VITAMINS

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The enzymes concerned with thiamin phosphates metabolism in Saccharomyces cerevisae were characterized by genetic and biological analyses. There exist two species of acid phosphatase in yeast periplasmic space ; one, repressible by Pi and the other, repressible by thiamin. Thiamin-repressible acid phosphatase encoded by PHO3 gene was ascertained to physiologically catalyze the hydrolysis of extracellular thiamin phosphates, thus participating in utilization of the thiamin moiety of the phosphates. Next, the THI80 gene encoding for a thiamin pyrophosphokinase and the THI6 gene encoding for a bifunctional thiamin-phosphate pyrophosphorylase/hydroxyethylthiazole kinase were isolated from yeast genomic library by functional complementation of thi80 and thi6 mutant, respectively. The THI80 gene contained a 957-bp open reading frame coding a 319-aa polypeptide (Mw : 36, 616), and the THI80 gene was proved to be essential for growth by a gene disruption study. The THI6 gene contained a 1,620-bp open reading frame coding a 540-aa polypeptide (Mw : 58, 058), and the functional domains of the two enzvme activities in the THI6 gene were determined to locate independently by a in vitro mutagenesis. Northern blot analysis demonstrated that PHO3, THI80 and THI6 gene expression are regulated at the mRNA level by intracellular thiamin pyrophosphate, and they require the positive regulatory factors encoded by the THI2 and THI3 genes in S. cerevisiae.

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Protein nutrition in vitamin B_<12> (B_<12>)-deficient rats was evaluated not only by examining the effect of dietary protein on B_<12>-deficiency but also by examining the utilization of dietary protein in B_<12>-deficient rats. Severe B_<12>-deficiency was confirmed in rats fed on a soybean protein diet by a remarkable increase in urinary methylmalonic acid (MMA) and a striking depression of hepatic B_<12>-dependent enzymes. Growth retardation and an increase in urinary excretion of MMA were prominent in the B_<12>-deficient rats fed on a high protein diet, and the amount of MMA excretion was proportionally increased to the amount of dietary protein. Therefore, it was obvious that the high protein diet caused severe and rapid B_<12>-deficiency. The severe B_<12>-deficient rats showed growth retardation even in pair-feeding. Plasma proteins significantly decreased in B_<12>-deficient rats than those in pair-feeding control rats , and the excretion of urinary nitrogen compounds was also significantly decreased. The activity of hepatic cystathionine β-synthase and hepatic S-adenosylmethionine content decreased in severe B_<12>-deficient rats which showed a distinct depression of hepatic methionine synthase activity, and these decreases could be alleviated partially by methionine supplementation. It was assumed that methionine resynthesis, availability and dietary protein utilization were depressed in severe B_<12>-deficient rats.

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The high-performance liquid chromatographic analysis for the determination of β-carotene content in human serum with retinyl myristate as an internal standard was improved with the aid of electrochemical detection in oxidation mode by a short and long column-swiching method with a species of mobile phase. The mobile phase was acetonitrile, 2-propanol, methanol (71 : 20 : 9) including 0.025 M NaClO_4. The inter-day coefficient of variation were 0.202 ± 0.014 μg/ml (n=6). Also the recovery rate of adding 0.04 μg/ml β-carotene was 92%. It is concluded that this method provided the stable chromatogram by a short-repeated interval measurement for detection of β-carotene in serum at the 10^2 nanomolar level.

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