Content of liver and brain ubiquinol-9 and ubiquinol-10 after chronic ethanol intake in rats subjected to two levels of dietary α-tocopherol

To assess the effect of chronic ethanol ingestion in the content of the reduced forms of coenzymes Q₉ (ubiquinol-9) and Q₁₀ (ubiquinol-10) as a factor contributing to oxidative stress in liver and brain, male Wistar rats were fed ad libitum a basal diet containing either 10 or 2.5 mg α-tocopherol/100 g diet (controls), or the same basal diet plus a 32% ethanol-25% sucrose solution. After three months treatment, ethanol chronically-treated rats showed identical growth rates to the isocalorically pair-fed controls, irrespectively of α-tocopherol dietary level. Lowering dietary α-tocopherol led to a decreased content of this vitamin in the liver and brain of control rats, without changes in that of ubiquinol-9, and increased levels of hepatic ubiquinol-10 and total glutathione (tGSH), accompanied by a decrease in brain tGSH. At the two levels of dietary α-tocopherol, ethanol treatment significantly decreased the content of hepatic α-tocopherol and ubiquinols 9 and 10. This effect was significantly greater at 10 mg α-tocopherol/100 g diet than at 2.5, whereas those of tGSH were significantly elevated by 43% and 9%, respectively. Chronic ethanol intake did not alter the content of brain α-tocopherol and tGSH, whereas those of ubiquinol-9 were significantly lowered by 20% and 14% in rats subjected to 10 and 2.5 mg α-tocopherol/100 g diet, respectively. It is concluded that chronic ethanol intake at two levels of dietary α-tocopherol induces a depletion of hepatic α-tocopherol and ubiquinols 9 and 10, thus contributing to ethanol-induced oxidative stress in the liver tissue. This effect of ethanol is dependent upon the dietary level of α-tocopherol, involves a compensatory enhancement in hepatic tGSH availability, and is not observed in the brain tissue, probably due to its limited capacity for ethanol biotransformation and glutathione synthesis.     

Fragility of Erythrocytes from Chicks and Rats Fed Dilauryl Succinate and Related Compounds

Susceptibility to hydrogen peroxide of the erythrocytes from chicks and rats fed dilauryl succinate and related compounds with and without supplementation of dl-α-tocopheryl acetate was determined.

Dilauryl succinate, lauryl alcohol, n-decyI alcohol, myristyl alcohol, and lauraldehyde were confirmed to make the erythrocytes from the chicks fragile. Supplemented dl-α-tocopheryl acetate of 200 mg per kg of diet completely prevented the hemolysis induced by these compounds. Dilauryl succinate also makes the rat’s erythrocytes fragile and supplemented dl-α-tocopheryl acetate prevented the hemolysis of the rats, but ethoxyquin was not. The symptoms of encephalomalacia in the chick is preceded by increased hemolysis value of the erythrocytes, and this hemolysis value dropped after the appearance of encephalomalacia.     

Studies on the Metabolic Activity of Muscle Proteins

The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others.     

Effect of α-Tocopherol on Endotoxicosis

The effect of α-tocopherol in endotoxicosis was studied. The α-tocopherol level significantly decreased in mouse liver 18 hr after endotoxin administration, thereafter tending to increase to approach the normal range. In endotoxin-tolerant mouse liver, the lipid peroxide level was reduced to less than half of that in nontolerant animals following endotoxin challenge. The liver lipid peroxide level and serum lactate dehydrogenase or acid phosphatase leakage were studied in mice fed a vitamin E-deficient (ED) diet and a vitamin E-supplemented (ES) diet for 40 days. ED mouse liver exhibited a higher formation of lipid peroxide after endotoxin was given while there was a markedly lower level in ES mouse liver. There was significantly more serum lactate dehydrogenase or acid phosphatase leakage in ED mice than in ES mice after endotoxin administration. There was about a 25% decrease in liver superoxide dismutase (SOD) activity in endotoxin-poisoned mice fed both the normal and the ED diets, while the activity was at a higher level in ES-fed mice. These results suggest that α-tocopherol may be helpful in preventing membrane instability in endotoxin poisoning.     

Observations on the mechanism of the oxygen/dialuric acid-induced hemolysis of vitamin e-deficient rat red blood cells and the protective roles of catalase and superoxide dismutase.

We have demonstrated that the hemolysis of vitamin E-deficient rat erythrocytes induced by ~1 mm levels of dialuric acid occurs in three distinct phases: (1) The red cell is modified in an unknown manner in the brief time (~2 min) during which dialuric acid is oxidized by O₂ to alloxan and H₂O₂. (2) Lipid peroxidation subsequently occurs. (3) When lipid peroxidation approaches ~75% of its maximal value hemolysis begins to occur. As measured by low-temperature EPR spectroscopy, free radicals, if formed, did not accumulate to a concentration greater than 0.1 μm. During the first phase, catalase or a mixture of catalase and superoxide dismutase (but not superoxide dismutase alone) offered considerable protection against hemolysis, while during the second phase external addition of these enzymes generally gave no protection against hemolysis and occasionally hemolysis was enhanced. Results are presented which strongly suggest that the species formed during the oxidation of dialuric acid which is active toward the cell is neither superoxide ion nor hydrogen peroxide nor a product of these substances. It is proposed that catalase reacts directly with the deleterious intermediate.     

Quantitative Significance of Plasma Albumin Metabolism in the Whole Body Protein Turnover in Rats

The amount of extra- and intravascular albumin was estimated in two groups of rats, i.e., those fed a 20% casein (20% protein) diet and a 3% casein (low protein or 3% protein) diet.

The fractional turnover rate of whole body plasma albumin was also measured in the two groups of rats, employing the constant infusion method of Waterlow et al. At the same time, the fractional turnover rate of the whole body protein was measured.

When the diet was changed from the 20% protein to the 3% protein diet, the amount of albumin mass in both extra- and intravascular spaces decreased significantly. During 7 days on the diet, the extra- and intravascular albumin mass per 100 g of body weight did not change significantly in the rats fed the 20% protein diet. On the other hand, rats fed the 3% protein diet lost almost 30% of the extra- and intravascular albumin per lOOg body weight.

The fractional turnover rates of whole body albumin were estimated to be 31.7 and 19.8%/day in the 20% protein and the 3% protein diet-fed rats, respectively. The fractional turnover rates of whole body protein were 16.1 and 10.6%/day in the 20% protein and the 3% protein diet-fed rats, respectively.

The leucine fluxes to albumin synthesis and whole body protein synthesis were calculated to be 5.9 and 83 μmol/hr, respectively, in the 20% protein diet-fed rats. The leucine fluxes in the 3% protein diet-fed rats were 2.5 and 54μmol/hr for the albumin synthesis and for the whole body protein synthesis, respectively.

These results demonstrate the quantitative significance of albumin metabolism in the whole body protein turnover in rats fed on two levels of protein intake.     

Distribution of α-Tocopherol Stereoisomers in Rats

The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on a diet containing all-rac-α-tocopheryl acetate was investigated by a newly revised HPLC. The concentrations of 2R-isomers of α-Toc in blood and tissues of the rats were significantly higher than those of 2S-isomers. In most tissues, the levels of 2S-isomers were in order SRS> (SSS +SSR)/2 > SRR.     

Carboxyl Proteinase Essential for Maturation of Basidiospores of Lentinus edodes (shiitake)

The effects of eicosapentaenoic acid (EPA, 20: 5n-3) on essential fatty acid (EFA)-deficient rats were studied. After low growth and scaly dermatitis in the hind legs due to dietary EFA deficiency were induced by feeding rats an EFA-free 25 % casein diet (25C) containing 30 % hydrogenated coconut oil with 1 % cholesterol (HCO ? CHOL) for 8 weeks, they received the 25C diet with 0.19 or 0.57 % EPA ethyl ester concentrate added, or 0.02 % or 0.38 % linoleic acid (LA, 18: 2n-6) concentrate (Exp. I), and the HCO ? CHOL meal including any one of 0.25, 0.50, or 1.00 % EPA concentrate, and 0.12 and 0.48 % LA concentrate (Exp. II) for an additional 6 weeks. When EFA-deficient rats were fed the EPA in both experiments, body weight was gained to almost reach those of the 0.38 or 0.48 % LA-fed group (control), and the dermal symptoms of the hind legs were relieved, though the degree of healing was less than those of the controls. The ratios of eicosatrienoic acid (20: 3n-9) to arachidonic acid (20: 4n-6) characteristically increased due to EFA deficiency were reduced to the level of the control in the liver and heart by addition of the EPA concentrate.     

Utilization of α-starch in ayu, Plecoglossus altivelis, relating to growth and body composition

To examine the possibility of dietary α‐starch in reducing feed costs in a practical diet, α‐starch was supplemented at 10, 20, 30 and 40% in a composed diet having the same protein level. The four diets were fed to ayu, Plecoglossus altivelis (initial weight 9.1 g) for 43 days. Growth and feed efficiency increased with the supplement, with values highest in the 30–40%α‐starch diet. The level of dietary α‐starch did not affect the proximate muscle composition; although the hepatosomatic index was not affected, liver glycogen increased with increasing dietary α‐starch. The dietary α‐starch did not influence evacuation time from the gut, and was well digested through passage in the gut, mainly between the stomach and the anterior part of the intestine. Ayu have an ability to adapt their metabolism to high dietary α‐starch, and can digest 40% or more in a composed diet. Although the muscle lipid content did not change, the fatty acid composition was influenced by dietary starch. With the elevation of dietary starch, a decrease of C18:2n‐6 and an increase of C22:6n‐3 occurred. These results indicate that at least 40%α‐starch can be used in practical diets for ayu.     

Lunatoic Acid A,a Morphogenic Substance Inducing Chlamydospore-like Cells in Some Fungi

The effect of the addition of 0.26 % free tryptophan (Trp) to a 20 % casein diet containing 6 mg of nicotinic acid per 100 g of diet on the ratio of N¹-methyl-2-pyridone-5-carboxamide (2-py) plus N¹-methyl-4-pyridone-3-carboxamide (4-py) to -methylnicotinamide (MNA) excretion was investigated in rats. The urinary excretion of MNA, 2-py and 4-py, respectively, increased statistically significantly with the feeding of a 0.26% Trp (the same as the content of the 20% casein diet) supplemented 20% casein diet, although it did not increase with the feeding of a 40% casein diet, compared with in the case of the 20 % casein diet [Agric. Biol. Chem., 52, 1765 (1988)]. So, the total urinary excretion of Nam and its metabolites was 1.8 times higher in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet. However, the ratio of 2-py plus 4-py to MNA excretion was much lower in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet (13.16 ± 3.75→5.49 ± 2.25). This decreased ratio was considered to be partially due to a decrease in the 4-py forming MNA oxidase, which decreased significantly with the feeding of the Trp supplemented diet. Furthermore, the metabolic fate of Trp was greatly affected by the form of Trp, free or bound.     

Studies on the Nutritional Value of Tempeh

1. The nutritional value of tempeh in comparison with that of unfermented soybeans was studied in rat feeding experiment. It was observed that the PER value of tempeh (fresh or stored) was not significantly different from that of the unfermented soybeans (fresh or stored).

2. The peroxide value of the oil of stored tempeh powder was only 10% of that of stored soybean powder. Red blood cells of rats receiving tempeh were less than 20% in hemolysis by dialuric acid whereas hemolysis was 100% for the rats receiving the unfermented soybeans. Serum tocopherol was 0.14±0.05 mg/dl in the former group, but 0.07±0.05 mg/dl in the latter. Liver TBA values were 0.20±0.05 O. D./g and 0.65±0.13 O. D./g in the tempeh and the unfermented soybeans group, respectively.

3. Subsitution of whole egg for tempeh to supply 30% of protein in the diet improved the quality of the protein as measured by the protein efficiency ratio. An equal improvement was accomplished by supplementation of tempeh with lysine, methionine, and threonine in such amounts as the level of these amino acids equal to that in the tempeh-egg diet.     

n-Hexanal and Some Volatile Alcohols

Both α-tocopherol and a 1: 1.7 mixture of α-tocopherol and tocotrienols at a 0.2% dietary level significantly depressed the age-related increase in the systolic blood pressure of spontaneously hypertensive rats (SHRs) after 3 weeks of feeding. The aortic production of prostacyclin was increased 1.5 times both by α-tocopherol and a tocotrienol mixture, suggesting a possible relevance to their hypotensive effect. These vitamins did not influence the Δ6- and Δ5-desaturase activities of liver microsomes, but fatty acid profiles of the liver phospholipids predicted a reduction of linoleic acid desaturation. These effects were in general more clear with tocotrienols than with α-tocopherol. Platelet aggregation by 5 μM ADP remained uninfluenced. Thus, tocotrienols may have effects on various lipid parameters somewhat different from those of α-tocopherol.     

Changes in Creatine and Urea Formation in Rats Fasted and Fed Low Protein Diets

Changes in the time course of the urinary excretion of creatinine, creatine and urea, and the activities of kidney transamidinase and liver urea-cycle enzymes were investigated in rats fasted and fed on a 10% casein diet and 10% casein diets supplemented with 10% glycine and/or 1.4% arginine.

The urinary total-creatinine of the fasted rats increased extremely during fasting for 7 days, while that of the animals given the 10% casein diet supplemented with glycine and arginine rose exceedingly on the 3rd day and thereafter no significant change was observed. Most of the increase of total-creatinine could be accounted for by the increase of creatine. The activity of kidney transamidinase in the fasted rats decreased in the 3rd day and thereafter kept nearly constant. The transamidinase activity of rats fed on the 10% casein diet after giving a protein-free diet for 5 days increased in the 3rd day. An inverse relation was observed between the urinary creatine and the transamidinase activity. The urinary urea increased in the rats fasted or fed on the 10% casein diets with the supplement of glycine and/or arginine. In fasting, the activities of liver urea-cycle enzymes, except arginase, had a tendency of increasing with the lapse of time. The arginase activity remained more or less constant. The reason of the extreme increase of urinary creatine during starvation was discussed.     

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.     

Inhibition of reproduction of Xyleborus ferrugineus by ascorbic acid and related chemicals

Effects of various dietary chemicals on the reproduction of the ambrosia beetle, Xyleborus ferrugineus were studied. Ascorbic acid, araboascorbic acid, dehydroascorbic acid, hydroquinone, catechol, cysteine, and α-tocopherol each inhibited progeny production by virgin females. Detailed studies of the effects of ascorbic acid showed that it inhibited progeny production by causing a nutritionally detrimental non-enzymic browning of the dietary casein. Amino acid analyses of such browned and unbrowned casein, after in vitro digestion with pepsin and pancreatin, showed that lesser amounts of certain amino acids were released from the browned material. Effects of the non-enzymic browning were overcome by increasing the casein component of the diet. It was further concluded that araboascorbic acid, dehydroascorbic acid, hydroquinone, and catechol probably inhibit reproduction of X. ferrugineus by the same mechanism as ascorbic acid. No explanation of the inhibitory action of α-tocopherol on X. ferrugineus is offered.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Distribution of Selenium in Bovine Milk and Selenium Deficiency in Rats Fed Casein-based Diets,Monitored by Lipid Peroxide Level and Glutathione Peroxidase Activity

A major proportion of selenium in bovine milk was found in fluorometric analysis to be associated with the casein fraction, largely in alkali-labile form, and the rest with the whey fraction mostly in free selenite form. This uneven distribution of milk selenium seems to provide an explanation for selenium deficiency in purified caseins. The activity of glutathione peroxidase, a selenoprotein, in the liver of growing male rats fed ad libitum low-selenium diets containing either vitamin-free casein or Torula yeast 0.065 ± 0.012 or 0.015 ± 0.004 μ g Se/g diet, respectively) for 3 weeks decreased to 4 to 6% of that of the control rats fed a commercial stock diet (0.185 ± 0.092 μ g Se/g diet). Selenium status was evaluated by three different parameters for the rats assigned under pair-feeding regimen to those vitamin-free casein-based diets which were supplemented with graded levels of selenium as sodium selenite. The hepatic levels of the thiobarbituric acid-reactive substance, an indication of lipid peroxidation, decreased to control level with selenium supplementation per g diet of 0.1 μ g and over. The hepatic glutathione peroxidase activity reached a plateau above a 0.1 μ g/g diet of selenium supplementation, whereas the erythrocyte enzyme activity increased with increasing levels of supplementary selenium. These results support the notion that semi-purified diets containing vitamin-free casein as a prime protein source would not satisfy the selenium requirement of growing animals unless deliberately supplemented with additional selenium.     

In Vivo Antioxidant Activity of Okara Koji,a Fermented Okara,by Aspergillus oryzae

The antioxidants in Okara Koji (OK), an okara (OC) fermented by Aspergillus oryzae, γ-tocopherol, δ-tocopherol, genistin, daizein, genistein, and 3-hydroxyanthranilic acid were identified by HPLC. OK’s extract with 80% methanol strongly inhibited linoleate peroxidation, much more than other OK’s extracts with hexane or hot water. The methanol extract accelerated 12-hydroxyeicosatetraenoic acid formation in membrane lipids at 10^?3 concentration, but inhibited the formation at higher concentrations than 10^?3 ex vivo. To confirm the total effect of all components of OK on lipid peroxidation in vivo, rats fed food deficient in vitamin E were put on diets containing OK or OC with oxidized oil. In rats fed the OK diet, no effect of oxidized oil feeding on the body weight gain, of the TBA value in plasma, or of glutathione peroxidase activities of plasma and liver was observed. But in rats fed the OC diet, the effect of oxidized oil feeding was apparently observed on all of those values. These results suggested that OK would scavenge lipid peroxides in vivo.     

Effect of casein and soy protein isolate on vitamin B6 nutritional status in rats

Two experiments were conducted to test the effect of casein and soy protein isolate (SPI) on the nutritional status of vitamin B₆ in rats. Adult Long-Evans rats were fed with a casein or SPI diet at a 40% protein level with (control) or without (B₆-deficient) 7 mg of pyridoxine/kg diet. Vitamin-B₆-deficient rats were depleted of B₆ with (experiment 1) or without (experiment 2) deoxypyridoxine. In experiment 1, each rat was loaded with 150 mg ofDL-tryptophan after 5 weeks of pair feeding. The rats on the vitamin-B₆-deficient SPI diet (SPI-B₆) excreted twice the amount of urine xanthurenic acid in 24 h than did the rats on the vitamin-B₆-deficient casein (casein-B₆) diet (p<0.05). In experiment 2,L-tryptophan was loaded in a 20-mg dose at the end of each week. The excretion of xanthurenic acid was higher in the SPI-B₆ group than in the casein-B₆ group over the 5-week period of the experiment (p<0.05). Erythrocyte transaminase (EGOT and EGPT) activities were lower, while EGOT and EGPT indexes were higher in the SPI-B₆ group than in the casein-B₆ group (p<0.05). The results suggest that the source of dietary protein significantly influenced the status of B₆ nutrition in these rats.