Purine Base Concentrations Research Articles

Effect of aα-galactosidase supplementation of cereal-soya-bean-pea diets on the productive performances, digestibility and lower gut fermentation in growing and finishing pigs

AbstractAn experiment was carried out to study the effect of addingα-galactosidase to the diet on the performance and digestive parameters of growing-finishing pigs. Fifty-four gilts, average body weight (BW) of 43 (s.e. 0·4) kg, were allocated to 18 pens and used in a production experiment divided in two consecutive periods of 28 days each (growing and finishing). In each period gilts were given a diet based on cereals, soya-bean meal and peas, supplemented (Enzyme) or unsupplemented (Control) with 200 units per kg ofα-galactosidase. All diets included 2 g Cr2O3per kg as a digestibility marker. Food intake and body weight were recorded every 2 weeks and faecal samples by pen were collected at the end of each period. Average daily gain (ADG), food: gain ratio and faecal digestibility of dry matter (DM), crude protein (CP) and neutral-detergent fibre (NDF) were calculated. At the end of the production experiment nine gilts from each treatment were selected and given the finishing diet for a further 10 days before being slaughtered. Samples of digesta from the ileum and caecum were collected and ileal digestibility of monosaccharides determined. Short chain volatile fatty acids (SCVFA ) and purine bases (PB) in caecal digesta were analysed. The use ofα-galactosidase improved the ADG (P< 0·01) and the food: gain ratio (P< 0·01) simultaneous to increases in the faecal digestibility of DM (P< 0·05), CP (P< 0·05) and NDF (P= 0·07) in the finishing period. Alpha-galactosidase supplementation also improved the ileal digestibility of some monosaccharides (galactose, rhamnose, mannose and fucose) of the dietary fibre fraction (P< 0·05); and reduced total caecal concentration of SCVFA (P= 0·15), proportion of branched-chain VFA (P< 0·001), and concentration of total PB (P< 0·001). It is concluded that addingα-galactosidase to a cereal-soya-bean meal-pea diet improves ADG, food: gain ratio and digestibility in fattening pigs, and reduces the amount of fermentable substrate flowing to the large intestine.

Digestion and microbial fermentation ofEragrostis curvulasupplemented with tallow

AbstractTwoin vitroexperiments were conducted in order to determine if microbial fermentation of Eragrostis curvula hay is depressed by high levels of added tallow. Two levels of tallow, to reach 0.06 (T6) and 0.12 (T12) of the ether extract (organic matter basis) in food were compared with a control (T0, 25 g ether extract per kg). The first experiment studied the pattern of gas production. From 24 h onwards, gas volume for T0 was higher (P < 0.05) than for T6 and T12. However, lag time was shorter with tallow, probably because of utilization of the released glycerol. Fermentation of a similar amount of fat included in T6 and T12 as the only substrate (fat-6 and fat-12) depressed gas production compared with the blank, irrespective of fat level. In a second experiment, characteristics of microbial fermentation were studied, including volatile fatty acid (VFA) production, bacterial adhesion to fibrous particles (measured according to purine bases concentration) and polysaccharidase and glycosidase activities, at 6, 12, 24 and 48 h incubation. Total VFA was higher (P < 0.05) in T0 compared with T6 but not with T12. Acetate: propionate ratio diminished with tallow in the food. Higher total xylosidase (P > 0⋅05) and glycosidase (P < 0⋅001) activities were observed for T0 than for T6 and T12 and similar responses were observed regarding specific activities. Bacterial adhesion was not different between T0 and T6 but it was smaller in T12. The inhibition of microbial fermentation by tallow addition is more related to specific polysaccharidase and glycosidase activities, rather than to a depressed bacterial adhesion.

Stimulation of caffeine biosynthesis in suspension-cultured coffee cells and the in situ existence of 7-methylxanthosine

Suspension-cultured cells of coffee were subjected to various conditions such as photoperiod, 1 mM adenine, 0.1–10 mM ethephon, or to the combination of both adenine and ethephon. Concentrations of purine bases, nucleosides, nucleotides as well as of purine alkaloids (PA; i.e. 7-methylxanthine, theobromine and caffeine) were measured by HPLC. In the dark, both adenine and ethephon drastically stimulated overall PA formation by a factor of 4 and 7, respectively. Their simultaneous application resulted in an additional increase yielding a stimulation factor of 11. In photoperiod, caffeine formation was, as compared to the control in the dark, enhanced by a factor of 21 without affecting theobromine and 7-methylxanthine pools; additional stimulation by ethephon was not possible. Conversely to light and ethephon, which had no effect on the accumulation of primary purine metabolites, adenine feeding resulted in persistently enlarged pools of nucleosides (xanthosine, guanosine, inosine) and 7-glucopyranosyladenine. 7-Methylxanthosine, the postulated precursor of 7-methylxanthine in caffeine biosynthesis could not be detected under any conditions at any time. Since no other methylated purine was found, it is not yet feasible to discard the ‘7-methylxanthosine hypothesis’. However, the present investigation provides a suitable system to study the early steps of caffeine biosynthesis by means of radiolabelling kinetics.

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.

INFLUENCE OF FREEZE-STORAGE ON NUCLEIC ACID CONCENTRATIONS IN BACTERIA AND DUODENAL DIGESTA

Separation of bacteria from duodenal digesta prior to storage (−25 °C) resulted in 17 and 27% loss of RNA at 1 and 2 weeks, respectively. Storage of total digesta, with bacterial separation immediately prior to analysis, prevented loss of RNA. Purine base concentrations were not influenced by storage. However, removal of free purine base in digesta prior to analysis is recommended. Key words: Nucleic acids, freeze-storage, bacteria, duodenal digesta

Effect of hypoxia and exercise on nucleotide metabolism in man.

Intrapartum asphyxia affects about 1% of all newborn; the damage to the nervous system has been related to the excretion of hypoxanthine (Harkness et al., 1982). To interpret the changes found in pathological hypoxia we have studied the duration of hypoxanthine release after reducing muscle ATP by severe exercise in 7 adult males using similar conditions to those of Sutton et al. (1980). The concentrations of purine bases, nucleosides and nucleotides in plasma, urine, leucocytes and erythrocytes have been determined using the methods of Simmonds and Harkness (1981).

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.

Purine transport and metabolism in man: the effect of exercise on concentrations of purine bases, nucleosides and nucleotides in plasma, urine, leucocytes and erythrocytes.

1. After decreasing muscle ATP by a 2 min period of intense exercise, we have studied purine metabolism by using high-pressure liquid chromatography. 2. A major increase in hypoxanthine concentration in plasma and urine was found with increases in xanthine and, in plasma, inosine. Erythrocyte hypoxanthine rose with the level in plasma, but there was no corresponding rise in IMP, the first intracellular metabolite of hypoxanthine. No rises in uridine or urate were found in plasma. 3. Plasma adenosine did not rise and fall significantly after exercise, but a small rise and fall in adenine nucleotide concentrations in plasma was found. 4. Running, swimming and games, which tended to be at the weekend, were associated with a rise in hypoxanthine and xanthine excretion; exercise was probably the cause of the higher excretion during the day than at night. Such activities do not produce changes in concentrations of ATP in muscle, although turnover must rise. 5. The results are consistent with widespread purine exchange between tissues and a 'circulating hypoxanthine pool'.

Purine nucleoside and purine base concentrations in bovine thyroid and plasma

1. 1. Concentrations of eight purine nucleosides and bases in bovine thyroid and plasma were determined by high pressure liquid chromatography. 2. 2. Plasma purines were metabolized to uric acid in the absence of inhibitors. 3. 3. The concentrations of these purines were 10–100 times greater in thyroid tissue than in plasma.

Nucleotide, nucleoside and purine base concentrations in human placentae

Nucleotides, nucleosides and purine bases in trichloroacetic acid extracts of freeze clamped samples of human placenta have been measured by high-pressure liquid chromatography. The concentrations of the nucleotides concerned with energy transduction, ATP, ADP and AMP, and especially the energy charge, are stable over periods of ischaemia of 30 min. Concentrations of 14 nucleotides, including UDPAG, GDP Man, UDP and CTP, have now been defined. In addition, the concentrations of hypoxanthine, xanthine, uridine, adenine and inosine are indicated. Concentrations of the vasodilator adenosine are similar to the apparent Michaelis constants of its main metabolizing enzymes adenosine kinase and adenosine deaminase. The availability of 'normal' values of adenine nucleotide concentrations in human placenta should permit the detection of 'placental insufficiency' of energy supply, if this condition exists.

Analysis of purines and their nucleosides by the reverse phase partition mode of high pressure liquid chromatography.

Although the analysis of nucleotides in blood is now being carried out routinely by high pressure liquid chromatography (HPLC) (1–4), and work is in progress to determine alterations casued by various disease states of free nucleotide concentrations in physiological fluids and cell extracts (5,6), less attention has been paid to free nucleoside levels in cells. Recent investigations, however, have focused attention on methylated nucleosides in cells as biological markers in cancer (7,8). Moreover, nucleoside concentrations are thought to be important in cardiac disease and birth defects. The intracellular level of adenosine, one of the physiological regulators of coronary blood flow, has been found to increase in cardiac hypertrophy and after brief periods of myocardial ischemia and hypoxia (9,10). In abnormalities in purine metabolic pathways caused by disease, the concentrations of purine bases and/or their nucleosides often are involved. For example in patients with adenosine deaminase deficiency, it has been observed that excess adenosine in cultured mammalian cells is toxic (11) and it has been postulated that accumulated adenosine is associated with severe immunological defects in children (12).KeywordsXanthine OxidaseHigh Pressure Liquid ChromatographyPurine BasePotassium Dihydrogen PhosphatePartition ModeThese 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.