Oxygen free radicals, in particular the hydroxyl radical (·OH), which is readily formed by the Fenton reaction, are known to initiate lipid peroxidation. In this study the effects of several buffer solutions and chelators on the generation of ·OH and lipid peroxidation were examined. The generation of ·OH was decreased significantly in phosphate buffer solution, and slightly in Tris-HCl buffer solution, compared to that in HEPES or MOPS buffer solution, both of which have low affinity for iron ions. Phosphate and Tris-HCl buffer solutions also decreased the formation of lipid peroxides. The generation of ·OH varied by the addition of several kinds of chelators at different ratios of chelator/Fe(II), 1/10-10/1. And conflicting effects of EDTA or diethylenetriamine pentaacetic acid (DETAPAC) were observed in HEPES buffer and in phosphate buffer. At the concentration ratios of chelator/Fe(II) at which EDTA or DETAPAC inhibited ·OH generation in HEPES buffer, ·OH generation was promoted by EDTA or DETAPAC in phosphate buffer. On the other hand ADP slightly enhanced ·OH generation at the ratios of ADP/Fe(II) of 5/1 and 10/1 in HEPES buffer, but did not affect ·OH generation in phosphate buffer. Lipid peroxidation was completely inhibited by the addition of DETAPAC and EDTA, while ADP promoted ·OH generation at the chelator/Fe(II) ratios of 5/1 and 10/1.
To determine free iron in human serum, we first determined total concentrations of iron (It μmol/liter) and protein (Pt μg/ml) in the serum precisely with bathophenanthroline and Coomassie Brilliant Blue G-250 (Bio-Rad Protein Assay) by colorimetry. Then, 1.0ml of the serum was centrifuged on a membrane cone filter (Centriflo CF-25) at 1, 050×g for 60min at 4°C, and the serum protein fraction on the membrane cone filter was dissolved in saline and centrifuged again in the same way. The washed serum protein fraction was dissolved in saline, and an aliquot of the solution was subjected to analyses of iron (Iw μmol/liter) and protein (Pw μg/ml). To correct for the free iron adsorbed on the membrane cone filter during the centrifugation and to obtain the free iron concentration (If μmol/liter) in the serum, we used the following equation: If(μmol/liter)=It-(Iw×Pt/Pw). The average values of total and free iron in 16 different samples of human fresh sera were determined to be 21.5±6.1μmol/liter and 1.24±0.89 μmol/liter, respectively, by the present standard method; and the values were compared with those of the same sera frozen for 1 week. Free iron in human serum was found to exist as Fe2+.
The effect of dietary oleic acid on lipid metabolism was investigated in SD rats. Different types of oleic acid (ethyl oleate, triolein, olive oil, and high-oleic sunflower oil) were compared with ethyl linoleate and fat-free diets for their efficacy in lowering the plasma cholesterol concentration. Plasma cholesterol concentrations were significantly higher in rats fed ethyl oleate and olive oil than in those fed triolein, high-oleic sunflower oil, ethyl linoleate, or the fat-free diet. The lowest plasma cholesterol concentration was observed in rats fed ethyl linoleate. Chylomicron plus VLDL and LDL-cholesterol concentrations were more markedly increased in rats fed the oleic acid-rich diets, especially ethyl oleate and olive oil, than in those fed the ethyl linoleate diet. HDL-cholesterol concentrations were not different among the experimental groups. LCAT activity in rats fed the ethyl linoleate was twice as high as that in any other group. These data suggest that oleic acid-rich oil does not induce a hypocholesterolemic response in rats fed hypercholesterolemic diets.
The purpose of these experiments was to determine whether resident murine peritoneal macrophages possess desaturase activity to convert stearic acid into oleic acid. Peritoneal macrophages were purified by adherence and incubated in a medium containing fatty acid-free BSA with [1-14C] stearic acid (18:0). Incorporation of [1-14C] 18:0 into macrophage lipids increased up to 4h (>55%) after incubation and then declined in a linear fashion over the next 20h. Majority of the [1-14C] 18:0 incorporated into macrophages remained as free acid throughout the incubation period. Incorporation of [1-14C] 18:0 and desaturated products into the phospholipid fraction increased linearly up to 16h and then decreased at 24h of incubation. A small amount of radioactivity was also associated with triacylglycerol and cholesterol ester fractions. The relative incorporation into phospholipid classes reached a plateau at 4h and remained unchanged throughout the 24-h incubation period. Resident macrophages possessed the capability to desaturase [1-14C] 18:0 into oleic acid, and this rate of desaturation was maximum at 8h. These results clearly demonstrate that murine peritoneal macrophages have the ability to synthesize oleic acid and to incorporate it into various lipid fractions.
Long-term effects of capsaicin, a pungent principle of capsicum fruits, on intestinal thiamine absorption were examined in mice. Inhibition of thiamine absorption was noted after oral administration of capsaicin (1mg/kg body weight/day) for 1, 2, 4, or 8 weeks. When given for 12 weeks at a dose of 2mg/kg body weight/day, the principle was also inhibitory; however, the inhibition of thiamine absorption was not observed if the dose level was 1mg/kg body weight/day. Reductions in the activity of intestinal Na+, K+-ATPase and intestinal mucosal ATP content were responsible for the inhibition of thiamine absorption by capsaicin. The decrease in the intestinal mucosal ATP content was accompanied by a decrease in the activities of mitochondrial NADH cytochrome c reductase and cytochrome oxidase. These results suggest that inhibition of intestinal thiamine absorption by capsaicin in mice is due to the inhibition of intestinal mucosal Na+, K+-ATPase and the reduction in mucosal ATP content.
WHT/Ht mice are known to have GM2 deficiency especially in the liver. In order to know the relationship of this abnormality to the general metabolism in organs or cells, we compared 17 hydrolytic enzyme activities in the liver, brain, spleen, and kidney between the WHT/Ht mice and control BALB/c mice or without administration of GM2 and GM3. Regardless of the GM2 or GM3 treatment, the galactosidase activity was markedly low in all of the tested organs in the WHT/Ht mice when compared with that in the control animals. This consistency stands in contrast with the known organ-dependent heterogeneity of ganglioside expression and thus indicates independency of the GM2 deficiency from the galactosidase deficiency in this model animal.
Effects of administration of 4G-β-D-galactosylsucrose (lactosucrose: LS) on fecal microflora, putrefactive products, short-chain fatty acids, weight, moisture and pH, and subjective sensation of defecation were investigated in 13 elderly patients with constipation. The LS mixture used in the present study consisted of LS (59.0%), lactose (22.7%), sucrose (8.4%), fructose (1.6%), glucose (6.8%), and non-identified polysaccharides (0.8%). Subjects were fed 0.32g of LS mixture per kg body weight per day (0.18g of LS per kg body weight per day) for 3 weeks after a one-week control period. The results obtained were as follows: 1. The number and percentage of Bifidobacterium remarkably increased. The frequency of occurrence of Bifidobacterium increased, while that of lecithinase-negative Clostridium decreased. 2. The concentrations of p-cresol, indole, skatole and ammonia in the feces (nmol or μmol/g wet feces) were decreased significantly, and the total fecal excretion of p-cresol and skatole (μmol/week) was also diminished significantly. 3. LS administration caused significant increases in the concentration (μmol/g wet feces) and the total fecal excretion (mmol/week) of acetic and n-butyric acids. 4. Fecal moisture (%) increased significantly, whereas fecal pH decreased. LS also tented to cause a gain in fecal weight (g/week). 5. The stool of patient was softened. The subjective sensation of defecation was improved in 11 out of the 13 patients and unchanged in 2 of them. These results suggest that LS ingestion corrects the fecal condition through improvement of the intestinal microflora.
A Japanese boy with fatal pyruvate dehydrogenase deficiency died at 93h of age despite intensive treatment for a progressive metabolic acidosis and hyperammonemia. Enzymatic studies of the patient's liver and kidney homogenates revealed a decreased PDH complex activity caused by the absence of PDH component activity. This was further confirmed in that there were no immunoreactive proteins in positions corresponding to PDHα and β. All exon segments of genomic DNAs of PDHα and β isolated from the patient's liver and control leukocytes were amplified by PCR. Sequences of PCR-amplified products were compared with those of normal PDHα and β genes. Two single base-pair substitutions in two exons of the PDHα gene, one, G to T, in exon 5 and the other, T to C, in exon 6, were detected. These mutations generate a Phe-116 in the mature PDHα in place of the normal Cys-116 and a Pro-162 in place of the normal Leu-162. PCR-amplified exon segments of PDHα genomic DNA from the patient hybridized only to the mutant allele-specific oligonucleotide probes.