyticum in response to purines, selenium, and molybdenum

The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by Clostridium purinolyticum was unexpected. In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH. PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously. In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates. The levels of PH protein in extracts were determined by immunoblot assay. The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium. PH levels were highest in the cells cultured in the presence of either adenine or purine. XDH activity increased dramatically in cells grown with either xanthine or uric acid. The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients.