Optimum conditions for the assay of thiamin were studied using a cyanogen bromide (BrCN) oxidation method. The adopted procedure included neither pre-purification of samples through an ion exchanger nor extraction of the thiochrome into an organic solvent. The 0.25M BrCN (the concentration before the addition of alkali) and the final NaOH concentration of approx. 1% gave the highest yield of thiochrome by a laboratory-prepared BrCN. To obtain the highest intensity of fluorescence, a concentrated BrCN (1.8M) was introduced in place of the conventional BrCN (0.11M), obtaining 300% or more intensity of fluores-cence. For the oxidation of thiamin diphosphate, 0.15-0.2M of laboratory-prepared BrCN gave the highest intensity of fluorescence instead of the 0.25M for free thiamin. For simultaneous oxidation of free thiamin and thiamin diphosphate, therefore, 0.23-0.24M of laboratory-prepared BrCN was deduced to give the best yield of fluorescence. With a solution of commercially obtained solid CNBr, optimum concentrations for the oxidation of thiamin were about 0.04M for CNBr and about 0.16% for NaOH. When the sample contained an inhibitor of oxidation, such as ascorbic acid, the percentage of inhibition decreased inversely pro-portional to the concentration of the sample in a rough approximation. The degree of inhibition was not reduced by the increased amount of BrCN reagent. Thus the possibility was indicated that thiamin in an ascorbic acid-contaminated sample could be determined accurately by extrapolating values for serially diluted samples.
The intestinal absorption of pyridoxal 5'-phosphate (PLP) at physiological levels (10-7-10-6 M) was studied in comparison with that of pyridoxal (PL) in rat, using in vitro everted sac and an intestinal preparation that permitted continuous in situ collection of mesenteric venous blood. After PLP administration (10-6-10-3 M) in situ, larger amounts of PLP were found in the mesenteric venous plasma than after PL administration at the same dose. The amount of PLP found in the mesenteric venous plasma was dependent on its dose at lower concentrations up to 10-4M but became independent at higher concentrations. After PL administration at various doses, the amount of PL found in the mesenteric venous blood increased linearly with the dose. When various concentrations of PLP were added to the mucosal side, under the in vitro condition with protection from alkaline phosphatase hydrolysis, PLP was detected in the serosal side and the extent of PLP transport was dependent on the initial concentration of PLP in the mucosal side. When various concentrations of PL were added to the mucosal side, the extent of PL transport was independent of the initial concentration of PL in the mucosal side. In rat pretreated with actinomycin D, PLP transport in vitro was inhibited but not that of PL. N2-induced anoxia and pyridoxamine 5'-phosphate and anion transport inhibitor (4, 4'-diisothiocyanostilben-2, 2'-disulfonic acid disodium salt) showed no effect on PLP transport. These results suggest that PLP can be absorbed in the phosphorylated form and imply the presence of a saturable process for direct absorption of PLP itself and a diffusive process for PL absorption. In addition, the result of the in vitro neonatal experiment suggests that the neonatal intestine also can transport PLP in phosphorylated form.
The effects of the addition of varying levels of arginine (Arg), Cystine (Cys), and glycine (Gly) to the bovine milk-simulated amino acid mixture on the levels of plasma and liver cholesterol were investigated in rats. The diets containing a high amount of Cys lowered significantly the level of plasma cholesterol as the amount of Cys in diets increased. The serum high density lipoprotei (HDL)-cholesterol level and fecal excretion of acidic steroids were higher in rats fed the Cys diets than in those fed the Arg and Gly diets. No difference, however, was observed in the content of liver cholesterol. Liver triglyceride contents elevated significantly on feeding the Arg and Cys diets. Furthermore, liver phospholipid contents elevated significantly on feeding the Arg diet but lowered on feeding the Cys diet. Therefore, these results indicated that the feeding of a high amount of Cys lowers the plasma cholesterol levels as the result of the enhanced conversion of cholesterol to bile acids.
In order to understand the function of Carrageenan, an indigestible polysaccharide, as a promoter of colonic tumors induced by 1, 2-dimethylhydrazine (DMH), molecular weight distribution of fecal carrageenan and amounts of fecal bile acids in rats given carrageenan and DMH treatment were examined. Gel filtration pattern on Sephacryl 5-300 of fecal carrageenan was very similar to that of feeding carragee-nan, and carrageenan ingested was quantitatively excreted in feces. Hexafluoroisopropyl ester-trifluoroacetyl derivatives of fecal bile acids were analyzed by gas chromatography on QF-l. Although there was a decreased concentration of deoxycholic acid and total bile acids in carrageenan-fed rats compared to control rats, no difference in the daily output was found because carrageenan ingestion increases fecal output. Significant increased concentration and daily output of lithocholic acid, a tumor-promoter, by feeding carrageenan were found. Thus, it was suggest-ed that the promoting effect of carrageenan on colon tumorigenesis by DMH may be mediated by increased excretion of lithocholic acid and may not participate in degradation of carrageenan ingested.
To clarify whether capsaicin or Isothiocyanate, which are found in common spices, activate brown adipose tissue (BAT) function, BAT temperature, mitochondrial guanosine diphosphate (GDP) binding (a thermogenic indicator), and mitochondrial oxygen consumption were measured in Interscapular brown adipose tissue (IBAT) of rats. Intramuscular injection of capsaicin (0.7mg/kg) or Isothiocyanate (3.0mg/kg) increased significantly IBAT temperature without affecting the rectal temperature, a possible side effect, and increased significantly GDP binding and mitochondrial oxygen consumption in IBAT, as did adminis-tration of ephedrine (0.375mg/kg). Therefore, the administration of capsaicin or Isothiocyanate activates BAT function in rats.
The effects of copper administration to neonatal male mice on the copper concentrations and activities of copper-containing enzymes in cerebrum, liver, and kidney were studied. Intraperitoneal copper injections at 7 and 10 days of age increased the activities of superoxide dismutase and cytochrome oxidase in cerebrum and liver, and also increased the copper concentrations in cerebrum, liver, and kidney at 13 days of age. Maternal copper administration during the late-gestational period (from 13 days gestation to delivery) decreased the activities of both enzymes and increased the copper concentration in cerebrum. This increased level of copper remained by 13 days of age after birth. Liver showed similar changes to those in cerebrum, but the renal responses were less remarkable. Maternal copper administration from the late-gestational through lactational periods affected neonatal growth, decreased the activity of cytochrome oxidase, and increased the copper concentrations in all tissues examined. It is known that the copper concentration and copper-containing enzyme activity are low in cerebrum of mottled mice as well as of patients with Menkes' disease. These results suggested that the cytochrome oxidase activity in cerebrum was decreased by not only copper deficiency but also excess. The combination of prenatal copper sup-plementation by means of maternal copper administration during the late-gestational period and Intraperitoneal copper injections after birth, while being careful not to overdose, is expected to be efficient for the copper supplementation to mottled mice.
An assay method for pyruvate kinase in rat plasma is described. Plasma samples were incubated with ADP and phosphoenolpy-ruvate in Tris buffer solution. The ATP produced by pyruvate kinase was measured by photocounting after the addition of a commercially available luciferin-luciferase preparation. Interference by ATP or adenylate kinase originally present in the sample was removed by a high degree of dilution. The assay is sensitive, reproducible, and rapid, especially when used for large numbers of samples. By this method, pyruvate kinase activity in normal rats was determined to be 0.51±0.05 (n=6) U/ml plasma. In rats fed a vitamin E-deficient basal diet for 7, 10, or 14 weeks, pyruvate kinase activities were 0.70±0.11, 1.64±0.51, and 4.28±0.85 (n=6) U/ml plasma, respectively. This method appears to be useful for the determina-tion of pyruvate kinase activity in nutritional or pharmacological studies.
The atherogenecity of corn oil, hydrogenated corn oil, and butter fat was studied using 57 fourty-day-old male Japanese quails. The animals were fed one of the following diets: basal diet, basal diet with corn oil plus cholesterol, basal diet with hydrogenated corn oil plus cholesterol, or basal diet with butter fat plus cholesterol. Each atherogenic diet contained 15% fat and 2% cholesterol. Marked hypercholesterolemia developed in all fat-fed groups after 3 weeks or 3 months, but no significant difference was seen among the groups. The degree of the luminal narrowing of the ascending aorta and brachiocephalic arteries was highest in the corn oil group and lowest in the hydrogenated fat group. Ultrastructurally, all the fat-fed groups showed similar cellular changes in their aortic lesions. The major cell type of the thickened intima was fibroblast-like cells with or without lipid droplets. Immunohistochemical studies disclosed that alpha-l-antichymotrypsin was strongly positive for fibroblast-like cells in the thickened intima, whereas it was negative for those in the tunica media of the ascending aorta and its branches. These data suggest that hydrogenated corn oil has a less potent stimulating effect on the thickening of the arterial intima than corn oil and butter fat.
Red blood cell (RBC) alpha-tocopherol and fatty acid levels in children undergoing long-term antiepileptic therapy were examined. Antiepileptic drugs included diphenylhydantoin (PHT), valproic acid (VPA), phenobarbital (PB), and carbamazepine (CBZ). RBC alpha-tocopherol levels were low in patients receiving multi-drug combination therapy, as compared with children receiving no treatment (controls), but plasma alpha-tocopherol levels were the same in both groups. With respect to fatty acid composition in RBCs, docosahexaenoic acid (DHA) level was decreased in children receiving antiepileptic therapy, while no changes were documented in the other fatty acids.
Recently, a diet enriched in oleate and moderately restricted in hexacosanoate (C26:0) was found effective to reduce the plasma very long chain fatty acid (VLCFA) levels in patients with adrenoleukodys-trophy (ALD), an X-linked disorder characterized by demyelination of the adrenal cortex and cerebral white matter, and accumulation of saturated VLCFA, particularly C26:0, in tissues of the demyelination. The infor-mation about the C26:0 content in Japanese food was, however, almost nil except for one report about foods in the USA, but this did not include some Japanese common foods. With the hope of treating an ALD patient in our hospital, 026:0 contents in Japanese common foods (42 items) were measured. In our case, a one-hour direct transesterification method was used to obtain methylesters of total fatty acids in foods and they were applied directly to a selected ion monitoring gas chromatography-mass spectrometry for the quantitative C26:0 analysis. The C26:0 content in nuts and seeds as well as in fats and oils was found to be significantly higher than in other foods; the content was highest in peanuts. The content in almost all kinds of examined fishes, the common protein foods in Japan, was relatively low. From these data and that in the national nutrition survey in 1986, the daily intake of C26:0 from the average Japanese diet could be estimated to be 12-36 mg. It can be recommended, therefore, that nuts and seeds as well as fats and oils should be restricted as severely as possible from the diet of ALD patients in Japan in order to keep daily C26:0 intake below 10 mg as recommended in the