4-Aminobenzoate hydroxylase(4ABH) is a FAD-dependent monooxygenase that catelyzes the decarboxylative hydroxylation of 4-aminobenzoate with the consumption of NAD(P)H and O_2. 4ABH was shown to utilize the proA hydrogen of NAD(P)H. The fact supports the idea that all of the external monoxygenases are involved in the transfer of the proA hydrogen atom of the nicotinamide nucleotide. In order to investigate the structural characteristics of 4ABH, four monoclonal antibodies (mAbs) against the enzyme were produced. Of the mAbs, three mAbs were shown to recognize the FAD-binding domain of the enzyme. The order of the epitopes to the four mAbs was determined by analyzing the immunoblotting patterns of the peptides obtained from the enzyme. The findings provide the evidence for the location of the FAD-binding domain of the enzyme. Interestingly, one of the mAbs recognizing the FAD-binding domain of 4ABH was shown to recognize those of salicylate hydroxylase and _D-amino acid oxidase, supporting the Rossman's hypothesis. The sequence of 37 N-terminal amino acid residues of the peptide fragments obtained by digestion with lysyl endopeptidase has been determined. The cDNA coding the 37 amino acid residues was produced by PCR. Using the probe prepared by random-priming of the cDNA, the mRNA of the enzyme was detected and its size was shown to be approximately 1.7 kb.

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To clarify the nutritional status of vitamin D in elderly subjects, the plasma levels of 25-hydroxyvitamin D (25-OH-D) and 1α, 25-dihydroxyvitamin D (1, 25-(OH)_2D) and the effects of single oral administration of multivitamin tablets (200 IU/day of vitamin D_2) on plasma levels were compared with those in young adults. Separative assay on the vitamins D_2 and D_3 compounds of 25-OH-D and 1, 25(OH)_2D was performed. When the concentrations of these compounds in the plasma were assayed before administration of multivitamin tablets, the plasma levels of total 25-OH-D (25-OH-D_2^+25-OH-D_3) and total 1, 25(OH)_2D(1, 25(OH)_2D_2^+1, 25(OH)_2D_3) in elderly subjects were significantly lower than those in young adults. No significant difference was found between those in male and female of elderly subjects. When the concentrations of 25-OH-D and 1, 25(OH)_2D in the plasma were assayed after single oral administration of 200 IU of vitamin D_2 in multivitamin tablets, neither 25-OH-D_2 nor 1, 25(OH)_2D_2 plasma levels was increased.

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Elderly patients in hospitals for a long term have been reported to have low levels of vitamin C in USA and Europe, but no such data on Japanese elderly patients have been published. We therefore determined the vitamin C status of long-term hospitalized patients aged 66-96 years by assessing plasma levels and the dietary intake of this vitamin. Vitamin C status was also determined in a control group of healthy young adults aged 19-35 years. The plasma vitamin C level (M±SD, mg/100ml) was 0.20±0.07 for elderly male (n=9), 0.39±1.12 for elderly female (n=10), 0.50±0.17 for young male (n=10), and 0.61±0.13 for young female (n=10). As expected, the vitamin C level was significantly lower in the elderly patients than in the young adults. The proportion of the elderly patients classified as being vitamin C deficiency was much greater than in the young adults. The dietary intake of vitamin C (M±SD, mg/day) was 61.3±7.4 for elderly male, 54.9±13.1 for elderly female, 92.2±67.5 for young male, and 85.6±50.6 for young female. Dietary intake of vitamin C was lower in the elderly patients than in the young adults. All subjects were given a multivitamin tablet containing 250mg of vitamin C after breakfast. In all cases the vitamin C levels at 24h after ingestion were significantly higher than before ingestion. Our findings suggest that low-dose supplementation of vitamin C should be considered for hospitalized elderly individuals.

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Nitroblue tetrazolium (NBT)-glycinate assay was proposed as a suitable method of identification as well as determination for PQQ and quinoproteins by Fluckiger et al.[Biochem.Biophys.Res.Commun.153, 353(1988)] and Paz et al.[Biochem.Biophys.Res.Commun.154, 1330(1988)]. Before trying to applicate this assay method to our choline dehydrogenase preparation from rat liver, we investigated specificity of this assay method. It was ascertained that PQQ was the most reactive compound among investigated (minimum sensitivity : 1 pmol). Some coenzymes and some quinone compounds were also positive to the method although they were less sensitive. Some enzymes were also positive to it but with no correlation with the amount of enzyme. As a result, low specificity of the method will elicit erroneous results if one would insure the existence of quinone compound in his preparation by only this criterion. Our choline dehydrogenase preparation did not show proper redox-cycling in the method indicating the absence of quinoid compound in it.

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An experiment was conducted with growing rats to investigate the effects of 0.1% myo-inositol, 0.1% p-aminobenzoic acid and 0.1% _<DL>-α-lipoic acid in food for 13-14 days on serum and liver lipids, liver thiobarbituric acid reactive substances (TBA-RS) and activity of serum glutamate-pyruvate transaminase (GPT) in rats fed 0.1% 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane(DDT). Feeding of myo-inositol and p-aminobenzoic acid significantly depressed the increase in serum activity of GPT due to DDT intake. In addition, the increases in liver weight, liver total lipids, liver cholesterol, liver triglyceride and serum phospholipid due to DDT feeding were suppressed by dietary addition of myo-inositol. These results suggest that dietary myo-inositol and p-aminobenzoic acid are important for animals exposed to xenobiotics such as DDT.

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