Alan M. Snoswell

Comparative studies on the methionine synthesis in sheep and rat tissues

The important features of the enzymes involved in methionine synthesis in sheep were found to be the low activity of betaine-homocysteine methyltransferase and the high activity of 5-methyltetrahydrofolate-homocysteine methyltransferase. The rate of the methionine synthesis in sheep liver was significantly lower than that in rats due to the low activity of hepatic betaine-homocysteine methyltransferase. The hepatic methionine recycling was stimulated by the addition of betaine in both species. These results indicate that in sheep 5-methyltetrahydrofolate-homocysteine methyltransferase plays a significant role in hepatic methionine synthesis along with betaine-homocysteine methyltransferase. In contrast, in the rat hepatic system methionine synthesis is virtually dependent on betaine-homocysteine methyltransferase.

Effects of dietary oat bran and diabetes on plasma and caecal volatile fatty acids in the rat

Abstract Diets high in plant fibre, particularly viscous gums, are of potential benefit in the control of diabetes in man. This study compares the effects of oat bran, one such fibre preparation, with purified cellulose on volatile fatty acid metabolism in rats fed a high carbohydrate diet. Concentrations of total volatile fatty acids were significantly higher both in hepatic portal venous plasma and caecal contents of rats fed oat bran. Major differences were also found in the relative contributions of individual acids with proportionately less acetate and more propionate and butyrate in these animals compared with rats fed cellulose. In rats fed a standard laboratory diet, diabetes induced by streptozotocin produced a sustained increase in caecal and hepatic portal venous concentrations of acetate, propionate and butyrate. Because of the high simple carbohydrate content of the experimental diet, the tolerance of diabetic rats for the bran and cellulose diets was poor and no differences in VFA metabolism were observed after 24 hr of diabetes. It is concluded that volatile fatty acid concentrations were in proportion to the probable digestibility of the source of dietary fibre and are significant metabolic fuels liable to altered metabolism by insulin insufficiency.

Metabolic effects of acetate in perfused rat liver. Studies on ketogenesis, glucose output, lactate uptake and lipogenesis.

Abstract 1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-14C]acetate or [U-14C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-14C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-14C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with 3H2O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].

Determination of creatinine in whole blood, plasma, and urine by high-performance liquid chromatography

A sensitive method for the specific determination of creatinine in whole blood, plasma, and urine with high precision and accuracy is described. Samples were deproteinized by addition of acetonitrile and analyzed by high-performance liquid chromatography using a cation-exchange column with a mobile phase of 9% acetonitrile in 40 mM ammonium phosphate (pH 4.0). The recoveries of creatinine added to blood and plasma were almost complete, ranging from 99 to 101%. The coefficients of variation were very small, 1.6% for blood and plasma and 1.5% for urine. Samples can be assayed in 11-min intervals subsequent to the initial injection. As little as 2 microliter of blood or plasma or 0.02 microliter of urine is sufficient for chromatographic analysis. The present method was successfully used for the accurate measurement of small arterial-venous differences of creatinine concentrations in blood across body organs and showed that in the sheep creatinine is produced in the muscles and is excreted by the kidneys. The method is also suitable for routine analysis of creatinine in clinical laboratories.

Oxidized nicotinamide-adenine dinucleotide-independent lactate dehydrogenases of Lactobacillus arabinosus 17.5

Abstract 1. 1. The object of this work was to investigate the nature and properties of an l - and a d -lactate dehydro g enase of Lactobacillus arabinosus1 and to establish the relationship of these two enzymes to the two lactate dehydrogenases purified from the same organism by Dennis and Kaplan 2. 2. 2. The d - and l -lactate dejydrogenases were partially purified from cell-free extracts by ammonium sulphate and protamine sulphate treatments followed by chromatography on DEAE-cellulose columns. Finally the two enzymes were completely separated on CM-cellulose columns. 3. 3. The l -lactate dehydrogenase was further purified (a total of 1200-fold) by chromatography on TEAE- and Ecteola-cellulose columns. This enzymes contained 1 molecule of FMN per molecule of enzyme and approx. 20% of the flavin was reduced by the addition of sodium l -lactate. 4. 4. Both enzymes were NAD+-independent and only reduced oxidation-reduction dyes. The Km and optimum pH values were quite different from those reported for the d - and l -lactate dehydrogenases isolated by Dennis and Kaplan , also the former enzymes readily separated by electrophoresis in contrast to the latter enzymes. 5. 5. Both the NAD+-independent d - and l -lactate dehydrogenases were separated from the NAD+-dependent enzymes on a TEAE-cellulose column. Thus, Lactobacillus arabinosus appears to contain two pairs of lactate dehydrogenases, two of which are NAD+-linked and another two which are NAD+-independent. Of these latter two enzymes the l -lactate dehydrogenase is a flavoprotein while the d -lactate dehydrogenase is probably a flavoprotein also.

Developmental changes in the activities of enzymes related to methyl group metabolism in sheep tissues

The activities of choline oxidase and betaine-homocysteine methyltransferase increased markedly in pre-ruminant lamb liver after birth and subsequently decreased when the lambs reached the ruminant state, while the developmental changes in hepatic 5-methyl-H4folate-homocysteine methyltransferase were negatively correlated with those of betaine-homocysteine methyltransferase. Hepatic phospholipid methyltransferase was elevated almost four-fold by the 10th postnatal day, but declined thereafter. Hepatic glycine methyltransferase in one-day-old lambs increased 55-fold, compared with that of fetuses, and thereafter decreased dramatically with age. Guanidoacetate methyltransferase, glycine methyltransferase and betaine-homocysteine methyltransferase in sheep pancreas increased markedly with age and were many times higher than the hepatic enzymes in adult sheep. Choline oxidase, betaine-homocysteine methyltransferase, cystathionine beta-synthase and glycine methyltransferase in adult sheep liver were much lower than those in rat. These results illustrate the conservative features of methyl group metabolism in postruminant sheep.

Methyl group metabolism in sheep

1. Sheep have a very low intake of methyl nutrients in the post-ruminant state, due to the almost complete degradation of dietary choline by rumen microorganisms, the lack of dietary creatine and the relatively low content of methionine in microbial proteins. 2. Methylneogenesis provides a major source of labile methyl groups in post-ruminant sheep and impairment of the methylneogenesis leads to a marked reduction of the labile methyl pool. 3. S-Adenosylmethionine (AdoMet) metabolism via transmethylation is most active in sheep liver and pancreas and is regulated by the availability of methionine and intracellular ratios of AdoMet to S-adenosylhomocysteine (AdoHcy). 4. Adaptive mechanisms which arise as a consequence of the poor methyl nutrition in post-ruminant sheep are a marked reduction of labile methyl catabolism and an increase in the capacity of methylneogenesis.

PLASMA AND CAECAL VOLATILE FATTY ACIDS IN MALE AND FEMALE RATS: EFFECTS OF DIETARY GUM ARABIC AND CELLULOSE

Abstract Adult male and female rats were fed synthetic diets containing 10% gum arabic or cellulose. Concentrations of total volatile fatty acids in caecal fluid and hepatic portal venous plasma of both sexes were higher on the gum arabic diet. Total and individual acids in caecal fluid did not differ between male and female rats fed gum arabic, but with cellulose concentrations of acetate and propionate were significantly lower in females than in males. Caecal pH correlated negatively with volatile fatty acid concentration in both sexes, but with gum arabic, pH was significantly lower in males than in females with the same caecal volatile fatty acid concentration. The concentration and distribution of plasma volatile fatty acids in the hepatic portal vein generally reflected that of caecal fluid although acetate concentrations were lower in male than in female rats fed gum arabic. Blood glucose and plasma triacylglycerols were unrelated to hepatic portal venous plasma volatile fatty acids but in males liver glycogen in each dietary group was positively correlated with volatile fatty acid concentration. This relationship was absent in females. It is concluded that there are differences in the production and absorption of volatile fatty acids between male and female rats and that these are affected by the source of dietary fibre.

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.

Carnitine and creatine content of tissues of normal and alloxan-diabetic sheep and rats

The concentration of carnitine in liver increased 28-fold and urinary carnitine excretion 5-fold in alloxan-diabetic sheep. In contrast there were no similar increases in alloxan-diabetic rats. The creatine content of liver decreased 3-fold and creatine excretion decreased 2-fold in diabetic sheep. In contrast the creatine content of liver increased nearly 4-fold in diabetic rats with no change in creatine excretion. The marked increased in production of carnitine by the liver of the diabetic sheep appears possible because of decreased production and excretion of creatine.