To elucidate the mechanisms of intracellular vitamin B_<12> (Cbl) transport and the coenzyme form synthesis in view of comparative biochemistry, the Euglena gracilis systems were studied. Euglena contained numerous Cbl-binding proteins in addition of Cbl-dependent enzymes. They were distributed in the extracellular, pellicular, cytosolic, mitochondrial, chloroplastic or microsomal fraction. Some of these binding proteins were purified to homogeneity and characterized. Euglena cytosolic binding proteins functioned in the intracellular Cbl transport. These observations suggest that Euglena can efficiently accumulate Cbl from a trace amount of Cbl in the environment using these Cbl-binding proteins. The Euglena system is different from the mammalian or the bacterial one. Activities of enzymes involved in the synthesis of the Cbl coenzymes were found in the Euglena mitochondria. Euglena aquacobalamin reductase was purified to homogeneity and characterized. The Euglena enzyme was a flavoprotein with Mr of 66,000 and was specific to NADPH and ON-Cbl. A novel enzyme, cyanocobalamin reductase (NADPH; CN-eliminating) required NADPH and FAD (FMN) for decyanation of CN-Cbl. Overall-conversion activity of OH-Cbl to AdoCbl was also found in the Euglena mitochondria, indicating that AdoCbl is synthesized only in the mitochondria. On the other hand, rat liver contained both NADH-and NADPH-linked aquacobalamin reductases. The NADH-linked enzyme was distributed in the mitochondrial outer and microsomal membranes. The distribution was identical to that of the NADPH-linked enzyme. The results suggest that the mammalian system for the coenzyme synthesis is distinct from the Euglena one.

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Two multipore-type sustained-release preparations (VC-SR-A and VC-SR-B) containing 500 mg of vitamin C were prepared, which were coated with mixtures of water-soluble polyethylene glycol and insoluble ethylcellulose. The preparations differed in the dissolution profile of vitamin C; the in vitro release rates being 7.3 %/h for VC-SR-A and 14.2 %/h for VC-SR-B. The bioavailability of vitamin C after the administration of a single dose was examined in six subjests (25-32 years) who had been saturated with vitamin C, and compared with an osmorelease-type sustained-release preparation (AcuSystem C) and a conventional immediate-release preparation (VC-IR) in a crossover trial. The mean T_<max> was 3 h for VC-IR, 6 h for VC-SR-B, 7 h for VC-SR-A and 8.5 h for AcuSystem C. Mean C_<max> (mg/100ml) were 0.28 for VC-SR-A, 0.40 for AcuSystem C, 0.48 for VC-SR-B and 0.74 for VC-IR. The mean AUC (mg.h/100 ml, 0-24 h) was 3.43 for VC-SR-A, 5.73 for AcuSystem C, 6.68 for VC-SR-B and 6.80 for VC-IR. The mean percentages of the dose excreted in the 24-h urine were 7.7 for VC-SR-A, 17.6 for AcuSystem, 23.4 for VC-SR-B and 39.1 for VC-IR. The results indicate that for VC-SR-B the rate of bioavailability is slower but prolonged and thus the extent of bioavailability is almost equal to that of VC-IR. Also, concerning retention of vitamin C in the body, VC-SR-B seems to be preferable to VC-IR.

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The biosynthetic routes of the pyrimidine moiety of thiamin differ between eucaryotes and procaryotes. In eucaryotes, the N-3, C-4, and amino-N at C-4 of the pyrimidine have been shown to originate from N-1, C-2, and N-3 of imidazole ring of histidine, respectively. Further C-6, C-5 and C-7 of the pyrimidine are C-8 of the pyrimidine remains unsolved. In procaryotes, N, C-1 and C-2 of glycine are incorporated into N-1, C-4 and C-6 of the pyrimidine, respectively. We studied the incorporation of glycine into the pyrimidine in a eucaryote, Saccharomyces cerevisiae. The results rerealed that [1-^<14>C]glycine was not incorporated into the pyrimidine, whereas [2-^<14>C]glycine was incorporated with a little dilution. The incorporation of [2-^<14>C]glycine was diluted by the addition of formate. The localization of the radioactivity in the pyrimidine labeled with [2-^<14>C]glycine was at C-4 of the pyrimidine. It was concluded that C-2 of glycine was cleaved into one-carbon unit, which was then incorported into C-4 of the pyrimidine via C-2 of the imidazole of histidine; indicating that glycine was not directly incorporated into the pyrimidine as a molecule.

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Women students were administered three times 50 mg of nicotinamide after meal, the 24-hour urine was collected and the urinary excretions of N^1-methylnicotinamide (MNA), N^1-methyl-2-pyridone-5-carboxamide (2-Pyr) and N^1-methyl-4-pyridone-3-carboxamide (4-Pyr) were measured. Furthermore, 10 μl of blood was taken from finger 2 hour after the lunch and the blood NAD and NADP values were measured. As the results, the urinary excretions of MNA, 2-Pyr and 4-Pyr was increased 8, 13- and 13-folds, respectively, compared with those of the control period. A recommended index of niacin nutrition, (2-Pyr+4-Pyr)/MNA excretion was increased by 1.7-fold by nicotinamide load. The blood NAD level was 1.7-fold higher in the loading period than in the control period, but the blood NADP level was almost the same between the two period. From these results, it was found that blood NAD level as well as (2-Pyr+4-Pyr)/MNA excretion would be used as an index of niacin nutrition in humans.

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Biotin determination in blood has been usually carried out with either microbiological methods or competitive protein binding methods (binding assay). Blood samples are either hydrolyzed with H_2SO_4 by autoclaving or treated by papain to release free biotin from protein bound biotin. Thus, we tried to determine serum biotin levels of Japanese women using these procedures. The biotin levels of women's serum treated with papain were 2.3±0.51 ng/ml (n=31) as measured by a microbiological assay and 3.6 ± 0.72 ng/ml (n=31) by a binding assay. We found that for the papain treatment much longer incubation time (4 days) was needed than the overnight as previously reported. We proposed a new binding assay by using polystyrene beads and biotinylated houseradish peroxidase. This assay is a sensitive (0.02-0.4 ng/assay tube) and simple method which does not need any special equipments.

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