Hypoxanthine Values Research Articles

Purine metabolites and lactate as parameters of hypoxia in the newborn infant.

In order to evaluate possible biochemical parameters of tissue hypoxia, arterial samples from 29 sick neonates were taken at 6-h intervals for lactate and blood gas analyses, as well as assays of purine metabolites in plasma. The state of the infant at each sampling was arbitrarily classified as severe, moderate, or mild on the basis of PO2, pH, FiO2, and transcutaneous PO2. Of the total of 289 samples, 72 were in the mild, 178 in the moderate, and 39 in the severe category. Elevated values for hypoxanthine (greater than 36 mumol/L), xanthine (greater than 11 mumol/L), and lactate (greater than 3.6 mmol/L) were defined as greater than 2 SD above the mean of infants with slight or no hypoxia. Hypoxanthine values were elevated in 7% of mild and in 13% of severe cases whereas the corresponding incidences for elevated xanthine values were 1 and 8%, and for elevated lactate values 7 and 39%. Conversely, when hypoxanthine was elevated (n = 21), mild hypoxia was present in 24% and severe in the same percentage of cases whereas the corresponding incidences for elevated xanthine (n = 7) were 14 and 43% and for lactic acid (n = 41) 12 and 37%. No consistent differences in plasma uric acid levels were noted between the mild and severe cases of hypoxia. We conclude that neither purine metabolites nor lactic acid concentrations in intermittent samples of arterial blood are reliable indicators of tissue oxygenation in the newborn.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaroytic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C 18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing pairedion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formyin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent K m of 18.2 μ m and an apparent V max of 32.4 mmol min −1 mg −1 were observed for the activity. A purine nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent K m of 236 μ m and V max of 2.78 μmol min −1 mg −1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent K i value of 136 μ m was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a K m value for GTP, IMP, and aspartic acid of 23, 34, and 714 μ m, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have K m and V max values for hypoxanthine of 55.5 μ m and 34.3 nmol −1 min −1 mg 1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures. The methodology is extended to interpreting a series of enzyme reactions in which the product of the first was the substrate for the second.

L’hypoxanthine-guanine phosphoribosyltransférase des pousses de Topinambour Helianthus tuberosus L.

Extracts from Jerusalem artichoke shoots exhibited hypoxanthine-guaninephosphoribosyltransferase activity. Only partial purification by DEAE-Sephacel chromatography was performed considering the instability of the enzyme. The HGPR Transferase showed an absolute requirement for divalent cations: the greatest activity was found with Mn++. The optimal pH for the enzyme activity was 7,5 to 8,5, in contrast to the optimal pH of the APR Transferase of the same species which was 5,5 to 6,5. The Km values for hypoxanthine, guanine and PRPP, at the optimal pH, were 6.1 x 10-6 M, 5.5 x 10-6 M and 8.8 x 10-5 M respectively, but the rate of phosphoribosylation of hypoxanthine was greater than that of guanine at the tested concentration. Competitive inhibition of the phosphoribosylation of hypoxanthine by guanine and inversely are consistent with the fact that only one enzyme catalyses the reaction. The product inhibition by IMP and GMP were competitive towards PRPP (Ki = 5.7 x 10-5 M and 6.3 x 10-6 M respectively) and uncompetitive towards hypoxanthine (Ki = 4.1 x 10-4 M and 5.3 x 10-5 M respectively). GMP was much more inhibitory than IMP; GDP and GTP were also inhibitory. The properties of HGPR Transferase of Helianthus tuberosus are discussed in relation to its physiological role.

OXYPURINE CONCENTRATION IN THE CSF IN CHILDREN WITH DIFFERENT DISEASES OF THE NERVOUS SYSTEM1

Oxypurine analysis was done in the CSF of 190 children with different diseases. The patients could be divided into four groups: Group A, serving as controls, consisted of 56 children suffering from diseases without neurological signs, for example, leukaemia. 16% of them had raised hypoxanthine values greater than 7.5 mumol/l and 32% raised uric acid values greater than 12.0 mumol/l. Group B comprised 97 children suffering from diseases with neurological signs, for example, meningitis. For these patients the frequency of raised hypoxanthine and uric acid values in the CSF was twice as high as in Group A. Group C comprised 31 patients with different forms of cerebral convulsions. Among these patients 52% had raised hypoxanthine and 70% raised uric acid values. The findings of these patients are described in a previous paper (Manzke et al. 1981). Group D comprises 6 patients from whom CSF samples were taken postmortally. All these deceased patients showed extremely high hypoxanthine + xanthine and uric acid concentrations in their CSF.

Increase of adenosine content in cerebral cortex of the cat during bicuculline-induced seizure.

In order to elucidate whether adenosine may be involved in the increase in cerebral blood flow (CBF) during functional hyperemia, cortical tissue levels of adenosine, inosine and hypoxanthine were measured in the cat following bicuculline (3 mg/kg)-induced seizure. In addition, the subcellular distribution of 5′-nucleotidase in the cortex was determined by histochemical techniques. Experiments were performed on anaesthetized and immobilized cats and tissue samples to be analyzed for the different purine compounds were obtained by freezing through the trepanized and non-trepanized skull. Control values for adenosine, inosine and hypoxanthine on the trepanized side were 1.31, 1.12 and 3.79 nmoles/g, respectively. The cortical content of adenosine increased to 3.85 nmoles/g, 15 s after beginning of seizure activity and remained elevated for 20 min. Inosine and hypoxanthine also increased, exhibiting highest values after 20 min. Values for adenosine and lactate were found to be generally higher when analysis was performed from tissue frozen through the intact skull bone. In all experiments the adenosine content correlated with lactate levels. From measurements of plasma concentrations of adenosine and inosine in the sinus sagittalis superior it is concluded that seizure is also accompanied by an enhanced release of adenosine from the brain. Localization of 5′-nucleotidase by electron-microscopic cytochemistry revealed that this enzyme is associated almost exclusively with plasma membranes of glial cell membranes including perivascular astrocytes. Thus high concentration of adenosine can be expected to accumulate locally in the brain cortex. In view of the well known increase in CBF during onset of seizure and the time course of adenosine formation, our findings support the view that adenosine may be involved in the initial phase of seizure-induced functional hyperemia of the brain.

MODIFIED ATMOSPHERE STORAGE OF ROCKFISH (Sebastes miniatus) AND SILVER SALMON (Oncorhynchus kisutch)

ABSTRACTRockfish (Sebastes miniatus) fillets and salmon (Oncorhynchus kisutch) steaks were held in atmospheres containing 20% or 40% carbon dioxide, with or without 1% carbon monoxide. Controls were stored similarly in air. At intervals of refrigerated storage up to 14 days, samples were removed for sensory, chemical, and microbiological analyses. At 7 days, all treatment groups were significantly different visually, with appearance of slime on the air controls, but not on samples in the gas treatments. Samples held in air were judged by panelists to have stronger aromas than others held under carbon dioxide at either level. The higher level of carbon dioxide was more effective. Values for thiobarbituric acid were low in all groups; hypoxanthine values varied widely, with no particular effect due to modified atmospheres. Storage under carbon dioxide was effective in reducing the formation of trimethylamine and ammonia, and markedly inhibited microbial growth.

Myocardial release of lactate, inosine and hypoxanthine during atrial pacing and exercise-induced angina.

The coronary venous efflux of lactate, inosine and hypoxanthine during pacing-induced angina has been compared with myocardial extraction of the catabolites during exercise-induced angina. Inosine and hypoxanthine were analyzed by enzyme assay after separation by column chromatography. Myocardial lactate extraction at rest (15 +/- 9%, mean +/- SD) was converted to production levels (-34 +/- 26%) during pacing-induced angina (p less than 0.0005) and increased (24 +/- 13%) during exercise (p less than 0.05). The arterial values at rest (850 +/- 330 mumol/1) were unchanged during pacing and increased five-fold during exercise (4380 +/- 1860 mumol/1). The mean myocardial inosine extraction at rest (33 +/- 10%) was transformed to release values (-41 +/- 30%) during pacing (p less than 0.0005) as well as during exercise (-20 +/- 27%) (p less than 0.0005). The hypoxanthine extraction at rest (25 +/- 11%) decreased during pacing (-7.8 +/- 29%) (p less than 0.0025) and exercise (10 +/- 25%) (NS). The slight increase of arterial inosine and hypoxanthine values was not significant. Myocardially produced lactate, a sensitive marker of pacing-induced ischemia, was obscured by elevated arterial concentrations during exercise. However, inosine significantly correlated with lactate during pacing, and was useful in detecting ischemic myocardial energy deficiency during exercise-induced angina.

A column chromatographic method for determination of plasma and erythrocyte levels of inosine and hypoxanthine

A column chromatographic separation of inosine and hypoxanthine in plasma and erythrocytic samples after deproteination by ultrafiltration, adsorption of the compounds onto charcoal, elution in pyridine/ethanol solution, and filtration by celite gel is described. Sephadex G-10 was used to separate the compounds with small but different molecular weights. Inosine and hypoxanthine values were determined by enzymatic spectrophotometric assay. In arterial, coronary venous, and erythrocyte samples the mean ± SD values for inosine were calculated as 1365 ± 560 nmol/liter of plasma, 915 ± 310 nmol/liter of plasma, and 8925 ± 6720 nmol/liter of blood, respectively. The corresponding values for hypoxanthine were 2525 ± 1950 nmol/liter, 1835 ± 1315 nmol/liter, and 11090 ± 7600 nmol/liter, respectively.

The effect of nitroglycerin on myocardial release of inosine, hypoxanthine and lactate during pacing-induced angina.

The efficacy of nitroglycerin as an antianginal drug has been evaluated by calculation of myocardial extraction and production values of lactate and the adenosine triphosphate (ATP) catabolites inosine and hypoxanthine. Coronary venous and arterial blood was sampled at rest, during pacing-induced angina and 4--6 min after nitroglycerin at identical paced heart rates for enzymatic assay of inosine and hypoxanthine after separation by column chromatography and for determination of lactate. Sublingual nitroglycerin given to 10 patients with coronary artery disease decreased coronary venous lactate values from 1175 +/- 320 mumol/l during pacing-induced angina to 950 +/- 240 mumol/l (p less than 0.05). The calculated myocardial lactate production during angina (-31 +/- 19%) diminished after nitroglycerin (-1.7 +/- 22%) (p less than 0.0025). Coronary venous inosine values during angina (1275 +/- 865 nmol/l) decreased after nitroglycerin (795 +/- 555 nmol/l) (p = n.s.), the arterial values (885 +/- 610 nmol/l) increased (960 +/- 580 nmol/l) (p = n.s.), the myocardial inosine release (-26 +/- 20%) changed to extraction values (19 +/- 19%) (p less than 0.0005). Coronary venous hypoxanthine values during angina (1540 +/- 1035 nmol/l) were reduced (1110 +/- 675 nmol/l) (p = n.s.); the arterial values (1625 +/- 1050 nmol/l) decreased (1510 +/- 935 nmol/l) (p = n.s.), the myocardial hypoxanthine extraction (0.3 +/- 29%) with a wide individual variability increased after nitroglycerin (24 +/- 13%) (p less than 0.025). The myocardial release of inosine and lactate during severe angina with significant positive correlation (r = 0.66, p less than 0.0025) demonstrates that anaerobic glycolysis is accompanied by ATP breakdown. The unchanged myocardial inosine and hypoxanthine extraction after nitroglycerin indicates that nitroglycerin is capable of attenuating this effect. In spite of reduced mean myocardial lactate production after nitroglycerin ischemic myocardial energy deficiency may be less marked. Thus, the enhanced myocardial inosine uptake may be one factor contributing the beneficial effects of nitroglycerin including the improvement of myocardial oxygen balance.

Uric acid lithiasis associated with altered kinetics of hypoxanthine-guanine phosphoribosyltranferase

1. A patient is described who had uric acid calculi which obstructed both kidneys. Serum uric acid was 34 mg/100 ml upon admission to hospital, and evidence of precipitation of uric acid in joints was obtained. 2. Total activity of hypoxanthine-guanine phosphoribosyltransferase in erythrocytes lysates from this patient was 44% of normal. Kinetic studies revealed that the apparent Michaelis constant of this enzyme for phosphoribosyl pyrophosphate was elevated 15-fold, while these values for hypoxanthine and guanine were in the normal range.

Beeinflussung der trennung von purinen und pyrimidinen an sephadex G-10

Factors which influence the separation of purine and pyrimidine compounds on Sephadex G-10Sephadex adsorption chromatography can be used for separating pyrimidine and purine compounds. The separation is affected by a number of conditions. This paper describes the variation of temperature, ionic strength, pH, degree of crosslinking, concentration of n-propanol, dioxna and urea, and their influence on the adsorption behaviour of compounds.The degree of adsorption decreases with increasing temperature. Theis relationship is valid for gel—solid interactions. The enthalpy values (δH°) for hypoxanthine and adenosine are −2.35 and –1.92 kcal/mol, respectively, and suggest the existence of hydrogen bonding between the compounds and the gel. For purine and pyrimidine compounds different mechanisms of separatio exist as a result of different δH° values. For the most of the compounds examined the basic heterocyclic nitrogen atoms are the primary sites of adsorption. Therefore, hydrogen bonding between the unshared pairs of electrons of the heterocyclic nitrogen atoms and the hydroxy group or the oxygen of the gel matrix is discussed.Dioxan n-propanol and urea decrease the degree of adsorption, while an increase in ionic strength increases the adsorption. Elution values of compounds correspond to their degree of dissociation.The adsorption chromatography of heterocyclic compounds on Sephadex gels is a useful technique for the separation of mixtures of compounds of synthetic or biological origin. A detailed paper on the chromatographic behaviour and chemical structure is published18.

On the effectiveness of EDTA in prolonging the shelf life of fresh fish

The purpose of this investigation was to ascertain whether EDTA might be useful in prolonging the keeping quality of fresh fish. The criteria of quality used were: the total bacterial count, the content of volatile basic nitrogen and trimethylamine, the degree of dephosphorylation of the nucleotides, the level of the hypoxanthine, and the organoleptic properties. The material consisted of rainbow trout (Salmo irideus) and Baltic herring (Clupea harengus var. membranus). The results showed that EDTA had a markedly favorable effect on the keeping quality of fish. Although the effect on the total bacterial count was only slight, a chemical and organoleptical comparison with control material revealed that spoilage in the EDTA-treated samples was definitely slower. In the case of fillets of rainbow trout, judged by the organoleptical method, the controls became unacceptable after storage for 18–20 days at +4°C, whereas after the same storage time the EDTA-treated samples were still free of off-odors and had a fair taste. Similarly, the values of volatile basic nitrogen, trimethylamine, and hypoxanthine increased at a slower rate, and the dephosphorylation of nucleotides proceeded less rapidly in the EDTA-treated fillets than in the controls. In the case of fillets of Baltic herring, sensory evaluation brought out the positive effect of the EDTA treatment. A corresponding difference was noted in the volatile basic nitrogen and trimethylamine values, and a slight difference in the hypoxanthine values. In this fish, the dephosphorylation of the nucleotides was rapid, and between the samples subjected to EDTA treatment and the control material there appeared only a small difference. With regard to the round Baltic herring, again, the differences between the two categories were less conspicuous; the chief difference was noted in the volatile basic nitrogen, but in the sensory evaluation the difference was not fully consistent.