Several subforms of Cytosolic aspartate aminotransferase (AspATc) in the crude extracts of rat liver and kidney were separated by isoelectric focusing using immunoblotting and staining of activity to detect the enzyme protein and activity, respectively. Vitamin B6-deficiency resulted in decrease in the subforms with higher isoelectric points and increase in those with lower ones both in the liver and in the kidney. When pyridoxal phosphate was added to those preparations from vitamin B6-deficient rats, the isoelectric focusing pattern of kidney was recovered to the similar one to that of the controls. However, the liver preparation was affected only partially by the addition of PLP. The pattern of subforms was altered during in vitro incubation at 37°C for 24 h in both liver and kidney preparations, and their patterns were very similar to that of liver preparation from VB6-deficient rats. The enzyme activity also decreased during this incubation, especially in preparations of the enzyme from the liver of vitamin B6-deficient rats. This loss of enzyme activity was not affected by addition of PLP alone, but was almost completely prevented by addition of substrate. The inactivation was recovered by addition of substrate and pyridoxal phosphate simultaneously. This finding suggests that the inactivation may be related with a conformational change around the catalytic site of the AspATc molecule.
Two distinct cellular retinol-binding proteins were detected in chicken small intestine. A predominant form was purified to homogeneity. The apparent molecular weight of this protein was estimated to be 17, 200. This form was larger than a second minor form partially purified (molecular weight of 15, 000). The absorption and fluorescence spectra of the bound retinol to the purified proteins were typical for the known cellular retinol-binding proteins. The results suggest that the purified binding protein corresponds to CRBP(II), previously identified in small intestine of rats and humans. To gain an insight into the possible role of CRBP (II) in chicken small intestine, the CRBP(II) contents in cytosols of small intestine of embryonic and post-hatch chicks were determined by enzyme-linked immunosorbent assay. The amount of CRBP(II) per unit DNA in small intestine was low at 15- and 17-day embryonic stage, but rapidly increased around the period of hatching. The increased level was still maintained in 6-week-old chicks, which accounted for 0.9% of total proteins in duodenum. The developmental pattern and the presence of abundant amount of CRBP(II) in chicken small intestine supports the hypothesis that CRBP(II) might play some role in the intestinal absorption of retinoids. Thus the involvement of a tissue-specific cellular retinol-binding protein in the intestinal absorption of retinoids appears to be common in mammalian and avian species.
Effect of proportion of three branched-chain amino acids (leucine, valine, and isoleucine: BCAA) for nitrogen utilization was studied in vivo by an intragastric administration of 15N-L-leucine to control rats and liver-injured rats treated with carbon tetrachloride (CCl4-rats). Following the administration of isonitrogenous dose of three amino acid solutions [Standard (i 5N-L-leucine, L-valine, L-isoleucine, and L-alanine; 11, 8, 6, 18 mg/ml), Low-Val (11, 2, 6, 23 mg/ml), and High-Val (11, 32, 6, 0 mg/ml)], 15N enrichments in serum albumin, liver, skeletal muscle, and brain proteins and non-protein fractions, and urea nitrogen were compared by using 15N-analyzer. In CCl4-rats, the 15N enrichment in liver protein fraction was significantly lower in the High-Val group than in the Low-Val group. However, the difference of 15N enrichment in serum albumin between Low-Val and High-Val groups in CCl4-rats was unclear. The 15N enrichments in non-protein fractions of the brains in CCl4-rats were about twofold those in the skeletal muscles and the highest 15N enrichment was recognized in the Low-Val group. In the non-protein fraction of skeletal muscle in CCl4-rats, significantly low 15N enrichment was shown in the High-Val group compared with the Low-Val group. The 15N enrichment in urinary urea was significantly higher in the High-Val group than in the Low-Val group in CCl4-rats. In the Standard group of control rats, 15N enrichments in serum albumin and protein fraction of skeletal muscle were higher than in other groups. In non-protein fractions of control rats, the lowest 15N enrichment in liver and the highest 15N enrichment in skeletal muscle were recognized in the Standard group. These results suggest that a large amount of valine supplement among BCAA is less useful for leucine utilization in liver-injured rats than in normal rats.
Chemiluminescence (CL) from tissue preparations of mice i.p. injected with and without the chemical carcinogen benzo[a]pyrene (BP) was detected by a single photon counting apparatus. The spontaneously emitted CL (spontaneous CL) and the CL after luminol addition (luminol-CL) were measured for mice with and without previous induction of their liver mixed-function oxidases (MFO) by phenobarbital. In MFO-non-induced mice, although the spontaneous CL was not notably modified by BP injection, the kidneys presented three times greater luminol-CL after BP injection. On the other hand, MFO-induced mice had higher spontaneous CL of plasma, liver, kidneys, and lungs, as well as higher luminol-CL of liver after BP injection, when compared with the respective MFO-induced but BP-non-injected mice. The luminol-CL of liver was suppressed by scavengers of active oxygen and free-radicals such as TIRON, butylhydroxytoluene and in a less extent by superoxide dismutase. The CL detected from tissue preparations of mice after BP treatment is thought to reflect the formation of oxygen radicals and electronically excited-species during BP metabolism.
An α-amylase inhibitor prepared from cranberry bean (Phaseolus vulgaris) was examined for its in vivo action on pancreatic α-amylase in rat small intestine. For this purpose, postprandial changes not only in intraluminal α-amylase activity but also in plasma glucose and insulin concentrations were measured at various times after administration of 2 g of 10% polyethylene glycol-containing experimental diets with and without the inhibitor. No considerable increase was observed in the levels of intraluminal α-amylase activity, blood sugar, and plasma insulin in the animals given the inhibitor at a dose of 10 mg each. These results suggest that the purified preparation from cranberry bean serves in fact as a potent inhibitor of rat pancreatic α-amylase.
It was previously reported that the hypercholesterolemia induced by polychlorinated biphenyls (PCB) was influenced by dietary protein quantity and quality. On the other hand, the supplementation of methionine and threonine to a nonprotein diet ameliorated the body weight loss and decreased the urinary urea excretion in rats. We examined the effect of methionine and threonine supplements on the hypercholester olemia induced by PCB in rats fed a nonprotein diet. The administration of PCB increased plasma cholesterol concentration and the supplements of methionine and threonine to the nonprotein diet significantly accel erated the elevation of plasma level of cholesterol due to PCB feeding. Liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase ac tivity in rats fed the nonprotein diet was also elevated by PCB administra tion and the supplementation of methionine and threonine caused further inducing effect.
Incorporation and metabolism of γ-linolenic acid (GLA) in both rat hepatocytes and Hep G2 cells were compared to those of oleic (OA), linoleic (LA), α-linolenic (LLA), and dihomo-γ-linolenic (DGLA) acids. The incorporation of GLA into both types of cells was higher than LLA and DGLA, but lower than OA and LA. It was efficiently converted into DGLA in both types of cells and increased the concentration of DGLA. LLA was converted to a small amount of C20 : 4 (n-3) only in Hep G2 cells. Incubation with LA, GLA, LLA, and DGLA did not increase the concentration of arachidonic acid (AA) in both types of cells. LA. GLA, LLA, and their metabolites were incorporated into phosphatidylcholine, but only GLA and its metabolite, DGLA, were also incorporated into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The coexistence of GLA and LLA during their catabolism diminished the amounts of respective metabolite in Hep G2 cells. The presence of GLA inhibited completely the formation of C20:4(n-3) from LLA. The results indicate that GLA is more effective in raising the ratio of DGLA/AA. Also, polyunsaturated fatty acids of n-3 and n-6 series have competitively catabolized in both types of hepatocytes.
The effects of methyl-group acceptors such as glycine, guanidinoacetic acid, and nicotinamide on cholesterol metabolism and phosphatidylcholine(PC) biosynthesis were investigated with rats fed a 25% casein diet containing cholesterol with or without methionine supplement. The effect of ethanolamine, an indirect methyl-group acceptor via phosphatidylethanolamine(PE) formation, was also compared with those of methyl-group acceptors. The methyl-group acceptors and ethanolamine decreased or tended to decrease plasma total cholesterol level when added to the 25% casein diet. These compounds also significantly depressed the methionine-induced enhancement of plasma cholesterol level. The activity of PE N methyltransferase was decreased by the addition of glycine, guanidinoacetic acid, and nicotinamide, but not ethanolamine, to the reaction mixture when assayed using the postmitochondrial fraction of liver homogenate, suggesting that PE N methyltransferase activity can be depressed by glycine N methyltransferase, guanidinoacetic acid N methyltransferase, and nicotinamide N methyltransferase systems. The PE N-methyltransferase activity in liver microsomes, however, did not decrease in response to the dietary addition of methyl-group acceptors. The in vitro incorporation of [CH3-14C]methionine into PC of liver slices was also significantly inhibited by the addition of glycine and nicotinamide, but not guanidinoacetic acid and ethanolamine, to the incubation medium. It is suggested that methyl-group acceptors can decrease plasma cholesterol level at least in part through the depression of PC biosynthesis via PE N-methylation pathway, and that the mechanism for the plasma cholesterol-lowering effect of ethanolamine is different from that of methyl-group acceptors.
Copper (6 ppm) was administered to pregnant heterozygous brindled and normal mice from 13 to 18 days gestation. The copper and zinc concentrations in the cerebrum, cerebellum, liver, and kidneys of mothers and their fetuses were determined. The placental concentrations in fetuses of heterozygous mothers administered copper were also determined. The heterozygous mothers had smaller numbers of live fetuses than the normal mothers, but had the same number as normal mothers when copper was administered. The hepatic copper concentration in the heterozygous mothers was lower than that in the normal mothers and was not increased by the administration. The body and tissue wet weights of all fetuses were unaffected by the maternal genotype or drinking fluid. The cerebral copper concentrations in hemizygous and heterozygous fetuses were increased by the copper administration but did not reach normal levels. The hepatic and renal concentrations remained unchanged. The cerebral copper concentrations in normal fetuses of both heterozygous and normal mothers were increased by the copper administration. The copper administration increased the copper concentrations in liver of normal fetuses of heterozygous mothers and in kidneys of normal fetuses of normal mothers. The placental copper concentration in hemizygous fetuses was higher than those in heterozygous and normal fetuses. These results suggested that oral copper administration to pregnant females could improve an abnormal copper distribution in hemizygous and heterozygous fetuses without affecting fetal growth.
To clarify the relationship between the pattern of diet intake and the circadian adrenocortical rhythm, we measured plasma cortisol levels at 4-h intervals over a 24-h period in 18 patients who were in the vegetative state and had been receiving total enteral nutrition (TEN) for 4 weeks. One group of 6 patients was given a liquid diet intraduodenally and continuously throughout a day (continuous TEN), whereas the two other groups received their daily enteral feeding during a restricted time of day, either in the daytime from 0800 h to 2000 h (diurnal TEN, 6 patients) or in the nighttime from 2000 h to 0800 h (nocturnal TEN, 6 patients). In patients with diurnal TEN, there was a clear cortisol rhythm with a peak at 0800 h, whose pattern was quite similar to the well-established cortisol rhythm in normal subjects. Patients with nocturnal TEN also showed a cortisol rhythm, but the peak appeared at 1600 h. There was no appreciable difference in the amplitude of the rhythm between the two groups. Patients with continuous TEN did not show any consistent circadian cortisol rhythms. Plasma levels of glucose, insulin, and free fatty acids also showed circadian fluctuations corresponding to the pattern of diet infusion in the groups with diurnal and nocturnal TEN, and remained almost constant throughout a day in the group with continuous TEN. We conclude from these results that the timing of diet intake may have a synchronizing effect on the circadian cortisol rhythm in man, as it does in laboratory animals.
The transfer of orally administered α-tocopherol (1.1 g) into breast milk reached a maximum value of 414μg/100 g milk, which was 6.6-fold the pre-supplemental level, after three days and declined to the base line level after five days. The amount of α-tocopherol recovered in the milk was 0.11%. The α-tocopherol equivalent/PUFA ratio (mg/g) was increased from 0.25 to values between 0.7 and 1.7 by the administration.
An experiment was conducted with growing rats to examine the influence of dietary addition of 0.1% polychlorinated biphenyls (PCB) and dietary level of protein on the distributions of α-tocopherol and cholesterol in serum lipoproteins and tissues. Addition of PCB increased serum α-tocopherol. This was mainly due to the increment in the fractions of chylomicrons/very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL). This chemical also increased serum cholesterol and HDL cholesterol. The contents of α-tocopherol and cholesterol in HDL fraction were higher in the high protein groups as compared to the low protein groups, regardless of PCB intake. PCB intake increased the concentrations of α-tocopherol in tissues including spleen, lung, kidney, testis, muscle, adipose tissue, and brain. High protein diet increased the levels of α-tocopherol in these tissues as compared to low protein diet. PCB intake also increased adrenal α-tocopherol, which was potentiated with low protein diet. The increment in liver α-tocopherol by PCB intake was observed only in the animals fed on low protein diet.
The production of vitamin B12 from carbohydrates, peptone, casamino acid, etc., by intestinal bacteria was investigated. Klebsiella pneumoniae IFO 13541 was the most efficient strain for vitamin Laboratory of Food Microbiology, Department of Food Science, Faculty of Home Economics, Mukogawa Women's University production, which depended exclusively on the concentration of yeast extract added to the medium. A concentrated solution of yeast extract (1 ml) was chromatographed on a Sephadex G-25 column (1×180 cm) and eluted with H2O (eighty fractions of 3 ml each were collected). It was found that fractions in which bacterial growth was most prevalent also exhibited the highest amount of vitamin production. The effectiveness of yeast extract was shown by the participation of pyrroloquinoline quinone and aspartic acid in the growth stimulation and in the vitamin B 12 production in this strain.
To clarify the physiological significance of circadian rhythmic changes in the activity of intestinal sucrase, the activity of digestion and absorption of sucrose in vivo were assessed during the daytime and the nighttime in rats fed ad libitum. When the jejunum was perfused at night with a solution containing sucrose in situ, the disappearance rate of sucrose from the lumen was higher than when perfused during the daytime, in parallel with the day/night difference in sucrase activity. The early response of plasma glucose to oral sucrose load in unanesthetized free-moving rats was also greater during the nighttime than the daytime. It was concluded that the digestion and absorption rate of sucrose shows circadian fluctuations under normal physiological conditions.