Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine

An HPLC procedure developed for the rapid and simultaneous determination of purine derivatives (PD) in ruminants' urine was investigated, since the adoption of a single method for the simultaneous detection of PD and creatinine was not carried out due to elution of polar co-extractives and also due to overlapping of the peaks of allantoin and creatinine. The experimental conditions chosen in the present study avoid the presence of chemically competitive compounds and afford a good separation of the peaks of allantoin and creatinine. The recoveries of the standard compounds added to urine samples were 94-104%. This method can be proposed as a possible reference method for the estimation of allantoin, uric acid and creatinine in cattle urine.     

Uric acid deposits and estivation in the invasive apple-snail, Pomacea canaliculata

The physiological ability to estivate is relevant for the maintenance of population size in the invasive Pomacea canaliculata. However, tissue reoxygenation during arousal from estivation poses the problem of acute oxidative stress. Uric acid is a potent antioxidant in several systems and it is stored in specialized tissues of P. canaliculata. Changes in tissue concentration of thiobarbituric acid reactive substances (TBARS), uric acid and allantoin were measured during estivation and arousal in P. canaliculata. Both TBARS and uric acid increased two-fold during 45 days estivation, probably as a consequence of concomitant oxyradical production during uric acid synthesis by xanthine oxidase. However, after arousal was induced, uric acid and TBARS dropped to or near baseline levels within 20 min and remained low up to 24h after arousal induction, while the urate oxidation product allantoin continuously rose to a maximum at 24h after induction, indicating the participation of uric acid as an antioxidant during reoxygenation. Neither uric acid nor allantoin was detected in the excreta during this 24h period. Urate oxidase activity was also found in organs of active snails, but activity shut down during estivation and only a partial and sustained recovery was observed in the midgut gland.     

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after treatment with oxonic acid and allopurinol

In our previous experiments on rat liver we found that 15' after intraperitoneal administration of 14C-formate the specific radioactivity of allantoin was always higher than that of uric acid. The present experiments have been carried out to interpret this unexpected result, which was only observed in liver and we studied: a) the incorporation of 14C-glycine into uric acid and allantoin; b) the effects of two competitive inhibitors of xanthine oxidase and uricase, oxonic acid and allopurinol respectively, on levels of uric acid and allantoin in liver and on their specific radioactivity after administration of labelled precursor. The results suggested: a) that under normal conditions, the formation of allantoin is so fast that it exceedes export from liver to serum, and thus the radioactivity of labelled precursors accumulates in allantoin; b) that when allopurinol or oxonic acid are administered, the rate of export exceeds that of allantoin formation and the incorporation of radioactivity into allantoin is lower; c) that not all the data, however, could be interpreted on this basis, but seems to require the existence of different pools of uric acid, which are transformed separately into allantoin.     

Determination of uric acid by an anion-exchange column chromatography

A column chromatography using a conventional anion-exchange resin for the separation of uric acid from other purine metabolites is described. It uses a HCl gradient, and the amount of uric acid is quantified directly by monitoring the absorbance of the effluent at 285 nm. The linear range of response is 0.5 to 100 nmol. The method was applied to the analysis of uric acid in urine and serum. Urine was injected directly into the system, while serum required removal of an interfering substance which absorbs the light and coelutes with uric acid. However, this substance was simply removed by heat coagulation of serum by heating in a boiling water bath for 2 min.     

New insights into antioxidant strategies against paraquat toxicity

Free radicals are implicated in many diseases including atherosclerosis, cancer and also in rheumatoid arthritis. Reaction of uric acid with free radicals, such as hydroxyl radical and hypochlorous acid (HOCl) results in allantoin production. In this study, we measured the serum allantoin levels, oxidation products of uric acid, as a marker of free radical generation in rheumatoid arthritis. Fasting blood samples were obtained from 21 rheumatoid patients and 15 healthy controls. In this study, the serum allantoin and uric acid levels were measured by a gas chromatography–mass spectrometry method and the ratios were calculated. The mean allantoin and uric acid levels and ratios in the patient group were 22.1±11.3, 280.5±65.0 and 8.0±3.7?μM, while in the control group they were 13.6±6.3, 278.3±53.6 and 4.9±2.1?μM, respectively. The effects of gender, age, menopausal status, duration of disease and medications on serum allantoin and uric acid levels of the patient and control groups were studied. Our results suggest that uric acid acts as a free radical scavenger and thus is converted to allantoin. Increased allantoin levels suggest the possible involvement of free radicals in rheumatoid arthritis.     

Oxidation of uric acid in rheumatoid arthritis: is allantoin a marker of oxidative stress?

Free radicals are implicated in many diseases including atherosclerosis, cancer and also in rheumatoid arthritis. Reaction of uric acid with free radicals, such as hydroxyl radical and hypochlorous acid (HOCl) results in allantoin production. In this study, we measured the serum allantoin levels, oxidation products of uric acid, as a marker of free radical generation in rheumatoid arthritis. Fasting blood samples were obtained from 21 rheumatoid patients and 15 healthy controls. In this study, the serum allantoin and uric acid levels were measured by a gas chromatography-mass spectrometry method and the ratios were calculated. The mean allantoin and uric acid levels and ratios in the patient group were 22.1±11.3, 280.5±65.0 and 8.0±3.7 μM, while in the control group they were 13.6±6.3, 278.3±53.6 and 4.9±2.1 μM, respectively. The effects of gender, age, menopausal status, duration of disease and medications on serum allantoin and uric acid levels of the patient and control groups were studied. Our results suggest that uric acid acts as a free radical scavenger and thus is converted to allantoin. Increased allantoin levels suggest the possible involvement of free radicals in rheumatoid arthritis.     

The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds

Uric acid is the main nitrogenous waste product in birds but it is also known to be a potent antioxidant. Hominoid primates and birds lack the enzyme urate oxidase, which oxidizes uric acid to allantoin. Consequently, the presence of allantoin in their plasma results from non-enzymatic oxidation. In humans, the allantoin to uric acid ratio in plasma increases during oxidative stress, thus this ratio has been suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of white-crowned sparrows (Zonotrichia leucophrys gambelii) at rest, immediately after 30 min of exercise in a hop/hover wheel, and after 1 h of recovery. The plasma allantoin concentration and the allantoin to uric acid ratio did not increase during exercise but we found a positive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine in the three activity phases. In the plasma, the slope of the regression describing the above positive relationships was significantly higher immediately after activity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rate increases as a result of higher production of free radicals. The present study demonstrates that allantoin is present in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be useful as an indicator of oxidative stress in birds.     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

Evidence that the slow conformation change controlling NADH release from the enzyme is rate-limiting during the oxidation of propionaldehyde by aldehyde dehydrogenase.

Free-radical attack upon uric acid generates allantoin [Ames, Cathcart, Schwiers & Hochstein (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6858-6862]. Methods are described for the accurate measurement of uric acid and allantoin in human body fluids. The concentrations of uric acid and allantoin in human serum and synovial fluid are reported. It is suggested that measurement of changes in allantoin concentration may be a useful index of free-radical reactions taking place in vivo.     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

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.     

Allantoin and allantoic acid titre in the faeces and tissues of the developing larva of the moth, Orthaga exvinacea Hampson

Allantoin and allantoic acid are investigated in the faeces and tissues of the developing sixth instar larva of the moth, Orthaga exvinacea. The nitrogen excreted as allantoin and allantoic acid is compared with nitrogen excreted as uric acid and ammonia. The larva excretes 2.35–5.14 μmol/g allantoin and 0.74–1.34 μmol/g allantoic acid which account for 0.83 to 2.39% and 0.23 to 0.53%, respectively, of the excreted total nitrogen. Allantoin and allantoic acid are found to be minor nitrogenous end-products of the larva. Allantoin and allantoic acid are also present in the haemolymph and fat body of the larva in varying concentrations. The level of allantoin in the haemolymph shows a negative correlation with the allantoin concentration of faeces and fat body. The allantoin is found to be stored in the fat body at a low level. The results of the present study also indicate the coexistence of uric acid storage and uricolysis.     

A problem on the uric acid value of rats for clinical evaluation

Routine monitoring on levels of serum uric acid in the rats has been widely employed as an important factor in the nucleic acid metabolism, despite of the presence of the uricase in them. In this paper, it is confirmed that the levels of allantoin in serum and urine of the normal rats were higher than those of uric acid. Therefore, the concentration of allantoin in serum and urine of the rats with abnormal nucleic acid metabolism caused by adenine administration were measured. The results indicated that the value of serum allantoin was more sensitive to abnormal nucleic acid metabolism.     

A new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate

A new spectrophotometric assay method of xanthine oxidase applicable to the crude tissue homogenate containing uricase was presented in this paper. By adding potassium 2,4-dihydroxy-6-carboxy-1,3,5-triazine (potassium oxonate) (0.1 mm) to the crude xanthine oxidase reaction system, uric acid was stoichiometrically formed from xanthine and detectable allantoin was not formed and the formation of uric acid was not influenced by uricase.Distribution of xanthine oxidase in various rat tissues was measured by this method, and it was shown that the activity was high in the liver, the small intestine, and the spleen. Uricase was shown to distribute mainly in the liver of rats.     

Quantification and kinetics of purine catabolism in Dalmatian dogs at low and high purine intakes

1. In eight Dalmatian dogs low and high purine intakes resulted in plasma urate levels from 25 to 185 mumol/l. 2. The relationship between purine intake and excretion of uric acid and allantoin per day was described by linear regression equations. 3. The elimination of endogenous purines was 1.8 mmol/day for urate and 1.7 mmol/day for allantoin. Exogenous purines increased renal excretion by 0.57 mmol/mmol. 4. Kinetic measurements with [2(-14)C]uric acid infused continuously into each of two dogs on low and high purine revealed increases of plasma pool (urate + allantoin) of 3.3 fold and entry rate of 4.0 fold. Conversion of urate into allantoin increased from 20 to 36%. 5. Renal elimination of catabolites increased 3.3 fold and exhalation rate of purine-CO2 379 fold. Extra-renal elimination at high purine intake was quantitatively similar to humans and closely related to pool size.     

Identification of Products from Oxidation of Uric Acid Induced by Hydroxyl Radicals

The aim of the present study was to separate and characterise products formed by oxidation of uric acid by hydroxyl radicals with a view to probing for these products in vivo in clinical contexts. Aerated solutions of 200 μM uric acid, or its oxidation products, allantoin or parabanic acid, were exposed to gamma radiolysis, (52.0 Gy/min), as a source of HO- radicals, at pH 3.4 and 7.4. Aliquots were taken every 5 minutes for 20 minutes and oxidation products were separated by HPLC and analysed with a diode array detector. Identities of oxidation products were confirmed on the basis of similarity of retention times and absorbance spectra and peak purity parameters of known standards. Hydroperoxides were measured by tri-iodide formation in the 20 minute sample. Exposure of uric acid to such HO fluxes produced a net loss of the parent compound with formation of a complex mixture of products with allantoin and parabanic acid being the predominant products at pH 3.4. The rate of uric acid degradation at physiological pH was slower and the distribution of oxidation products was different. A small but significant amount of uric acid hydroperoxide was detected at both pHs. A mechanism for uric acid oxidation under these conditions is presented.     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

Uric acid and allantoin uptake by Bacillus fastidiosus spores

Uric acid enters Bacillus fasitidiosus spores by a constitutive carrier-mediated mechanism. The extent of uptake was proportional to the external uric acid concentration up to the limit of solubility. Most of the uric acid taken up after 2 min of incubation was not exchangeable with cold uric acid, suggesting that the uric acid was being quickly metabolized. Allantoin (an uric acid degradation product) was not incorporated by spores unles they were triggered to germinate with uric acid and the induced by allantoin. The induction of this uptake system was inhibited by chloramphenicol. The inability of spores to germinate in the sporulation medium was found to be due to the high pH of the sporulation medium after growth and sporulation.