In an attempt to clarify the mechanism of LDH protein reaction with FMN, amino acid residues in the protein moiety such as histidine, arginine, tyrosine and tryptophan were chemically modified so that their effects on LDH activity and FMN reaction could be in-vestigated. Although it was recognized that histidine and arginine residues participate in the catalytic action, and that these residues exist at the binding site of coenzyme and .substrate, no reaction of FMN with these residues was noted. LDH was slightly inactivated when tryptophan residue was modified, but inhibition of this activity was completely prevented when FMN was allowed to react with zymoprotein in advance. As is true with native LDH, LDH with a modified histidine residue or tyrosine residue reacted with FMN, but LDH with a modified tryptophan residue did not so react. It was therefore shown that the tryptophan residue of native LDHM4 zymoprotein reacts with FMN.

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The combined use of CoQ₁₀ with adriamycin has beenrecommended for reduction, of the cardiotoxicity that occurs during cancer chemotherapy. Vitamin B₂-butyrate was also investigated in order to determine anti-oxidative effects on adriamycin cardiotoxicity. This vitamin analysis prevented enhanced lipid peroxidation and rectified the respiratory disorders of heart mitochondria induced by adriamycin, however, the deficiency of the CoQ₁₀-pool was not rectified. The combined approach of using CoQ₁₀ for rectifying the deficiency of this component and of using B₂-butyrate for reducing lipid peroxidation was indicated for adriamycin cancer chemotherapy.

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Vitamin B₂-aldehyde-forming enzyme isolated from Schizo-phyllum commune with 1, 400-fold purity was found to require NADPH under aerobic but not anaerobic conditions. Both NADPH- and DCIP-requiring enzyme activities which could not be separated by our enzyme purification system were found in the same single protein band on polyacrylamide disc gel electrophoresis. The cation, Cu²⁺, markedly stimulated the NADPH-dependent enzyme reaction under the aerobic conditions. The reagents, EDTA, pyrazole, N ethylmaleimide, PCMB, and arsenite, inhibited the formation of vitamin B₂-aldehyde to various extents, whereas azide did not.

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The effect of the feeding of a vitamin A-free diet on vitamin A metabolism was studied in rats by following changes in tissue distribution of the vitamin for 24 days. The serum level of vitamin A was maintained at a constant level until the last day of the experiment, by which time the liver reserve had declined to as low as one-seventh of its initial value. In contrast, the concentration of vitamin A in the kidney increased gradually in rats fed on a vitamin A-free diet and rose to levels higher than those in the liver after 24 days, whereas the kidney vitamin A concentration in the control animals remained at a constant level. In the testis, total vitamin A content increased in both the control group and that fed on a vitamin A-free diet. However, the magnitude of increase was smaller in the latter than in the former. Replenishment of the vitamin-A depleted rats with the vitamin provoked a prompt restoration of all changes produced by feeding of the depleted diet. Possible roles of the kidney are discussed with respect to homeostatic maintenance of the vitamin in vitamin A-depleted animals.

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The biological aspects of Cobalamin were studied in autopsy brains of eight elderly subjects who had lacked marked neurologic symptoms. Separation of the gray and white matter of the frontal and temporal lobes, and subsequently, subcellular fractionation of each matter were attempted. The contents of cobalamin and its binders and cobal-amin concentrations were measured in each subcellular fraction and whole homogenate.
The contents of Cobalamin were in the ranges of 50 to 60ng/g wet tissue. No marked differences in contents were found between the frontal and temporal lobes. The mitochondria-rich fraction contained more coba-lamin than did the microsomal fraction.
Two binders were found; a large-sized and a small-sized. The molecular weight of the larger binder was ca. 120, 000, coinciding with the elution region of transcobalamin I, and the molecular weight of the smaller binder was 40, 000, coinciding with the elution region of transcobalamin II, in gel filtration respectively. More of the larger binder than the smaller was found in the mitochondria-rich and microsomal fractions.
These data are useful for the study of various aspects of cobalamin in brains with various types of neurologic pathology or aging changes.

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A glucosyltransferase, catalyzing the transfer of D-glucose from UDP-glucose to the 5'-hydroxyl group of pyridoxine, was isolated as a particulate enzyme from seedlings of podded pea (Pisum sativum L, cv. Kinusaya). The enzyme required additional Mg²⁺ for its function. The pH optimum for glucosylation of pyridoxine was between 7.8 and 8.8. The enzyme showed high specificity for UDP-glucose and relative specificity for glucosyl acceptor : pyridoxine was replaceable by pyridoxamine. Several compounds tested other than vitamin B₆ failed to serve as the acceptor. It was shown that a methylene group on C-4 participated in the formation of enzyme-substrate complex, and that the rate of glucosylation was dependent upon the C-4 substituent. From the results of kinetic studies and an experiment in vivo, the enzyme was inferred to be UDP-glucose: pyridoxine 5'-O-β-glucosyltransferase.

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Studies were made on whether glucose starvation causes fatty liver in pyridoxine-deficient male Wistar rats. Pyridoxine deficiency resulted in significantly lower levels of liver glucose than in pair-fed controls but no significant change in the serum glucose concentration. In non-starving animals, serum immuno-reactive insulin (IRI) was signi-ficantly lower in pyridoxine-deficient rats than in pair- or ad libitum-fed controls. Liver glucokinase activity in pyridoxine-deficient rats was also significantly lower than in ad libitum-fed controls. The extent of insulin deficiency was evaluated by examining the effect of administration of insulin on pyridoxine-deficient rats. Administration of insulin had no effect on the activity of liver glucokinase in pyridoxine-deficient rats, but induced the enzyme in ad libitum-fed controls. In response to a decrease in the activity of liver glucokinase or hexokinase in the deficient group, glycolytic activity, estimated as lactate production from glucose in the liver supernatant spun at 100, 000×g, was reduced to half the control level in pyridoxine-deficient rats. The effects of glucose administration on the liver lipid content, serum insulin and serum glucose were investigated. The serum glucose concentration was not significantly different in pyridoxine-deficient and control rats at any time after the glucose load. The level of serum IRI after the load was similar in the two groups after 30 min but then gradually decreased in the deficient group. The liver lipid content of the deficient rats tended to decrease whereas that of the controls remained unchanged throughout the experiment. Thus glucose starvation in pyridoxine-deficient rats is one factor responsible for fatty liver formation. Possible mechanisms of this phenomenon are discussed.

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When weanling rats were given 70% casein diet deficient in pyridoxine, they showed marked accumulation of liver lipid consisting mainly of triglyceride (TG) and cholesterol ester (CE). The serum concentrations of phospholipid (PL) and cholesterol (CH) in pyridoxine-deficient rats were significantly lower than those in pair-fed controls, but their serum levels of TG and free fatty acid (FFA) were not significantly different from those of controls. A significant inverse relation was shown between the concentration of liver TG and PL of pyridoxine-deficient rats. Measurement of incorporation of radioactivity from [1-¹⁴C]acetate into lipid in vivo showed that lipogenesis in the liver of pyridoxine-deficient rats was depressed, although the acetate pool was significantly higher than that in pair-fed controls. The incorporations of radioactivity from [1-¹⁴C]acetate into liver TG and PL in vivo were also lower in pyridoxine-deficient rats than in the pair-fed controls, but the ratio of the radioactivity in liver TG to that in liver PL was higher in pyridoxine-deficient rats than in pair-fed controls. This higher ratio was not due to increased mobilization of serum FFA from extra-hepatic tissues or decreased hydrolysis of liver TG, because labeling of serum FFA and the activity of liver lipase were not significantly different in deficient and control rats. The ratio of total radioactivity in serum TG to that in liver TG was lower in pyridoxine-deficient rats than in pair-fed controls in the latter period of the experiment. Studies were made on the components of PL contributing to liver TG accumulation in pyridoxine-deficient rats. The concentration of liver phosphatidylcholine (PC) was significantly lower in pyridoxine-deficient rats than in pair-fed controls. In pyridoxine-deficient liver, only PC showed an inverse correlation with the liver lipid concentration. These results suggest that a decreased concentration of liver PC and impaired secretion of TG from the liver into blood may be responsible for the inverse relationship of liver TG and PL concentrations in pyridoxine-deficient rats.

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In order to examine the effect of dietary protein and fat on DDT metabolism and liver lipid concentration, rats were supplied with calory adjusted diets consisting of various amounts of protein and fat. The results suggested that dietary protein and fat changed the liver lipid concentration. They also showed that dietary protein and fat affected the residual concentration of DDT and its metabolites in the liver and adipose tissue. The change of the concentration of lipids in liver accompanied a change of the residual concentration of DDT in liver. This fact indicates that one effect of dietary protein and fat on the metabolism of DDT is attributable to the metabolic change of lipids in liver. Dietary protein accelerated the metabolism of DDT and reduced its residual concentration in liver. The results suggest that regression equations exist between the residual concentration of DDT in liver and (1) dietary factors and (2) lipid concentration in liver;
ln(DDT)=a⋅ln(χ₁)+b⋅ln(χ₂)+c⋅ln(TL)+d (1)
ln(DDD or DDE)=a'⋅ln(TL)+b'⋅ln(PL)+c' (2)
where x₁, x₂, TL, and PL are the dietary protein, dietary fat content, total lipid, and phospholipid concentration in liver, respectively, a, a', b, b', c, c', and d are constants. The concentrations of DDT and its metabolites estimated from equations (1) and (2) agrees well with the measured concentrations in liver.

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Young male mice of the C3H inbred strain were kept inside or outside a Trexler vinyl isolator and fed on three autoclaved purified diets which contained 0%, 1% or 5% sodium phytate. The feed N efficiency, the apparent digestibility of gross components of the diet, the balances of N, Ca, Mg and P, and the ash, Ca, Mg and P contents of bone were investigated for each group of mice. No significant differences in these parameters were found with respect to the location of the mouse cages inside or outside an isolator. Mice fed on 5% phytate diet, as compared with mice fed on 0% or 1% phytate diet, had a higher apparent digestibility of crude fat, and a lower apparent digestibility and, retention rate of Mg. Urinary excretion and apparent digestibility of P in mice fed on 5% phytate diet were higher than in other groups of mice.

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In studies on human adaptation to low protein intake, sera from 18 male highlanders of Kalugaluvi in Papua New Guinea, where the main protein supply is from vegetables, were analyzed for concentrations of serum protein, triglyceride, β-lipoprotein and cholesterol, using sera from Japanese males as controls. The serum protein concentration of the Papua New Guinean highlanders was slightly higher, albumin (A) lower and globulin (G) higher, as compared with the Japanese controls. The ratio of (A) to (G) was 1.4 for Papua New Guinean highlanders and 1.9 for Japanese. The concentrations of IgG, IgM, IgA, haptoglobin, prealbumin, α₁-acid glycoprotein, ceruloplasmin and transferrin were also determined by immunological technique. In comparison with Japanese sera, the concentrations of IgG and IgM of the sera of highlanders were significantly higher and the haptoglobin concentration was slightly higher. The concentrations of α₁-acid glycoprotein, ceruloplasmin, transferrin and IgA were similar to those of Japanese sera. A significant difference was noticed between Papua New Guinean highlanders and Japanese in serum total cholesterol concentration.

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Tyypsin inhibitors were isolated from wheat endosperm, and a major inhibitor (wheat endosperm trypsin inhibitor-I, WETI-I) was purified by ion-exchange chromatographies on CM-Sephadex and SP-Sephadex, gel filtration on Sephadex G-75 and chromatofocusing on Polybuffer exchanger PBE 94. This inhibitor was a polypeptide composed solely of amino acids, and its pl value was 9.35. It was found to be homogeneous in gel electrophoresis and velocity sedimentation. It showed strong inhibition on bovine trypsin but weak inhibition on bovine a-chymotrypsin. The molecular weight of the inhibitor was approximately 7, 800 as judged from SDS-gel electrophoresis. This finding, along with the trypsin inhibition data, suggested that the inhibitor bound trypsin in the molar ratio of 1:1. Certain other properties of the inhibitor, including amino acid composition and UV spectral characteristics are presented.

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Studies were made in August 1978 on the N-balance and hematological characteristics of 18 men living in the village of Kalugaluvi (altitude: 1, 500m) near Lufa, which is 60km from Goroka, in the Eastern Highland Province of Papua New Guinea. The average daily protein intake on 3 consecutive days was 95.2 + 29.3 mgN/kg (35.2±10.7g protein/day), and 32.6±18.7% of the total protein intake was in the form of animal proteins. The calculated nutritional value of the dietary protein, scored according to the 1973 FAO/WHO pattern, was 83.7±10.6. Urinary and fecal N excretions during the same period were 81.9± 18.7 and 26.3±11.8mgN/kg, respectively, giving a nitrogen balance of -13.5±24.9 mgN/kg. From N-balance data on individuals, the N-intake for maintenance of a zero N-balance was estimated as 116.3±48.6 mgN/ kg (mean 95% confidence interval). Hematological data showed normal or rather high values for hemoglobin (16.90±1.05 g/100 ml) and serum proteins (8.05±0.40g/100 ml). The in-creased level of serum proteins was associated with an increased globulin level.

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Japanese are unique among the peoples of developed count-ries in having a high intake of eicosapentaenoic acid (C 20:5) from fresh fish and this may in part contribute to their low incidence of cardiovas-cular diseases. Mass spectroscopic analyses of eicosapolyenoic acids (C 20:3, C 20:4 and C 20:5) were carried out on the serum of aged persons living on Kohama island in Okinawa and known to have the lowest incidence of cardiovascular diseases in Japan. All but 4 of the 77 persons examined (73.94±7.81 years old) led active fishing-farming lives. The total amount of eicosapolyenoic acids in the serum of persons on Kohama island (46.77±7.46 mg/ 100 ml) was higher (p<0.001) than that in people on mainland Japan, owing to the higher intake of fresh fish (147.7g/day). A positive correlation (p<0.01) was found between serum C 20: 5 concentration (6.82+2.54 mg/100 ml) and high density lipoprotein concentration (55.38±13.83 mg/100 ml). In addition, there were positive correlations (p<0.01) between serum C 20: 3 concentration (6.58+1.61 mg/100 ml) and total cholesterol (188.60±32.30 mg/ 100 ml), and triglyceride and skinfold thickness. The blood pressure level (p<0.01), incidence of abnormal ECG (p<0.05), and salt intake (6.2-8.3g/ day) estimated from urinalysis, were all lower than the average figures for Japanese of similar ages. No persons examined showed Q-wave on ECG. The percentage of smokers and drinkers were similar for Kohama island and mainland Japan.

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This paper describes experiments designed to characterize the effect of dietary antioxidants on lipid peroxide formation and vitamin A reduction in the liver of rats fed on diets containing polychlorinated biphenyls (PCB). Rats were given 0.025% PCB diets supplemented with dietary antioxidants, for 2 weeks. The antioxidants used were as follows: 10mg% (basal and usual level), 50mg% and 100 mg% of vitamin E, and 50mg% of DPPD and tinoridine respectively. A marked liver enlargement and a significant increase of total liver lipid content were observed in the PCB-fed groups irrespective of the levels of vitamin E and kinds of antioxidants, suggesting that antioxidants were ineffective in preventing the development of fatty liver. Endogeneous lipid peroxide contents in the liver of rats receiving the diets containing 10 mg% vitamin E, DPPD, and tinoridine with PCB increased significantly, whereas no increase was found with the 50 and 100 mg % vitamin E diets with PCB. Hepatic glutathione peroxidase activity was unaffected by PCB and dietary antioxidants. No increase in hepatic vitamin E content occurred in the PCB groups with the addition of 10mg% vitamin E, DPPD, and tinoridine. However, dietary supplementation of vitamin E at higher levels caused an elevation of hepatic vitamin E content and a further increase was observed on the addition of PCB. These results suggest that a sufficiently high level of vitamin E suppresses the increment of the endogeneous lipid peroxide content in the liver of rats fed PCB. On the other hand, the administration of PCB to rats resulted in a significant decrease in hepatic vitamin A content regardless of the levels of vitamin E and kinds of dietary antioxidants. The antioxidants used in this experiment failed to suppress the hepatic vitamin A reduction caused by PCB administration. In addition, an absence of a significant difference in hepatic vitamin A contents among PCB-fed groups was observed. This suggests that the hepatic vitamin A level was independent of PCB-induced lipid peroxidation. Thus lipid peroxidation probably did not mediate the hepatic vitamin A reduction caused by PCB, especially in vivo.

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