The photooxidation of glutathione (GSH) sensitized by the riboflavin and its derivatives under the illumination of ultraviolet light was investigated. The photooxidation of GSH was sensitized by riboflavin, flavinmononucleotide (FMN) and lumiflavin in an atmosphere containing suitable concentration of oxygen, and glutathione disulfide was found as the end product. This photooxidation was highly promoted by the rises of temperature and pH of solution, and followed roughly the time course expected for a first-order reaction. FMN and lumiflavin also promoted the photooxidation of GSH, but as to the flavinadeninedinucleotide these action was not found in both pH of 6.0 and 8.0. At the time of photooxidation of GSH, the photolysis of added riboflavin was observed at pH 7.0 and resulted lumiflavin and trace of lumichrome were found chromatographically.
In the studies of biogenesis of pyridoxal by Candida albicans 4888,the availability of glyoxylic acid as a source of carbon was investigated. In the case of glyoxylic acid as a sole source of carbon did not supported the growth of yeast but glyoxylic acid plus acetic acid caused the growth of yeast and the remarkable production of pyridoxal was observed. It was supposed that the main intermediate pathway of biogenesis of pyridoxal from organic or amino acids by Candida albicans 4888 go through the glyoxylic acid cycle.
The incorporation of radioactive carbon from succinic acid-2,3^<14>C into vitamin B_6 produced by the growing cell of Candida albicans 4888 was investigated. Vitamin B_6-^<14>C was isolated with Amberlite CG-50 ionexchange chromatography, thin layer chromatography, and repeated paper chromatography. Radioactive and bioautographic peaks of produced vitamin B_6 was in agreement with pyridoxal. On the other hand, it was recognized by radioautography that fumaric, malic, and citric acids were produced in the culture medium, especially remarkable accumulation of fumaric acid was observed.
A fluorometric determination method of pyridoxal-5-phosphate or pyridoxal in blood or organ has been developed. Bonavita's reaction of KCN with either pyridoxal-5-phosphate or pyridoxal was used in this fluorometric method, and was compared with the enzymatic methods, it was found to be convenient and has remarkable sensitivity for pyridoxal. This method is also applicable to some animal experiments, though it is somewhat inferior to the enzymatic methods in sensitivity for pyridoxal-5-phosphate.
The specificity of the previously reported fluorometric determination method of pyridoxal-5-phosphate in biological materials was investigated. The fluorescence excitation and emission spectra, the change in fluorescence intensity with pH, the electrophoresis, the paper chromatography, and the hydrolysis with acid phosphatase were examined with the pyridoxal-5-phosphate fraction obtained from mouse liver. The results showed that the method had high specificity for a determination of pyridoxal-5-phosphate in liver.
For determination of CoA, the method of Kaplan and Lipmann was used. The determinations of CoA were made on the liver, kidney and brain of rats ranging from the neonatal period to 40 days of age. The concentration of hepatic CoA was lower in neonatal period, thereafter, it gradually increased with growth and reached the constant level at 30 days after birth. In the kidney, the concentration of CoA was also lower in neonatal period, but it increased with the growth of rats by 10 days after birth. The brain CoA concentration approached to the adult level when rats were 20 days of age. The total content of CoA in the liver and kidney increased with the growth of rats from birth and in the brain it reached the constant level by the time rats were 20 days old.
Experiments were performed to determine the nutritional interrelationship between hepatic CoA and dietary protein or some essential amino acids in young rats. Rats were given either 18% casein diet (standard diet) or 5 % casein diet (low protein diet) during 4 weeks. Body weight and total hepatic CoA of the standard diet group were markedly increased throughout the feeding period, but such a tendency was not found in low protein diet group. The concentration of hepatic CoA in standard diet group was higher than that of low protein diet during the first 2 weeks of feeding, thereafter, it was observed that the concentration of CoA in low protein diet group was increased at the 4th week. In the next, young rats were divided into 7 groups ; standard, low-protein, control, methionine-deficiency, lysine-deficiency, tryptophan-deficiency and threonine-deficiency diet groups. The hepatic CoA concentration was reduced as compared with control when the methionine deficiency diet was administered for 10 days. The effect of lysine and tryptophan deficiency on hepatic CoA concentration was slight and threonine deficiency was hardly effected the hepatic CoA concentration.
When the weaning rats were fed on 20% casein diet for 14 days and were injected 0.2mg or 1.0mg of NPP (19-norandrosterone phenyl propionate) in 4 divided dose, the body weight and hepatic CoA concentration were unchanged or slightly reduced as compared with control. Weaning rats, fed on 5% casein diet for 10 days and on 20% casein for another 10 days, were injected of 0.15mg or 0.75mg of NPP in 3 divided dose. In these groups body weight and hepatic CoA were also unchanged. But in both dietary groups the serum protein and liver nitrogen were increased by the injection of 1.0mg or 0.75mg of NPP. In the group, fed on 5% casein diet alone for 14 days, the body weight, serum protein and liver nitrogen were not altered by administration of NPP. By the injection of 1.0mg NPP under the low potein condition, it was observed that the hepatic CoA concentration revealed a tendency to increase. Oral administration of HMD (2-hydroxymethylene-17α-methyl-dihydro testosterone) did not show a remarkable effects on the body weight, serum protein, liver nitrogen and CoA.
Oxalic acid has long been known to be a product of the oxidation of ascorbic acid. Since large doses of ascorbic acid are being prescribed by physicians at present, an investigation of the effect of ascorbic acid on the urinary oxalate excretion of man is warranted. This report is concerned with such an investigation. In the first experiment, 10 healthy adults ingested 1g of ascorbic acid daily for 90 days. In the second experiment, 10 healthy adults ingested 2g of ascorbic acid daily for 180 days. In the third experiment, 10 healthy adults ingested a special diet for consecutive 3 days before the test day and 2g of ascorbic acid daily for 90 days. In each case, any remarkable increase of urinary oxalic acid was not observed with oral administration of an indicated amount of ascorbic acid as compared with the control. When a large amount of ascorbic acid was administered, ascorbic acid was excreted mainly as reduced ascorbic acid in urine.
The results of experiments on the absorption of benzoylthiamine disulfide (BTDS) labeled in thiazole ring S were reported. The urinary excretion of ^<35>S after intraperitoneal injection of BTDS (990μg as thiamine-HCl) was 42% of the administered doses in 48 hours. BTDS, O-benzoylthiamine and thiamine were found in ligated intestinal cannal and wall, respectively 5 minutes after introducing 500μg of BTDS-^<35>S. In liver homogenate, O-benzoylthiamine and thiamine were recovered 5 minutes after addition of 50μg BTDS-^<35>S. O-benzoylthiamine was found in urine as one of the radiometabolites. Therefore, at the time of absorption from intestinal cannal, reduction of -S-S- of BTDS (formation of O-benzoylthiamine) was thought to be the first step. O-benzoylthiamine may be enzymatically converted to thiamine in the liver. Formation of thiamine diphosphate-^<35>S in the liver after intravenous injection of BTDS-^<35>S (500μg) were 20-30μg, the amounts of thiamine pyrophosphate-^<35>S, thiamine monophosphete-^<35>S, and thiamine-^<35>S were 1-2μg, respectively.
Thiamine disulfide O-acyl derivatives were heated at 100℃ in buffer solution of pH 3,5 or 7,and the conversion of them into thiamine active compounds and their decomposition were microbiologically determined using Lactobacillus fermenti 36. The results indicate that remarkable changes were observed in the compounds having not less than C_5-substituent, and in straight chain compounds than in branched chain ones when the number of C-atom is same. It was most labile at pH 7.
Investigations were carried out on the pharmacological effects of cyclocarbothiamine (CCT), a thiamine derivative newly developed in our laboratory. When orally administered in rat and mouse, LD_<50> were 7.6 g/kg and 6.2 g/kg, respectively, and when injected intravenously, they were 0.58 g/kg and 0.36 g/kg. On the cardio-vascular system, the blood pressure, respiration and EKG of rabbit, isolated heart of guinea-pig, and vascular preparation of rabbit ear, CCT acted similiarly to thiamine. CCT had little effect on the isolated intestine of rabbit. The motility of atrium preparation of guinea-pig was enhanced by CCT, but not by thiamine. On the nerve-muscular preparation of rat and on the EEG of rabbit, its effect resembled that of thiamine.
Chronic toxicity of cyclocarbothiamine was investigated on the Wistar rats of both sexes. The sample was mixed into food at the concentration of 30,100 and 300mg%, and administered orally to the test animals for 3 and 6 months. No deleterious effect was observed on their behavior, growth curve, food intake, and organ weights, nor were there any abnormal finding on autopsy and hematology. Histopathological examination of the lungs, liver, kidneys, adrenals, thyroid, pancreas, spleen, bone marrow, gastro-intestinal tracts, brain, and other 5 organs showed no observable chronic toxicity effect of cyclocarbothiamine, given orally in larger dosis for 3 and 6 months continuously.
Cyclocarbothiamine (CCT) was administered orally to pregnant mice and rats and its effect on the fetus was investigated according to the test method admitted by the Ministry of Public Health and Welfare. CCT were given orally at the daily doses of 60,1200,2400 mg/kg to pregnant ICR-JCL mice from 7th to the 12th day of gestation and 60 and 1200 mg/kg to pregnant Wistar rats from 9th to the 14th day. No teratogenic influence was observed on the fetus nor on the born babies.
To prevent loss of riboflavin from riboflavin tetrabutylate-enriched polished rice by washing, we examined to fix riboflavin tetrabutylate on rice by gelatininization. By treating with a definite amount of hot water at a definite temperature, riboflavin tetrabutylate was fixed on polished rice and the amount of riboflavin solubilized in water by washing was within 5 percent. By microscopic observation of tissue slice of the enriched rice, it was revealed that riboflavin tetrabutylate was localized in the surface layer of the rice.