A method for enzymatic determinationn of riboflavin was developed by enzymatic reduction of 2, 6-dichlorophenolindophenol using a vitamin B₂-aldehyde-forming enzyme prepared from Schizophyllum commune. The enzymatic method enabled us to determine riboflavin in a mixture containing riboflavin, FMN and FAD without separation of riboflavin. Moreover, the total amount of flavins in the mixture was shown to be determined by the enzyme in the presence of phosphatase.

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The effects of α-tocopherol and related compounds on the membrane fluidity of liposomes composed of dipalmitoyl-lecithin or egg lecithin were studied by measuring the fluorescence polarization of DPH (1, 6-diphenyl-1, 3, 5-hexatriene), a hydrophobic fluorescent probe distributed in the hydrocarbon region of lipid bilayers. The transition temperature of dipalmitoyl-lecithin liposomes was lowered on incorporation of α-tocopherol into the membranes. A similar effect was observed on incorporation of phytol or phytanic acid into liposomes, but not on incorporation of 2, 2, 5, 7, 8-pentamethyl-l-hydroxychroman as an α-tocopherol model compound (TMC) which lacks an isoprenoid side chain. Above the phase-transition temperatures, incorporation of α-tocopherol or TMC dose-dependently increased the polarization values of DPH in dipalmitoyl-lecithin and egg lecithin liposomes, while incorporation of phytol or phytanic acid had little effect or slightly lowered the polarization. These results suggest that the effect of α-tocopherol in lowering the phase-transition temperature depends on its hydrophobic side chain, and that its effect in decreasing the membrane fluidity above the phase-transition temperature depends on its hydrophylic chroman ring portion. Cholesterol had an effect similar to α-tocopherol on membrane fluidity, αTocopherol and cholesterol had independent effects in increasing the polarization of DPH above the phase-transition temperature when the two compounds were incorporated together into liposomes.

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An amino acid mixture diet (AA) simulated with purified whole-egg protein and a purified whole-egg protein diet (WE) were given to ICR strain male germ-free (GF) and conventional (CV) mice for three weeks from five to eight weeks of age. All mice were killed at eight weeks of age and the gastrointestinal tracts were removed. The lengths of the small intestine were measured and six parts, i.e., the stomach; upper, middle and lower parts of the small intestine; cecum; and colon and rectum were separated, and each part (with contents) was weighed immediately. The contents were removed from each part by washing with distilled water. For the gut without contents, only total nitrogen(TN) was estimated and for the gut contents, TN, protein nitrogen (PN) and water insoluble nitrogen (WIN) were estimated. The fresh weight of cecum with contents per 100g of body weight of GF mice fed on AA diet, 2.07±0.11g (mean±SE), was lighter than that of GF mice fed on WE diet, which was 4.51±0.28g. The weight and length of the small intestine of AA diet groups were smaller than those of WE diet group. TN and PN (mg) in whole gut contents per 100g of body weight in the WE diet group were higher than those in the AA diet group and high TN and PN were observed in GF mice. The PN per TN and WIN per PN of gut contents were affected more by the presence of the flora than by the difference in diets, and CV mice showed high values. The TN of the intestinal tract excluding contents showed no differences in GF and CV mice fed on either diet.

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The regulation of tryptophan-niacin metabolism was studied in vivo with regard to the activity of liver α-amino-β carboxymuconate-ε-semialdehyde decarboxylase (picolinic carboxylase) using non-diabetic and diabetic rats. Non-diabetic rats and rats with diabetes induced by alloxan were fed on a commercial diet for 80 days, followed by a niacin-free experimental diet for the next 16 days. Protamine zinc insulin (5 units/head) was injected into 3 diabetic rats every 12 hr from the 10th to 16th day of the start of administration of the experimental diet. At the 14th day of the experimental diet, 24-hr urine was collected and urine collection repeated after the oral administration of L-tryptophan (100mg/rat). At the end of the metabolic experiments, the content of total nicotinate in liver was determined and the enzyme activity of liver picolinic carboxylase was assayed. The urine was analyzed for N-methylnicotinamide (MNA).
The amount of urinary excreted MNA per diet or per administered tryptophan was significantly reduced in diabetes, and insulin treatment rather lessened the amount in spite of normal blood glucose levels. Although the activities of liver picolinic carboxylase in diabetic and insulin-treated diabetic rat groups were higher than that of non-diabetic groups, no difference was observed between the former two groups. As the content of total nicotinate in liver was found to be almost constant among these three groups, the reduction of the urinary MNA in the insulin-treated group is considered to be attributable to the elevated retention of nicotinate that is associated with the increased weight gain. The activity of liver picolinic carboxylase was found to be inversely proportional to the amount of urinary excreted MNA and appeared to be roughly proportional to the ratio, of the amount of L-tryptophan administered to the urinary MNA. Thus, the amount of tryptophan required to produce a constant amount of MNA is expected to increase proportionally according to the elevation of the activity of liver picolinic carboxylase.

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Feeding of rats on a low casein diet supplemented with a small amount of methionine caused an enlargement of liver size and an accumulation of lipids in the liver. Experiments were conducted to examine the effects induced by the amino acid-imbalanced diet on the metabolism of dietary amino acids with special reference to protein synthesis. Protein and DNA contents of the liver per 100g body weight with rats fed on the imbalanced diet were apparently higher than those of rats fed on the basal diet. Ingestion of the imbalanced diet clearly stimulated hepatic ribosome aggregation but not skeletal muscle ribo some aggregation. Incorporation of [¹⁴C]leucine into liver protein was markedly increased in the group consisting of rats fed on the imbalanced diet (imbalanced group) and that into skeletal muscle was similar to the result with the group receiving the basal diet (basal group). Relative content of [¹⁴C]leucine in plasma albumin was higher in rats of the imbalanced group, whereas that in plasma of very low-density lipoprotein was reduced. These results indicate that the unbalanced inter-organ or intea-organ utilizations of dietary amino acids for protein synthesis were produced by the condition of the amino acid imbalance. Such metabolic effects of the amino acid imbalance may result in the enlargement of liver size and the accumulation of lipids in the liver.

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Effect of food intake at various ages (fetal, suckling and weaning periods) on adipose tissue cellularity was studied.
1) Rats were given a restricted diet on weaning and later allowed free access to diet. Fat pads of these animals recovered normal weight on refeeding and no differences in cell number or cell size were observed.
2) Food intake was varied by changing the litter size during the suckling period and, after weaning, animals were given free access to diet. Fat pads of rats raised in small litters were heavier than those of rats raised in large litters. The differences in adipose tissue weight were accounted for by differences in cell size and cell number.
3) Maternal rats were given a restricted diet during pregnancy. The pups were irregular in their weight. Pups of the restricted group which were smaller than pups of the control group at birth were chosen and raised normally. Fat pads of these animals were lighter than those of control animals and could be explained in terms of differences in cell size. From these findings, it was suggested that cellular effects of early feeding depended on the phase of growth in the rat.

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The effects of anionic surface-active agents or of a laxative agent and dietary fiber added to a 20% casein high sucrose diet on changes in body weight, food consumption and gastrointestinal functions of rats were studied and compared with those of polyethyleneglycol 4000(PEG) and polyoxyethylene sorbitan monolaurate (Tween 20) reported in the previous study by Kimura, T., Furuta, H., Matsumoto, Y:, and Yoshida, A. (J. Nutr., 110, 513(1980)). And the effects of these chemicals on intestinal enzyme activities, localized in the brush border membrane of the small intestine, in vitro were investigated. The effects of sodium laurylbenzenesulfonate(LBS) added to the diet on changes in the body weight, food consumption, feces and intestinal sucrase and alkaline phosphatase activities were more adverse than those of PEG or Tween 20, and the adverse effects of the former were not prevented with the concurrent feeding of Gobo dietary fiber(GDF) or soybean dietary fiber(Okara), although those of the latter two were prevented with the concurrent feeding of these dietary fibers. The massive addition of sodium stearate or sodium oleate to the diet did not produce any adverse effects on changes in the body weight, food consumption, feces and intestinal enzyme activities. The addition of magnesium sulfate, a laxative agent, to the diet induced diarrhea without significant changes in the body weight, food consumption and intestinal enzyme activities, and the induced diarrhea was not prevented with the concurrent feeding of GDF. Intestinal alkaline phosphatase activity in vitro was not affected by PEG, Tween 20, LBS or magnesium sulfate, but intestinal leucine aminopeptidase activity in vitro was inhibited by PEG, Tween 20, LBS or sodium oleate. Although intestinal sucrase and maltase activities were inhibited by LBS alone, the inhibition-effect of LBS on the sucrase activity was more severe than that of LBS on the maltase activity. From the experimental results, the mechanism by which these chemicals and dietary fiber exerted their effects on physiological functions was discussed.

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In order to develop a simple and exact analytical method for cholesterol determination in foods by gas-liquid chromatography, several experiments were carried out in collaboration with several universities and institutes. For the extraction of lipids from foods, it was decided that chloroform-methanol (2:1) extraction was the most suitable procedure. Since pretreatment procedures using methods such as thin layer chromatography and florisil column chromatography to purify the unsaponifiable matters reduced the recovery of cholesterol, and good results were obtained without applying the pretreatment, these pro cedures were concluded to be unnecessary. Gas chromatograms obtained with free sterol showed results similar to those of the trimethylsilyl ether derivative and acetate. 5-α-Cholestane is used as a good internal standard.

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Transketolase (TK) activity was assessed by incubating hemolyzed whole blood with ribose-5-phosphate both in the presence (TK_sat.) and in the absence (TK) of thiamine pyrophosphate (TPP). The amount of pentoses utilized between 5 and 60 min after the start of the incubation was taken as a measure of the TK activity. The TPP effect was expressed as (TK_sat. -TK)/TK×100. The coefficient of variation for the TPP effect determination was 9.6% within the TPP effect range from 6 to 22%. Within the TPP effect range from 24 to 46%, the coefficient of variation was 8.7%.
The mean TPP effect as measured in a group of healthy individuals amounted to 13.5%, with a range from 8.3 to 18.5%. In a group of obese women submitted to an energy-restricted diet, the TPP effect ranged from 12.7 to 30%. In patients suffering from Wernicke's encephalopathy, the TPP effect varied from 28 to 67%.

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