The activity of an enzyme involved in decyanation of cyanocobalamin was found in the cell homogenate of Euglena gracilis. The enzyme essentially required FAD or FMN, and NADPH as cofactors. The apparent Km for cyanocobalamin and NADPH were 7.1μM and 0.2 mM, respectively. The enzyme reaction obeyed allosteric kinetics towards FAD ([FAD]0.5=30μM, n=2.7; as calculated by the Hill plots). The Euglena enzyme was located in the mitochondria.
Treatment of cultured rat hepatocytes with 10μM 1-chloro-2, 4-dinitrobenzene (CDNB) resulted in an acute loss of cellular glu-tathione (GSH) within 30 min and a marked increase in spontaneous lactic dehydrogenase (LDH) leakage to the culture medium after 24 h, with obvious cellular degeneration as viewed by phase-contrast microscopy. Simultaneous treatment of the cells with α-tocopherol markedly protected the cells not only against LDH leakage but cellular degeneration in a dose-dependent manner. The EC50 of α-tocopherol was 0.1μM, ca. 200 times less than normal plasma levels in the rat. In response to the inhibitory effects of α-tocopherol on the cytolysis as measured by LDH leakage, GSH biosynthesis was stimulated by CDNB, and cellular GSH levels returned to control levels. The recovery was inhibited by 0.2mM buthionine-SR-sulfoximine (BSO), a specific inhibitor of γ-glutamylcys-teine synthetase. However, the stimulation of GSH biosynthesis ap-parently was not essential for the protection from cytolysis by GSH depletion during the experimental period, because treatment with 0.2mM BSO and 20μM tocopherol completely protected the cells against the lysis induced by BSO up to 32h without cellular GSH recovery. The results suggest that α-tocopherol may be a primary natural inhibitor of the cytolysis induced by xenobiotics which consume the cellular GSH in vivo.
The action of α-tocopherol in the phosphatidylcholine liposomal membranes was studied by spin label technique in order to elucidate the role of vitamin E in the membranes. The fluidity of the liposomal membranes decreased with increasing concentrations of satu-rated phosphatidylcholine, cholesterol, and α-tocopherol. However, the physical effect of α-tocopherol was quite small at its physiological concentrations. The effects of structure of various kinds of chromanols on the membrane fluidity were studied. The fluidity of the liposomal mem-branes decreased with increasing length of the side chain at 2-position of 6-chromanols. This effect was smaller when the side chain at the 2-position did not possess branched methyl group. The effect of tocopherols on the membrane fluidity was larger in the inner part of the membranes. Ascorbic acid residing in the aqueous phase interacted with the nitroxide spin probe incorporated into the membranes and the rate was smaller as the nitroxide group was buried deeper in the phospholipid bilayer. The spin probes incorporated into liposomal membranes were consumed as the oxidation proceeded. α-Tocopherol suppressed both the oxidation of membranes and the decrease of spin probe, but when α-tocopherol was depleted, the spin probe decreased and the membranes became more rigid as the oxidation proceeded.
We observed the cholesterol metabolism of a colony of Wistar rats with a hereditary defect in vitamin C synthesizing ability (the ODS (osteogenic disorder-Shionogi) rats) in six kinds of experiments. Female ODS rats aged 36 days had a low HDL (high-density lipoprotein)-cholesterol level in serum as compared with age-matched control rats in spite of the absence of scorbutic symptoms. Female ODS rats aged 63 days which revealed severe scorbutic symptoms had a very low HDL-cholesterol level (mean value; 17 mg/dl). And male ODS rats, whose lives had been prolonged by supplementing with L-ascorbic acid, also had lower serum HDL-cholesterol and had increased total cholesterol in serum and liver when the acid supplement dose was relatively insufficient. On the other hand, we examined HDL2- and HDL3-cholesterol levels in serum to determine the mechanism of low HDL-cholesterol. As a result, we observed a low HDL2-cholesterol level in ODS rats but normal HDL3-cholesterol level. But the authors observed no decrease of LCAT (lecithin: cholesterol acyltransferase) activity in serum of ODS rats. These results could be due to disturbance of lipid metabolism in a vitamin C-deficient condition, that is to say, there might be abnormalities of the cholesterol excretion pathway of bile acid from liver, and maturity of the HDL-cholesterol particle due to other factors except that of LCAT activity.
The effects of high calcium and/or high protein diet on bone growth were examined in growing rats kept at high ambient temperature. Thirty-five Wistar strain male rats aged 45 days were divided into 5 groups; room temperature, 24°C and a normal diet (CN), 34°C and a normal diet (HN), 34°C and a high calcium diet (HCa), 34°C and a high protein diet (HPr), and, 34°C and a high calcium-protein diet (HCaPr). The animals were given the same amount of feed. On day 35 of experiment, blood and femurs were collected from all rats. Physical traits, calcium and phosphorus contents of right femur were measured. The number of chondrocyte and the thickness of epiphyseal growth plate were measured in distal epiphysis of left femur. Alkaline phosphatase activities and protein contents were measured in the homogenate of proximal epiphysis and diaphysis of left femur. Ultrafiltrable calcium concen-trations and total concentrations of calcium and phosphorus were mea-sured, in serum. None of the treatments significantly affected the elon-gation and densities of calcium and phosphorus in femur. The bone growth in width was reduced at high ambient temperature and high protein diet furthermore reduced the bone growth in width at high ambient temperature. Total calcium and phosphorus concentrations were lower in serum at high ambient temperature. Total calcium concentration in serum was increased by high calcium diet and decreased by high protein diet in hot environment, , while ultrafiltrable calcium concentration in serum tended to be higher at high temperature. Since the number of chondrocyte and the thickness of epiphyseal growth plate were greater at high ambient temperature, there was a possibility that the length of femur might become longer at high ambient temperature when the animals were kept at high ambient temperature for a longer period than in the present study.
Model diet samples of eight groups (3, 16, 40, and 70 years old for both sexes), were analyzed for sodium, potassium, phosphorus, calcium, magnesium, iron, zinc, manganese, aluminum, copper, barium, nickel and molybdenum by using inductively-coupled plasma atomic-emission spectrometry (ICP-AES). Mineral intakes for 3 year olds were lower than those of other age groups because total food consumption was less. The intake of major elements, Na, K, P, Ca, and Mg, varied within about 20% of each other. The intake of other elements except aluminum fluctuated within 30% or more of each other. For most of the elements studied, the mineral intakes of 40-year-old males agreed within 16% with results previously obtained by a duplicated portion study.
We examined hair properties from determinations of diam-eter, amino acid composition, trace metal elements, and tensile strength of the hairs from protein-malnourished rats. The coarse or medium hair diameters of the experimental protein-malnourished rats had a tendency to decrease more than those of the control. Total contents of amino acids in the hairs had a tendency to decrease in the protein-deficient rats. Cystine content of rat hairs definitely decreased only in 5% wheat- and 5% rice-pattern amino acid mixture diet groups but not in 5% gluten diet and 5% casein diet groups. And many of the low-protein diet groups were significantly lower in methionine content than the control except for the 5% casein diet group. The Mg, Zn, and Fe contents in the hairs considerably increased in protein-deficient rats against the control. Tensile strength of coarse hairs in the experimental protein-malnourished rats was significantly lower than that of the control. The changes in amino acid composition of rat hair proteins are more likely to be influenced by various qualities of dietary protein or different compositions of amino acid mixtures in the diets at a similar dietary protein level. It should seriously be considered from our data whether the reduced cystine content in the hair can be regarded as an indicator of nutritional status.
Urinary and renal inhibitory activities of calcium oxalate monohydrate crystal growth in normal or vitamin B6-deficient rats were investigated. Renal inhibitory activity in vitamin B6-deficient rats was lower than that in normal rats. Renal inhibitory activity in vitamin B6-deficient rats was about 74% of normal level. Urinary inhibitory activity did not show a significant difference between normal and vitamin B6-deficient rats.
The effect of branched-chain amino acid (BCAA) on protein synthesis and nitrogen metabolism in liver and skeletal muscle was evaluated by an intraperitoneal injection of [15N]ammonium chloride (15NH4Cl) to carbon tetrachloride (CCl4)-intoxicated rats. The 15NH4Cl was bolusly injected at a dose of 6mg/100g body weight one hour after an amino acid solution containing leucine and valine (150mM each, abbre-viated as BCAA), phenylalanine and alanine (150mM each, PA), 120mM leucine and 30mM valine (Leu-rich) or 120mM valine and 30mM leucine (Val-rich) was administered intragastrically at a dose of 2.5ml each 100g body weight. The 15N-enrichment in the protein fraction of the liver was higher in CCl4-BCAA group than CCl4-PA group. The Leu-rich solution was found more effective in enhanced incorporation of 15N into liver and skeletal muscle proteins. The disappearance rate of [15N]urea from the plasma, which was influenced by the synthesis from 15NH3 and the excretion into urine, was much faster in the CCl4-BCAA group than CCl4-PA group. In Leu-rich group, both 15N incorporation into non-protein fraction of skeletal muscle and disappearance rate of plasma urea-115N were greater than those in Val-rich group. The results suggest that BCAA, particularly leucine, has beneficial effects on protein synthesis and am-monium detoxification in liver-injured rats.
Autoxidized linoleic acid (AL) having 800meq/kg of per-oxide value and 1, 700 meq/kg of carbonyl value was given in repeated oral doses at a daily dose of 0 (control)-7.5ml/kg to male Wistar rats for 5 successive days. The effect of increasing AL dose on the drug-metabolizing system was investigated in rat liver microsomes and S-9 fractions. All the rats of a daily dose of 5.0-7.5ml/kg died after the third day of consecutive oral doses. The cytochrome P-450 and b5 contents, enzyme activities in electron transfer system, aminopyrin-N-demethylase activity and S-9 activity (metabolic activation of 2-acetylaminofluorene) in the drug-metabolizing system changed essentially in a similar manner, that is, both the contents and the activities were increased by a small dose of AL, and were decreased by a large dose of AL. These results strongly supported the findings in a previous report wherein we observed the periodical effect of AL dose on the drug-metabolizing system.
The present investigation was conducted to determine if rats can recognize the essential fatty acid (EFA) in the diet. Weanling rats were pre-fed a 30% hydrogenated coconut oil (30HCO) diet containing 1% cholesterol for 2 or 4 weeks to produce EFA deficiency, then allowed to choose between the fat-free (FF) diet and the 0.5, 1.0, or 2.O% corn oil diet, and between the 0.5% HCO diet and 0.5% corn oil diet. Rats pre-fed the 30HCO diet for 2 weeks selected the corn oil-containing diets for a few days at the beginning of the experiment, but the rats pre-fed for 4 weeks could not recognize the EFA-containing diet. The non pre-fed animals continued to prefer the EFA-containing diet for about 2 weeks. From these results we could demonstrate that rats have a mechanism to distinguish the EFA in their diet, but this EFA selection ability was only in the early period of the experiment, and diminished soon after.
The effects of varying the ratio of polyunsaturated/saturated fatty acids (P/S) and ω3/ω6 polyunsaturated fatty acids (PUFA) of dietary fats on lipid metabolism were studied in rats using safflower oil (SF0), linseed oil (LSO), palm oil (PLO), and a 1:1 com-bination of these oils. The hypocholesterolemic and hypotriglyceride-mic effects depended on the P/S ratio of dietary fats, LSO (ω3 PUFA) being more effective than SFO (ω6 PUFA). A similar pattern of the response was observed on liver cholesterol and triglyceride. The liver cholesterol-lowering effect of LSO, but not SFO, remained even when they were combined with PLO. The activity of liver Δ6-desaturase tended to be higher while that of liver phospholipase A2 was significantly lower in the LSO group than in the SFO or PLO groups. The aortic PGI2 production and the production by platelets of thromboxane A2 were significantly low in rats fed LSO accompanying a distinct reduction of arachidonate in tissue phospholipids. The depressing effect of LSO disappeared when it was combined with SFO but not with PLO. There were no significant differences in enzyme activities and eicosanoid production between SFO and PLO in spite of a large difference in their P/S ratio. Thus, lipid parameters examined were complicatedly regulated by the ratios of ω3/ω6 as well as P/S, suggesting an existence of an appropriate ratio for these variables.
The hydrolyzing activities in rat small intestine for newly developed sugar substitutes, α-D-glucopyranosyl-l, 6-sorbitol (GPS) and α-D-glucopyranosyl-l, 6-mannitol (GPM), both of which are produced by hydrogenation of palatinose, were characterized. GPS and GPM were hydrolyzed in mucosal homogenate as well as in brush border membranes of rat small intestine at a slower rate than the rate of hydrolysis of palatinose (30%) and sucrose (6-7%). Gel filtration column chromatog-raphy of disaccharidases solubilized from brush border membranes revealed that GPS and GPM were hydrolyzed mainly by sucrase-isomaltase complex and its degradation product, i.e. isomaltase monomer. The isomaltase monomer, purified from small intestine of rats, possessed similar Km values for GPS (2.47mM) and GPM (5.38mM) as compared to those of purified sucrase-isomaltase complex. The Vmax values of isomal-tase monomer for GPS and GPM were twice as high as those of sucrase-isomaltase, suggesting that GPS and GPM are hydrolyzed by the active site of isomaltase. On the other hand, a small amount (up to 17%) of GPS-and GPM-hydrolyzing activities was ascribed to glucoamylase, which possessed relatively high Km values for GPS (18.7 mM) and GPM (32.9mM). To examine a physiological significance of GPS- and GPM-hydrolyzing activities, the transmural potential difference (ΔPD) evoked by Na+ -dependent active transport of glucose, produced by the hydrolysis of these disaccharide alcohols, was measured in everted segments of rat jejunum. The relative rates of absorption of glucose produced by the hydrolysis of GPS and GPM were 36% and 27% of that of palatinose, directly reflecting the hydrolyzing activities determined in jejunal ho-mogenate. These results suggest that the process of hydrolysis is the rate limiting step in digestion-absorption process of palatinose, GPS and GPM in small intestine.
The addition of 1, 4-diazabicyclo-[2, 2, 2] octane (DABCO) (100mM) or 1, 2, -dihydroxybenzene-3, 5-disulfonic acid (Tiron) (1mM) to a reaction mixture containing 10mM linoleic acid (LA), 20% EtOH, and 135μM dehydroascorbic acid (DHA) as a catalyst suppressed LA per-oxidation, but the addition of mannitol (100mM), uric acid (100μM), and catalase (6.5 units) did not. DHA or 2, 3-diketo-L-gulonic acid (DKG) accelerated LA peroxidation, but the splitting products of DHA did not affect LA peroxidation. These results suggest that some specific radicals are liberated in the degradation of DHA or DKG.
The effect of a supplementary food mix prepared from roasted mungbean, groundnuts, and sugars on maternal nutrition and birth weight of newborns was studied. The results indicated that sup-plementary food provided to the pregnant women had a significantly positive effect on the birth weight of newborns. In addition, a significantly higher weight gain by the mothers was observed as compared to the control group.