VITAMINS

As the first step for the application of nicotinamidase (EC 3.5.1.19) to construct nicotinamide specific biosensor for nicotinamide determination or to prepare bioreactor for nicotinic acid production or to know the regulatory mechanism in niacin metabolism, the properties of the enzyme from Flammulina velutipes were investigated with dialyzed cell-free extract which had very high specific activity. As the results, the following properties of the enzyme were found: optimum pH for the reaction, 7.3; the temperature at which the maximum velocity was attained, 45℃; activation energy, 10.96 kcal/mol; Km value for nicotinamide, 1.4μM; inhibitors which inhibited more than 50% at 1mM, nicotinaldehyde, 3-cyanopyridine, nicotinic acid hydrazide, pyrazinamide, benzamide (all these are competitive inhibitors, Ki=0.2μM-0.6mM), nicotinic acid (product inhibition), heavy metal ions, NADP, NaAD, NAD, NMN; reverse reaction, not detectable. The possibility for application of this enzyme to biosensor or bioreactor is suggested.

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Using the 2,4-dinitrophenylhydrazine (DNPH) method, the α,α'-dipyridyl (DP) method and the high-performance liquid chromatography (HPLC) method, the total ascorbic acid contents in 20 kinds of food were determined. All values obtained by the three methods were compared. In the case of tomato juice, potato, alfalfa sprout, soybean sprout and powdered milk, there was no difference in the values using the three methods. However, the value obtained by the HPLC method for black gram sprout was higher than those by the other two methods. And the values of black tea and fungi such as shiitake obtained by the DP method showed higher values as compared to the DNPH method. Furthermore, the total ascorbic acid contents in black gram sprout, black tea and fungi were studied using the DNPH-HPLC method. The values of black gram sprout showed almost the same as for the DNPH and the DP methods, but the value of the black tea and fungi were different. Further study is required for black tea and fungi.

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To clarify the difference in the bioavailability of vitamin B_1(B_1) and vitamin B_2 (B_2) after oral administration of tablets containing thiamin tetrahydrofurfuryl disulfide (TTFDT) or multivitamin tablets (MVT), two tablets of each preparation were given to six healthy male subjects with age of 20-23 years old. In a single dose experiment of trial I, the peak blood B_1 concentration occurred at 2hrs after administration of 10mg of thiamin tetrahydrofurfuryl disulfide (TTFD) and approximately 16-17% of the dose was excreted into the urine in 24hrs. No differences were observed in the blood concentration-time profiles and in the urinary excretion between TTFDT and MVT. The peak blood concentration of B_2 was also reached at 2hrs after administration of 7mg of riboflavin and approximately 11% of the dose was excreted into the urine in 24hrs. After repeated oral administration of 10mg of TTFD and 7mg of riboflavin per day, in a form of a multivitamin tablet, for 1 week in trial II, B_1 and B_2 concentrations in blood and urine were also determined. B_1 concentrations of 7th administration at each point have shown higher values than those of 1st administration, while the concentration-time patterns have shown little difference between the 1st and the 7th administrations. And daily amounts of B_1 excreted have also been increased with the repeated administrations. On the other hand, the peak blood B_2 concentration has not changed greatly between the 1st and the 7th day administrations, although B_2 concentrations of each starting, point have risen with repeated administrations. However, daily amounts of riboflavin have also increased gradually.

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The method of high-performance liquid chromatography with fluorometric detection for the determination of K vitamins (Ks) and their epoxides (Kepo) in human serum was developed by using a platinum black column and/or two electrodes with two potentiostats as a reduction reactor instead of two electrodes with ESA 5100A Coulochem. The detection limits of Ks and Kepo in the present method were comparable to those with the Coulochem methods, and the costs of the reduction reactors in the present method are more cheaper than those with the Coulochem methods.

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