VITAMINS

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For the understanding of the molecular basis of the flavoenzyme catalyses in general terms, studies on old yellow enzyme (OYE) have been carried out from various points of view. We have found the microheterogeneity of OYE and separated the enzyme, which had been treated as a single entity, into five different subforms, OYE-I, II, III, IV and V, by means of anion exchange HPLC. These subforms exhibited similar NADPH oxidase activities but distinct microenvironments surrounding FMN as revealed by ^<13>C NMR with ^<13>C-enriched FMN. The limited proteolysis of OYE with α-chymotrypsin cleaved at a specific site yielding two fragments of molecular weights of 14,000 and 34,000. These fragments are tightly associated with each other forming a complex termed "nicked OYE" except under stringent denaturating conditions. Nicked OYE still retained FMN as the coenzyme and exhibited slightly modified kinetic and spectral properties as compared to native OYE. Covalently-flavinylated OYE was obtained by a combination of the limited proteolysis with α-chymotrypsin and reconstitution of OYE with 8-fluoro-8-demethyl-FMN (8F-FMN). The flavinylation took place between the amino group of the N-terminal leucine of the 14K fragment and the 8-position of the 8F-FMN. That the flavinylation took place within the 14K fragment indicated that in native OYE the 14K domain is the FMN binding domain. The amino acid sequence around the flavinylation site shows homology with a certain stretch of sequence in flavodoxin arguing in favor of the 14K domain being the FMN binding domain.

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The simultaneous radiodilution assay of serum vitamin B_<12> and folate was studied on the so-called "non boil methods", in which these two vitamins were released from their endogenous binding proteins with alkaline denaturation and separated the bound vitamins from the free ones with the magnetic iron particles coated these vitamin binders (purified hog intrinsic factor and β-lactoglobulin from cow milk), compared with other radiodilution assays. 1. The reproducibilities of this method were found 3.42〜5.25% of CV on B_<12> and 3.68〜7.89% on folate, with satisfaction. 2. The recovery tests of the two serum vitamins with the use of CN-cobalamin and folic acid (J.P.) were finely showed the ratios of 99.4〜100.4% on B_<12> and 99.3〜101.8% on folate, respectively. The values of both vitamins measured by this method were found slightly lower than those by boil method, and higher than those by non boil charcoal absorption method. 3. Normal range of serum total B_<12> concentration was 229〜847 (465±155, n=325) pg/ml and that of folic acid was 2.34〜7.08 (4.23±1.19, n=310) ng/ml. 4. The values of serum B_<12> measured by this method were found remarkably low in malignant lymphoma cases with gastorectomy during antitumor drug therapy, compared with its hematological findings of bone marrow.

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Bioavailability of calcium carbonate, DL- or L-calcium lactates and powdered oyster shell as dietary calcium sources was investigated .Vitamin D-deficient rats were fed ad libitum a diet with normal calcium contents (0.44%) adjusted with one of the above four kinds of calcium compounds under vitamin D-deficient or -replete conditions for a week. During and after feeding, the concentrations of calcium, phosphorus and parathyroid hormone in plasma were measured. In addition, plasma alkaline phosphatase activity and femoral mineral contents were also measured. Among the compounds, the bioavailability of L-calcium lactate and powdered oyster shell appeared to be slightly better than the other two compounds in the increase of body weights, plasma calcium and phosphorus levels and femoral mineral contents, and the decrease of plasma parathyroid hormone level and alkaline phosphatase activity in vitamin D-replete condition. However, there was no significant difference among the compounds in vitamin D-deficient condition.

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The catabolism of nicotinamide in riboflavin-deficient rats was compared with that in control rats. In riboflavin-deficient rats, no activities of N^1-methyl-2-pyridone-5-carboxamide (2-Pyr)-forming N^1-methylnicotinamide (MNA) oxidase and N^1-metyl-4-pyridone-3-carboxamide (4-Pyr)-forming MNA oxidase were not detected in vitro, however, the nicotinamide methyltransferase activity was not changed compared with the control rats. As a result, the excretion of MNA was higher in riboflavin-deficient rats than in control rats and the excretion of 2-Pyr and 4-Pyr was lower in riboflavin-deficient rats than in the control rats. Therefore, the resulting excretion ratio of (2-Pyr +4-Pyr )/MNA was greatly lower in the riboflavin-deficient rats than in the control rats.

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