Allantoin Production Research Articles

Computer-assisted in vitro reconstitution of purine degradation pathway to lower the purine content in food.

With the increasing prevalence of gout and its etiological hyperuricemia, dietary control of gout based on low-purine food according to patients' eating habits is becoming a better choice compared to the existing drug treatment such as allopurinol with notorious side effects. Reconstructing the purine metabolic pathway in vitro to degrade purine substances in food into natural functional allantoin appears to be an innovative method for preparing nutritious and healthy food of low purine content. The present study reports a computer-assisted in vitro reconstruction of four purinolytic enzymes metabolizing adenosine into allantoin to reduce purine content in food for personalized dietary control of hyperuricemia and gout. Under the optimum reaction conditions of 40 °C and pH7, 0.1U of enzymes and 0.5 mmol L-1 adenosine determined by an orthogonal test design, 16 different enzyme complexes were experimentally tested. The tested enzyme composition and allantoin production values were used as input and output to build a three-layer back propagation artificial neural network (BP-ANN) model, which was further optimized by a genetic algorithm (GA). The optimum enzyme complex predicted by the GA-BP-ANN model produced 248.08±7.832 μmol L-1 allantoin, which was 19.9% higher than equimolar mixture of enzymes, and also more efficiently lowered purine contents in beer, as well as beef and yeast extracts. This is the first in vitro reconstitution of complete purine metabolic pathway by combining ANN and GA technologies, with successful application with respect to lowering the purine content in food, indicating a promising application of computer-assisted in vitro reconstitution of purinolytic pathway in low-purine food to prevent hyperuricemia and gout. © 2022 Society of Chemical Industry.

Biosynthesis of allantoin in Escherichia coli via screening a highly effective urate oxidase.

Allantoin is an important fine chemical that can be widely used in pharmaceutical, cosmetic and agricultural industries. Currently, allantoin is mainly produced by plant extraction or chemical synthesis. Due to the cost and environmental concerns, biosynthesis of allantoin from renewable feedstock is much more desirable. However, microbial production of allantoin from simple carbon sources has not yet been achieved so far. In this study, de novo biosynthesis of allantoin was achieved by constructing an artificial biosynthetic pathway. First, screening of efficient urate oxidases and xanthine dehydrogenases enabled allantoin production from hypoxanthine, a natural intermediate in purine metabolic pathway in Escherichia coli. Then, assemble of the entire pathway resulted in 13.9 mg/L allantoin from glucose in shake flask experiments. The titer was further improved to 639.8 mg/L by enhancing the supply of the precursor, redistribution of carbon flux, and reduction of acetate. Finally, scale-up production of allantoin was conducted in a 1-L fermentor under fed-batch culture conditions, which enabled the synthesis of 2360 mg/L allantoin, representing a 170-fold increase compared with the initial strain. This study not only demonstrates the potential for industrial production of allantoin, but also provides a bacterial platform for synthesis of other purines-derived high-value chemicals.

Allantoin reduces cell death induced by cisplatin: possible implications for tumor lysis syndrome management.

Massive lysis of tumor mass in cancer patients under chemotherapy regimens generates high levels of uric acid, leading to what is known as tumor lysis syndrome (TLS). Rasburicase, a recombinant urate oxidase, converts urate to allantoin, which is readily excreted by the kidneys. Even though there is a high production of allantoin from urate in cancer patients following rasburicase treatment, there are no studies on how allantoin excess could interfere with chemotherapy. We have evaluated allantoin interference with cisplatin efficiency on the lung cancer cell line H460 in vitro. The cells were treated with cisplatin (33µM), with or without allantoin, for 48h, in the presence or absence of UV light, and N-acetyl-L-cysteine (NAC) for 24h. Cell viability, cell cycle, ROS production, apoptosis and immunoblot assays were performed. We showed that allantoin reduced the apoptosis induced by cisplatin in the H460 cell line. However, the activity of carboplatin and oxaliplatin, betulinic acid, TIBA, UV and H2O2 was not affected by allantoin. NMR spectroscopy showed that allantoin reduces cisplatin activity through direct interaction with cisplatin.

Urate Oxidase produced by Lucilia sericata medical maggots is localized in Malpighian tubes and facilitates allantoin production

Lucilia sericata maggots are the only species currently approved for maggot debridement therapy (MDT), an alternative treatment for chronic and recalcitrant wounds. Maggots promote wound debridement, disinfection and healing by producing a complex mixture of proteins, peptides and low-molecular-weight compounds in their secretions and excretions, but the individual components are not well characterized at the molecular level. Here we investigated the purine catabolism pathway in L. sericata, focusing on the production of allantoin by Urate Oxidase (UO), which is thought to promote wound healing. We produced recombinant L. sericata UO in Escherichia coli, and characterized the properties of the pure enzyme in terms of the optimum pH (7–10) and temperature (20–25 °C), its stability, sensitivity to inhibition and ion dependency. We used quantitative RT-PCR and RNA in situ hybridization to monitor the expression of the UO gene, and we used a guinea pig anti-UO antibody to detect the native enzyme by western blot and by florescence immunohistochemistry in larval tissues. We found that L. sericata UO is exclusively present in the larval excretion organ (the Malpighian tubes) and is freely available in the cytoplasm rather than restricted to a specific subcellular compartment. Allantoin is a final product of L. sericata purine catabolism. It is produced by UO in the Malpighian tubes to remove uric acid from the hemolymph and is consequently excreted via the hindgut. Our findings confirm the hypothesis that both actively secreted molecules and excretion products contribute to the beneficial effects of MDT.

Allantoin Increases Cadmium Tolerance in Arabidopsis via Activation of Antioxidant Mechanisms.

Plants apply various molecular, physiological and morphological strategies in response to undesirable environmental conditions. One of the possible responses which may contribute to surviving stressful conditions is the accumulation of ureides. Ureides are recognized as important nitrogen-rich compounds involved in recycling nitrogen in plants to support growth and reproduction. Amongst them, allantoin not only serves as a transportable nitrogen-rich compound, but has also been suggested to protect plants from abiotic stresses via minimizing oxidative damage. This work focuses on the effect of cadmium (Cd) on ureide metabolism in Arabidopsis, in order to clarify the potential role of allantoin in plant tolerance to heavy metals. In response to Cd treatment, allantoin levels increase in Arabidopsis thaliana, ecotype Col-0, due to reduced allantoinase (ALN) gene expression and enzyme activity. This coincides with increases in uricase (UO) transcripts. UO and ALN encode the enzymes for the production and degradation of allantoin, respectively. ALN-negative aln-3 Arabidopsis mutants with elevated allantoin levels demonstrate resistance to soil-applied CdCl2, up to 1,500 μM. Although aln-3 mutants take up and store more Cd within their leaf tissue, they contain less damaging superoxide radicals. The protective mechanism of aln-3 mutants appears to involve enhancing the activity of antioxidant enzymes such as superoxide dismutase and ascorbate peroxidase.

Effects of feeding goats with Leucaena leucocephala and Manihot esculenta leaves supplemented diets on rumen fermentation profiles, urinary purine derivatives and rumen microbial population

ABSTRACTThe effects of different levels of Leucaena leucocephala and Manihot esculenta leaves’ supplementation on in vitro gas production, rumen fermentation and microbial populations and urinary purine derivatives (PD) were investigated. Seven treatment groups – T1:C/rice straw (RS) (40:60) (Control); T2:C/RS/leucaena leaves (40:45:15); T3:C/RS/leucaena leaves (40:30:30); T4: C/RS/leucaena leaves (40:15:45); T5:C/RS/cassava leaves (40:45:15); T6:C/RS/cassava leaves (40:30:30) and T7:C/RS/cassava leaves (40:15:45) – were used in this experiment. In the in vitro study, acetate, propionate, butyrate and total VFA were found to increase significantly in T7. No significant difference was observed in in vitro gas production except the control diet although in vitro dry matter digestibility (IVDMD) was recorded significantly decreased. Determination of urinary PD, rumen fermentation and microbial population were done using 21 local Boer goats. Rumen NH₃-N, acetic and total VFA production were found to improve (P < .05) in all supplemented group. Significant (P < .05) reduction was noted in the urinary allantoin production and total PD at T7. The populations of total protozoa and Ruminococcus flavefacien had significantly increased (P < .05) while Ruminococcus albus, and Fibrobacter succinogenes were significantly reduced (P < .05) in supplemented group. In conclusion, the result from the present study suggested that 25% of L. leucocephala leaves diet and 50% of M. esculenta leaves diet can be incorporated in the goat diet so as to improve the nutritive value of poor quality diet.

Ureide metabolism under abiotic stress in Arabidopsis thaliana

Ureides are nitrogenous compounds derived from purine catabolism which contribute to nitrogen recycling in plants. Accumulation of ureide compounds has been reported in a number of plant species under stress conditions, suggesting their involvement in plants’ response to stress. In this research a biochemical and molecular approach was applied to address the ureide accumulation under abiotic stress conditions in Arabidopsis thaliana. Ureide concentration and changes in expression of ureide metabolic genes were examined in response to drought, NaCl and mannitol treatments. Additionally, an Arabidopsis allantoinase (ALN) mutant with constitutive accumulation of a ureide compound, allantoin, was used to investigate the impact of high levels of this compound on drought and NaCl stress responses. In the leaf tissue of adult plants allantoin accumulated in response to soil drying. Transcription of urate oxidase (UO), involved in allantoin production, was highly up-regulated under the same conditions. Allantoin and allantoate also accumulated in seedlings following treatment with NaCl or mannitol. aln mutants with enhanced levels of allantoin exhibited higher tolerance to drought and NaCl. Hydrogen peroxide and superoxide did not accumulate in the aln mutant leaves to the same degree in response to drought when compared to the wild-type. Our results suggest that ureide metabolism and accumulation contribute to the abiotic stress response which is regulated, at least in part, at the transcriptional level. Higher concentrations of allantoin in the mutant elevates abiotic stress tolerance, possibly by reducing oxidative damage.

Response and relation of allantoin production in different rice cultivars to competing barnyardgrass

The production of secondary metabolites and biomass of plants may be altered by coexistence and competition. Rice has coexisted with barnyardgrass in paddy systems for centuries and represents a model system for understanding plant–plant interactions. Despite increasing knowledge of allelochemicals produced and released by rice involved in coexistence of these two species, relatively little is known about the role and production of beneficial chemicals of rice in response to competing barnyardgrass. The purpose of this study was to quantify the growth-stimulating allantoin (5-ureidohydantoin) and its effect on barnyardgrass growth in a rice–barnyardgrass system. In this system, rice produced and released allantoin but barnyardgrass did not. The production of allantoin by rice responded to competition from barnyardgrass, and its concentration varied among rice cultivars tested. At 1:1 barnyardgrass and rice mixture proportion, allantoin concentration in allelopathic cultivars was obviously lowered by competing barnyardgrass, while no significant variation of allantoin concentration was observed in non-allelopathic cultivars. Barnyardgrass biomass was reduced in mixed-culture with rice cultivars, in particular, allelopathic cultivars. There was a positive relationship between allantoin concentration in rice cultivars and barnyardgrass biomass. Furthermore, allantoin stimulated the growth of barnyardgrass once released from rice or added to soil. The results suggest that rice plants of allelopathic cultivars appear to be able to detect the presence of competing barnyardgrass and respond by decreasing production of growth-stimulating allantoin, regulating the growth of barnyardgrass. In this manner, given rice cultivars may alleviate the competition of barnyardgrass and provide greater benefit to their own growth.

Relationship between serum allantoin and urate in healthy subjects and effects of benzbromarone in gout patients

Although hyperuricemia is suggested to increase allantoin production in both pro- and antioxidant manners, it remains undetermined whether it increases the serum concentration of allantoin. In addition, since uric acid has both pro- and antioxidant actions, a decrease in the serum concentration of uric acid may have an effect on the pro-oxidant-antioxidant balance. To examine whether serum allantoin is correlated with serum urate, we measured those levels as well as other parameters in 63 healthy subjects. In addition, to determine whether serum allantoin is correlated with reactive oxygen species (ROS) biomarkers, we measured 8-hydroxy deoxyguanosine and 15-F2t-isoprostane, markers of ROS, in urine samples from 30 gout patients before and 1 year after benzbromarone treatment (50 mg/d). The serum concentration of allantoin was correlated with that of urate in healthy subjects (R = 0.27, p < 0.05). Benzbromarone treatment in the patients decreased the concentrations of allantoin and urate in serum by 17% (p < 0.05) and 49% (p < 0.05), respectively, and the benzbromarone-induced change in serum allantoin was correlated with that in serum urate (R = 0.39, p < 0.05). However, benzbromarone treatment did not change the ratios of 8-hydroxydeoxyguanosine/creatinine or 15-F2t-isoprostane/creatinine in urine. Our findings suggest that hyperuricemia contributes to an increase in serum concentration of allantoin, though they do not indicate that hyperuricemia is a major factor for controlling oxidative stress in vivo.

Increased plasma homocysteine and allantoin levels in coronary artery disease: possible link between homocysteine and uric acid oxidation

Objective — Homocysteine increases the damage to the cardiovascular system in different ways, one of them is the formation of reactive oxygen species resulting from the auto-oxidation of homocysteine. At the same time, uric acid is one of the major antioxidants in the plasma and protects the cells towards increased ROS activity. In humans, allantoin is only formed from non-enzymatic oxidation of uric acid by free radicals.We aimed to determine the levels of homocysteine, uric acid and allontoin in patients with coronary artery diseases, and to evaluate the possible correlation between homocysteine and allantoin.Methods and results — Plasma total homocysteine, uric acid and allantoin levels of 50 patients with coronary artery diseases and 23 healthy controls were determined by HPLC methods. Commercial diagnostic kits were used for the determination of other biochemical parameters.We obtained higher homocysteine, uric acid and allantoin levels in patients than in controls (p<0.0001). Homocysteine levels were positively correlated with uric acid (r=0.435, p<0.0001) and allantoin (r=0.583, p<0.0001) levels in the whole study population.This correlation was persistent between allantoin and homocysteine after adjustment of these parameters for age, sex and creatinine.We accepted 15.0 mol/l as a cut-off value between normal and mildly elevated homocysteine levels for patients and controls. Twenty-five patients showed moderate hyperhomocysteinaemia. The mean allantoin and uric acid values of the moderate hyperhomocysteinaemic group were significantly higher than that of the group having lower homocysteine levels than this cut-off value (p<0.0001 for allantoin, p<0.02 for uric acid).Conclusion — Results imply that there is increased allantoin production resulting from uric acid oxidation by free radicals in hyperhomocysteinaemic patients with coronary artery disease. The possible significance of the relationship between homocysteine and allantoin warrants further study.

Urate oxidation during percutaneous transluminal coronary angioplasty and thrombolysis in patients with coronary artery disease

Thrombolysis and percutaneous transluminal coronary angioplasty (PTCA) are kinds of procedures that can be used to restore the blood flow of previously ischemic myocardium that can be the result of excessive production of reactive oxygen and nitrogen species, such as superoxide and hydroxyl radical, hypochlorous acid and peroxynitrite. Reaction of urate with some of these potent oxidants results in allantoin production. In this study, we measured the serum allantoin levels, an oxidation product of urate, and “in vivo” marker of free radical generation in reperfusion of ischemic myocardium. After an overnight fasting state, blood samples were collected from 35 patients with coronary occlusive diseases (7 women and 28 men) and 31 healthy subjects (8 women and 23 men). Serum allantoin and urate levels were measured by a GC-MS method. Serum allantoin levels of patients on PTCA therapy (mean±SD, 27.4 ± 15.2 μmol/l) and thrombolytic therapy (24.6 ± 8.6 μmol/l) were significantly higher than those of the patients without therapy (15.8 ± 6.2 μmol/l, p < 0.05 with PTCA and p < 0.006 with thrombolysis) and healthy controls (12.6 ± 6.3 μmol/l, p < 0.002 with PTCA and p < 0.0001 with thrombolysis). Although serum urate levels in PTCA (380.1 ± 72.6 μmol/l) and thrombolysis (359.5 ± 60.0 μmol/l) were higher than those in the non-therapy patients (336.6 ± 53.8 μmol/l) and controls (318.3 ± 81.0 μmol/l), there were no significant differences among groups ( p > 0.05). The results of the study are consistent with others which have demonstrated, higher urate levels are associated with coronary occlusive diseases. Our data support the hypothesis that generation of ROS occurs during myocardial reperfusion. Increased allantoin levels may be used as an index of increased oxidative stress during reperfusion.

Determinación de la excreción total de alantoína en suero de leche por Cromatografía Líquida de Alta Eficiencia (HPLC) en vacas Holstein alimentadas con pasto Kikuyo (Pennisetum clandestinum)

Se realizó un experimento con vacas Holstein con el fin de medir la excreción de alantoína en la leche. Se utilizaron tres hembras en la fase final de la lactancia, pertenecientes al hato de ordeño del C.I. Tibaitatá, que se alimentaron bajo confinamiento con pasto Kikuyo cortado diariamente de praderas pastoreadas por el hato completo. El experimento tuvo una duración de 14 días: siete de acostumbramiento y siete de muestreo, durante los cuales las vacas se alimentaron dos veces por día. Se medía el forraje ofrecido y el rechazado al día siguiente; así mismo, se tomaron 200 ml de leche de cada hembra, en los dos ordeños, durante los siete días de muestreo. Se midió el valor diario de alantoína en suero de leche, valor que se utilizó para calcular la cantidad digerida de nitrógeno microbial y para relacionarla con la materia orgánica digerida (MOD). Estos resultados se compararon con los generados en simulación por el programa CNCPS. Con el método colorimétrico los resultados fueron variables y poco satisfactorios. Con HPLC, la concentración de alantoína en suero de leche varió entre 2.74 mMol y 7.99 mMol L-1. Los valores calculados de flujo de N microbial al intestino fueron de 260.4 ± 32 g día-1, equivalentes a 1.628 g de proteína microbial digerida. El consumo de forraje fue de 12.1 kg de MS vaca día-1 (2.35% del peso vivo). La eficiencia de la síntesis de proteína microbial estimada fue de 55.3 g kg-1 de MOD. La cuantificación cromatográfica de alantoína se acercó a los valores reportados por la literatura y a las estimaciones de NM obtenidas por simulación con CNCPS (18 % de desviación). Cuando se incluyó el consumo de MS, la estimación de NM se acercó al valor de simulación (13% de desviación), considerándose que la cuantificación de alantoína por HPLC es un método aceptable.Abreviaturas usadas: CNCPS, Cornell Net Carbohydrate and Protein System; HPLC, High Performance Liquid Chromatography; MS, materia seca; MOD, materia orgánica digestible.

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.

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential sigma(54)-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the sigma(54)-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced (14)CO(2) from minimal medium containing [(14)C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated (14)CO(2) production from [(14)C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.

The effects of synchronizing the rate of dietary energy and nitrogen supply to the rumen on the metabolism and growth of ram lambs given food at a restricted level

AbstractThe effects of diets formulated to have two rates of organic matter (OM) release and to be either synchronous or asynchronous with respect to their hourly release of OM and nitrogen (N) in the rumen on N retention, microbial N production, growth and metabolism of ram lambs offered food at a restricted level was studied in two experiments. Four diets were formulated to differ in their rate and extent of OM and N release in the rumen based on the sum of in situ degradability data of the ingredients. All diets were formulated to have a similar metabolizable energy (10·4 MJ/kg dry matter (DM)), crude protein (140 g/kg DM) and daily ratio of N : OM released (34 g N per kg OM) and were either synchronous or asynchronous with respect to their hourly N : OM release. The four diets were slow energy, synchronous (SS), slow energy asynchronous (SA), fast energy, synchronous (FS) and fast energy asynchronous (FA). In both experiments the diets were offered at a rate of 1·75 maintenance energy requirements as two equal meals at 09:00 and 17:00 h. In experiment 1 there was no significant effect of diet on OM apparent digestibility or N retention. Lambs given diet FS had the highest daily production of allantoin (7·82 mmol per day; P &lt; 0·05) and microbial N (7·80 g/day; P &lt; 0Ό5). Hourly plasma urea and ß-hydroxy butyrate (3-OHB) concentrations exhibited a cyclical trend between meals with maximum concentrations occurring within 3 h of feeding and were higher in lambs given diet FA.In experiment 2 the four diets were offered to 32 growing ram lambs. Animals given synchronous diets (SS and FS) had a significantly higher live-weight gain than those given asynchronous diets (SA and FA; (132 g/day v. 107 g/day respectively; P &lt; 0·001). Food conversion efficiency (FCE; kg gain per kg DM intake) was improved proportionately by 0·23 in animals offered synchronous diets compared with asynchronous diets (P &lt; 0·001). There was little effect of diet on carcass composition except kidney fat, which was greater in lambs offered the synchronous diets (P &lt; 0·01). Plasma 3-OHB concentrations were higher throughout the growth period in lambs given diet FA whilst plasma urea concentrations were greater in lambs given the diet SS. In conclusion, greater attention to the formulation of diets to be synchronous for their hourly release of N : OM in the rumen can improve the growth rate and FCE of lambs. This effect may be due to an improvement in energy rather than N metabolism.

Identification of the True Product of the Urate Oxidase Reaction

The O2-dependent oxidation of urate catalyzed by urate oxidase has been examined in order to identify the immediate product of the enzymatic reaction. Specifically labeled [13C]urates were utilized as substrates, and the time courses were monitored by 13C NMR. On the basis of chemical shift values and 18O labeling, the product of the reaction was identified as 5-hydroxyisourate. This identification was substantiated by calculation of the 13C NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomers of urate and allantoin in aqueous solution were identified from 13C NMR titration data; the ionization behavior of urate and 5-hydroxyisourate were also examined by computational methods. The nonenzymatic pathway for production of allantoin from 5-hydroxyisourate was delineated; the reaction proceeds by the hydrolysis of the N1−C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allantoin.

Uric Acid and Ascorbic Acid Redox Ratios in Plasma and Tracheal Aspirate of Preterm Babies with Acute and Chronic Lung Disease

This study compared plasma redox ratios of uric acid and ascorbic acid in well preterm babies with those with respiratory distress syndrome (RDS) and chronic lung disease (CLD), and investigated the relationship between these ratios and their respective measurements in tracheal aspirate. On day 1 after birth, plasma allantoin and allantoin/uric acid ratio were elevated in CLD ( p < .05), and both markers of oxidative stress enabled early prediction of development of CLD (sensitivity and specificity: 54 and 83%, respectively). The relation between allantoin production and oxidative stress is supported by the correlation between the allantoin level and oxygen therapy in both RDS and CLD ( p < .05). Reduced and oxidized ascorbic acid in plasma decreased postnatally in all groups and their redox ratio remained stable. Uric acid and ascorbic acid redox ratios were significantly elevated in tracheal aspirates compared to plasma samples ( p < .05), and there was a strong positive correlation between both ratios ( p < .005). These markers may be useful in monitoring babies with respiratory distress. © 1997 Elsevier Science Inc.

Inhibition of purine catabolism by benzbromarone in isolated rat liver cells: Comparison with allopurinol and probenecid

Benzbromarone, a potent uricosuric agent, inhibited allantoin production in isolated hepatocytes at concentrations half to ten times greater than therapeutic plasma levels of the drug. In addition, the drug at these concentrations also markedly inhibited xanthine oxidase (EC 1.2.1.37), an enzyme involved in the regulation of this pathway. We found that allopurinol is several times superior to benzbromarone in the lowering of allantoin production (if they are compared in terms of their relative therapeutic levels), and that probenecid had no effect on it.

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.

Influence of ethanol on the production of allantoin by the perfused rat liver.

Ingestion of alcohol is traditionally considered a predisposing or precipitating factor in the gouty attack. Studies performed in man by Lieber et al. (1962) have shown that the administration of ethanol provokes an increase in the serum concentration of uric acid, which was attributed to a decrease in its renal excretion. More recently, ethanol infusion has been shown to enhance the production of uric acid by the liver, as measured from arteriovenous concentration differences during hepatic catheterisation in human volunteers (Grunst et al., 1973).KeywordsUric AcidAdenine NucleotideAdenosine DeaminasePerfuse LiverPerfusion MediumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.