8-Hydroxydeoxyguanosine (8-OH-dG) was formed in deoxyguanosine (dG) solution by the addition of Fe2+, ascorbic acid (AscA), EDTA, and H2O2, and was then detected by ECD after separation by HPLC. 8-OH-dG was not formed in significant amounts when either Fe2+, AscA, or H2O2 was added separately, or when two of these were added in combination. When Fe2+, AscA, and H2O2 were added simultaneously, however, 0.87nmol of 8-OH-dG was formed from 2.5μmol of dG. And the addition of EDTA (in the same concentration as Fe2+) to the Fe2+/AscA/H2O2 system caused a remarkable increase in the amount of 8-OH-dG formed (up to 2.89nmol from 2.5μmol of dG). The amount of 8-OH-dG formed increased as the concentration of AscA increased up to 1mM (the same concentration as Fe2+). However, higher concentrations of AscA inhibited the formation of 8-OH-dG.
The present study was carried out to isolate and identify the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase)-inhibiting agents in the lipids of fermented soybeans, Tempeh, since oral feeding of an alcohol extract of Tempeh to hypercholesterolemic rabbits reduced their level of serum cholesterol. Fermented soybeans were extracted with hexane:ethanol, and the extract was separated into different fractions by silica gel column chromatography. One fraction obtained, SF1, inhibited the rat liver microsomal enzyme 94% at a dose of 0.625μg lipid/μl of incubation contents; 0.17μg/μl lead to 50% inhibition. Analysis of SF1 by gas chromatography/mass-spectrometry and fast-atom-bombardment (FAB)/mass-spectrometry revealed the presence of palmitic, stearic, oleic, linolenic, arachidic, and behenic acids. Oleic acid inhibited HMG-CoA reductase 94% at a dose of 0.625μg/μl of incubation contents, and 0.055μg/μl inhibited 50% of the enzyme activity. Enzyme kinetics showed the inhibition was of a mixed type. Linolenic acid, 0.625μg/μl of incubation contents, inhibited the enzyme 100%; and 0.085μg/μl lead to 50% inhibition. The inhibition was competitive. The other fatty acids were less inhibitory (1-18%). Methyl esters of oleic acid and linolenic acid maximally inhibited the enzyme 20 and 15%, respectively.
We examined the inhibitory effects of “group A saponin” and “group B saponin” fractions, which were extracted and separated from soybean seed hypocotyls, on water-soluble 2, 2′-azobis(2-amidinopropane) (AAPH)- and lipid-soluble 2, 2′-azobis(2, 4-dimethylvaleronitrile)(AMVN)-initiated lipid peroxidation reactions that were conducted with mouse liver microsomes. The simultaneous addition of the group A saponin fraction dose-dependently inhibited AAPH- or AMVN-initiated lipid peroxidation in microsomes more strongly than that of the group B saponin fraction. The group A saponin fraction inhibited the AAPH-initiated lipid peroxidation with a lag phase, while it immediately blocked the AMVN-initiated lipid peroxidation. The group A saponin fraction inhibited microsomal AAPH-initiated lipid peroxidation even when added to the reaction mixture after the lag phase period. Microsomes pretreated with the group A saponin fraction showed inhibition of AAPH-initiated lipid peroxidation with a prolonged lag phase, and the saponin fraction-pretreated microsomes showed inhibition of the AMVN-initiated lipid peroxidation in which a lag phase was found. These results indicate that in mouse liver microsomes, the group A saponin fraction from soybean seed hypocotyls, which is present outside and/or near the microsomal membranes, inhibits AAPH-initiated lipid peroxidation by inhibiting the initiation and propagation of this reaction, while it prevents microsomal AMVN-initiated lipid peroxidation mainly by inhibiting the propagation of this reaction. In addition, the present results indicate that the group A saponin fraction can inhibit AAPH- or AMVN-initiated lipid peroxidation in mouse liver microsomes by its presence within the membranes and/or by binding to them.
In order to clarify the role of albumin in the transport of L-tryptophan (Trp) into the liver, we examined the effect of bovine serum albumin (BSA) on the disappearance of Trp from the perfusate into isolated perfused rat livers. In liver perfusion with the perfusion medium containing 100μM Trp and 0.5, 2, or 4% BSA, the disappearance of Trp from the perfusate was depressed with a decrease in the concentration of free Trp (albumin-unbound form). The perfusion of livers with the medium containing 1mM Trp and 0.5, 2, or 4% BSA had little effect on the disappearance of Trp from the perfusate, though free-Trp concentrations decreased with perfusion time. In the perfusate of liver perfusion with the medium containing 100μM Trp and 0.5% BSA, total Trp concentrations correlated well with free Trp concentrations, whereas when the perfusion medium contained 100μM Trp and 2 or 4% BSA, total Trp concentrations correlated well with bound Trp concentrations. When liver perfusion was conducted with the medium containing 1mM Trp with 0.5, 2, or 4% BSA, there was a good correlation between total Trp and free Trp concentrations in the perfusate. These results suggest that under physiological conditions (ca., 100μM total Trp and 4% albumin), albumin contributes to the maintenance of total serum Trp concentrations and to the constant supply of serum Trp to the liver by lowering changes in free serum Trp concentrations through its binding to the amino acid.
The protective effect of two types of vitamin E (α-tocopherol and γ-tocotrienol) in rats treated with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were studied by determination of plasma alkaline phosphatase (ALP), plasma and liver microsomal γ-glutamyl transpeptidase (GGT) activities, and blood glutathione (GSH). Rats treated with DEN/AAF had significantly elevated plasma and microsomal GGT, plasma ALP activities, and blood GSH levels compared with the normal controls (p<0.05). Supplementation with vitamin E of normal controls did not affect the enzyme activities or blood GSH. In rats treated with DEN/AAF, vitamin E supplementation attenuated GGT and ALP activities and blood GSH levels. The optimum dose required for highest attenuation of the tumor marker enzyme activities was 34mg/kg diet for α-tocopherol and 30mg/kg diet for γ-tocotrienol. Higher doses of the vitamin did not show further attenuation in the level of the tumor marker enzyme activities.
The protective effects of taurine on hyperlipidemia associated with puromycin aminonucleoside-induced nephrotis were assessed in male Wistar rats. Administration of puromycin aminonucleoside to rats resulted in hyperlipidemia at the end of 10 days, as indicated by significant elevation of cholesterol, triglycerides, and phospholipids in plasma and liver. A significant increase in plasma lipid peroxides associated with lipoprotein fractions was observed in nephrotic rats. Liver lipids and lipid peroxides were also higher in these animals. On the other hand, reduced lipoprotein lipase activity was noted in liver of the puromycin-treated rats. In addition, urinary excretion of cholesterol was also high in them. However, treatment of the nephrotic rats with taurine modified hyperlipidemia with reduction in lipid peroxide and all major lipoprotein fractions. Taurine significantly prevented the decrease in lipoprotein lipase activity induced by puromycin. Therefore, our results suggest that prevention of hyperlipidemia by taurine contributes to its therapeutic effects in attenuating the puromycin-induced nephrotic syndrome.
The present study was undertaken to clarify the difference in the intestinal microflora between biotin-deficient and control groups of osteogenic disorder Shionogi rats (ODS). Thirteen four-week-old, male ODS rats weighing 70g on average were used. Both the biotin-deficient and the control rats were fed a biotin-deficient diet, but the control rats were administered biotin (100μg) intraperitoneally once a week and the deficient rats were administered a comparable volume of saline. After feeding for 101 days, the cecal microflora was compared between the two groups by microbiological assay. The number of total aerobes (p<0.01) and total anaerobes (p<0.02) was higher in the biotin-deficient group than in the control group. The overall total anaerobes/total aerobes ratio was about 1, 000 for the control group and 100 for the deficient group. This was mainly due to an increase in the numbers of the aerobic species Staphylococcus (p<0.005) and Enterobacteriaceae (p<0.02) in the deficient group. In the present study biotin was administered intraperitoneally to the control rats, but the composition of the experimental diet (20% egg white diet) was exactly the same for both the control and the biotin-deficient groups. Therefore, the results of the present experiment indicate that peripherally administered biotin can modify the intestinal microflora, probably via humoral changes.
The effect of an orally administered, glutamine-enriched, elemental diet on the regeneration of small bowel mucosa and hepatic steatosis following massive small bowel resection was examined by determination of the levels of the bromodeoxyuridine labelling indices, alkaline phosphatase activity in the residual jejunal mucosa, and histological change of the liver. The serum glutamine level was significantly higher in the glutamine-enriched diet group than in the glutamine-free diet group, as were the alkaline phosphatase activity in the homogenate of the residual jejunal mucosa and the bromodeoxyuridine labelling index in the residual jejunal mucosa. The histological findings showed that the fat infiltration in the liver was more severe in the glutamine-free diet group than in the glutamine-enriched diet group. These findings suggest that an orally administered, glutamine-enriched, elemental diet promotes the regeneration of the intestinal mucosa and prevents the liver from fat infiltration following a massive small bowel resection.