The data in our previous paper demonstrated that some atocopheryl esters administered orally were absorbed in their unchanged form through the lymph, while some other esters were absorbed after being hydrolyzed individually to different degrees. The hydrolysis during absorption seems to be related to the structure of the ester group of α-tocopherol at the 6-position.
The purpose of this work is to study the metabolism and biological effect of tocopherol esters in vitamin E-deficient rats. Three esters were used on the basis of their behavior during absorption through the lymph, as follows; α-tocopheryl acetate (an easily hydrolyzable ester), the nicotinate (a moderately hydrolyzable one) and the pivalate (a scarcely hydrolyzable one).
The easily hydrolyzable esters will suffer the same metabolic fate through absorption as α-tocopherol. The moderately and scarcely hydrolyzable ones have a tendency to show different physiological effects from α-tocopherol due to absorption of the unchanged ester.
The effect of these esters on the microsomal enzymes in the liver such as cytochrome P-450, cytochrome b₅, aniline and hexobarbital difference spectra and NADPH-dependent cytochrome c reductase was determined. It was shown that the pivalate inhibited the release of NADPH-dependent cytochrome c reductase activity to supernatant in spite of low distribution in the 105, 000×g sediment. The result suggests that the pivalate as a model compound may be interesting to examine for its membrane stabilizing effect of α-tocopherol.

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The serum levels of vitamin E, cholesterol and lipoproteins, α₁-lipoproteins and β-lipoproteins in pregnant women were investigated. It was found that serum vitamin E, cholesterol and the lipoproteins had a tendency to increase throughout gestation. The mean serum α₁-lipoprotein concentration was higher in pregnant women than in healthy control women. The ratios of the vitamin E concentration to the sum of α₁-lipoproteins and β-lipoproteins concentration in sera was constant. Furthermore, it was found that serum vitamin E was correlated closely to the lipoprotein content (γ=0.814, p<0.001), except in a few subjects with abnormally high vitamin E levels.

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A methanol-utilizing bacterium, Protaminobacter Tuber, required cobalt ion or vitamin B₁₂ as its growth factor, which could be replaced by succinate among various additions to the cobalt-deficient medium. The presence of adenosylcobalamin (adenosyl-B₁₂)-dependent methylmalonyl-coenzyme A (CoA) mutase was demonstrated in the cellfree extracts of P. ruber. The specific activity of this mutase was not only fairly high in comparison with that reported with other organisms but also detected at a similar level throughout the cultivation period. The cell-free extracts of P. ruber grown on non-C₁ compounds as a sole carbon and energy source also had methylmalonyl-CoA mutase activity. Furthermore, the extracts of this microorganism catalyzed the reactions from propionyl-CoA to succinyl-CoA and from α-ketoglutarate to a-hydroxyglutarate.

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The mechanisms of proteolysis of two apo-forms of pyridoxal enzymes, cystathionase [EC 4. 2. 1. 15] and ornithine aminotransferase [EC 2. 6. 1. 13] by serine protease from the rat small intestine were compared.
The apo-forms of these two pyridoxal enzymes are susceptible to the serine protease, whereas the holo-forms of the enzymes are not. Pyridoxal phosphate, the coenzyme of these two enzymes, prevented their inactivation by the serine protease. The difference in susceptibility of the apo and holo-forms to the serine protease was due to the difference in their conformations.
The time course of inactivation of cystathionase by the protease was apparently biphasic. During the first phase of inactivation, disappearance of the band corresponding to the native protomer, with a molecular weight of 47, 000, was accompanied by accumulation of new material with a molecular weight of 39, 000. In the late stage of proteolysis, extensive degradation of the large molecular weight intermediate was observed. This large intermediate product was isolated and found to compete with intact cystathionase as a substrate for the protease. Proteolysis of cystathionase was accompanied by both dissociation of the enzyme molecule and loss of its antigenicity.
Limited proteolysis of ornithine aminotransferase apoenzyme by the serine protease resulted in formation of a large molecular weight product similar to the native apoenzyme, but with nicks in the molecule. Dodecylsulfate-polyacrylamide gel electrophoresis showed that the apoenzyme is degraded to intermediate forms (molecular weight 41, 500 and 15, 000) and later, to stable forms (molecularr weight 25, 500 and 13, 500). and D have no-activity. The intermediate product of ornithine aminotransferase of large molecular weight is formed rapidly in the initial reaction of the serine protease, but the subsequent proteolytic events proceed much more slowly.

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The metabolism of flavins in mouse was studied with [F-(2)-¹⁴C, A-(2, 8)-¹⁴C] FAD and [F-(2)-¹⁴C, ³²P] FMN. Ninety minutes after injection, radioactive isoalloxazine nucleus of double-labeled FAD was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas a small amount of radioactive adenine nucleus of double-labeled FAD was found in FAD in the liver. In the case of FMN, radioactive isoalloxazine nucleus of double-labeled FMN was markedly incorporated into FAD, FMN and riboflavin in the liver, whereas only a minute amount of radioactive phosphorus was incorporated into FMN and FAD in the same organ. These results indicate that FMN and FAD injected are rapidly hydrolyzed and resynthesized in animal body.

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The intestinal calcium transport activity and serum calcium and phosphorous concentrations of vitamin D-deficient rats were increased by irradiation with an ultraviolet (UV) lamp. The existence of 25-hydroxyvitamin D₃ (25-OH-D₃) in their bloods and livers was physicochemically confirmed by high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) and mass fragmentography, whereas the compound could not be detected in the tissues of nonirradiated rats. The results strongly suggested that vitamin D₃ in vivo generated in irradiated rat skin might be normally metabolized 'and utilized to prevent rickets. The level of 25-OH-D₃ in the tissues was determined by a HPLC method.

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The effect of vitamin D on the change of bone contents of cyclic nucleotides and DNA was examined in rats fed a vitamin D deficient, low calcium diet. Daily administration of vitamin D₃ (0.25 μg) to the animals for a week induced a significant increase of plasma calcium concentration as well as of bone DNA and cyclic AMP contents. After a single administration of vitamin D₃ (0.25 μg), the earliest change observed was the elevation of plasma calcium levels, followed by an increase of the cyclic AMP content which was significant at 48 hr. Significant increase of the DNA content in bone was first observed at 72 hr. In contrast to the increase of cyclic AMP contents in bone, the level of cyclic GMP was almost constant. When the animals were switched to the same vitamin D deficient, high calcium (3%) diet containing lactose, their serum calcium concentrations were markedly elevated, while neither the cyclic AMP nor DNA contents were changed. These results indicate that although vitamin D administration leads to a change in the cyclic AMP and DNA contents of bone, these changes are not directly related to the early calcium mobilizing action of the vitamin. The bone change in the cyclic AMP and DNA contents appears to be due to a direct effect of vitamin D rather than to the elevation of serum calcium concentration.

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Experiments were performed to investigate the mechanisms of the nutritional anemia caused by the excess methionine in rat, and the alleviative effects of glycine on it. After two months of feeding with 2.5% L-methionine on 9% casein diet, a moderate degree of anemia was manifested, but, the addition of glycine to the excess methionine diet prevented this phenomenon. The activity of heme-α-methenyl oxygenase, the key enzyme in the metabolic pathway of hemoglobin degradation, in the liver was not altered by the supplementation of either methionine or glycine to the diet. In order to investigate the mechanisms of the alleviation of the toxic effects of excess methionine by glycine, and also, whether glycine is the only amino acid which has the alleviative effect on the toxicities of excess methionine, two amino acids other than glycine, Lleucine and L-alanine had been chosen as the supplemental amino acid to the excess methionine diet. Deposition of iron in the spleen was greatly increased in the rat fed excess methionine diet, but not in the rat fed methionine + glycine diet. Activity of 5-aminolevulinic acid synthetase (ALAS), the key enzyme in .the hemoglobin biosynthesis, in the bone marrow was remarkably elevated by the addition of methionine, and only glycine could prevent this phenomenon. According to the results obtained, it is considered that the excess methionine would lower the biosynthesis of globin because of the amino acid imbalance. But the addition of glycine enhanced the metabolism of excess methionine, and this helps the accerelation of globin synthesis in the rat to promote the biosynthesis of hemoglobin.

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The time course of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity and lipogenesis and cholesterogenesis from 1-¹⁴C-acetate and 2-¹⁴C-mevalonate was examined in the liver of rats refed diets containing different fats at the 10% level after 48 hr fasting. Fasting caused a profound depression of the reductase activity and sterol and fatty acid synthesis. In rats refed for 30 hr, the activity of HMG-CoA reductase was restored to about one-half of the level observed in prefasting rats, irrespective of the type of dietary fats. When safflower oil and trilaurin were dietary fats, the activity remained this level until 78 hr, then declined, whereas with tristearin, activity progressively increased until 78 hr. On refeeding for 174 to 222 hr, the reductase activity was significantly higher in the tristearin than in the trilaurin group. Similar patterns were demonstrated in cholesterogenesis either from acetate or mevalonate, though extents of activation after refeeding were markedly different in these precursors. Dietary fat dependent changes in the content of hepatic cholesterol and in the concentration of plasma cholesterol were also observed.

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