The authors proved that the green fluorescent substance found in the culture filtrate of Cl.acetobutylicum was identical with G-substance (8-N-ribityl-6,7-dimethyllumazine) (I) found in Er.ashbyii by Kuwada and Masuda. Using the enzyme preparations of several microorganisms, the authors succeeded to synthesize riboflavin enzymatically from this compound (I) in the presence of adequate 4C-donor and cofactors. It was also proved that (I) was synthesized enzymatically from 4-ribitylamino-5-aminouracil (II) and these two enzyme systems, (II)→(I) and (I) →riboflavin, were differed each other.

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It was found that FAD or FMN formed complexes equimolecularly with metal ions such as Ca^<++>, Mg^<++>, Ba^<++>, Cd^<++>, Mn^<++>, Co^<++> and Ni^<++>. The stability constants for these complexes were determined at pH 7.2 and 0.1 of ionic strength. It was shown that the stability constants for FMN complexes containing transition metals such as Mn^<++>, Co^<++>, Ni^<++> were greater than those for the other metal complexes. However, these relations were not observed in the stability constants for FAD complexes.

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A chromatographical study was made on possibility for existence of nucleotides in FAD preparations as the impurities which were hardly eliminated from the preparation. The results were as follows : cyclic-FMN and two other substances were detected as the impurities in FAD with 75% purity. It was found that the latter substances absorbed ultraviolet rays at 260mμ. This fact suggested that the substances were nucleotide relating compounds. However, a very slight amount of cyclic-FMN was only detected in more purified FAD preparations. It was previously reported that cyclic-FMN was produced from FAD during preservation. In this paper, it was, furthermore, confirmed that occurrence of the substance was observed also in the course of purification procedure of FAD.

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Studies were made on behaviors of riboflavin, FMN and FAD on various anion exchange resins as a preliminary experiment for elimination of cyclic-FMN from FAD preparations. It was found that FMN and FAD were easily adsorbed on any kind of anion exchange resins, while adsorption of riboflavin on these resins hardly occurred. A remarkable difference was, however, observed between behaviors of FMN and FAD on such resins as Dowex-2 (Cl), Amberlite IRA-411 (HCOO) and IRA-400 (CH_3COO). This results suggests that these resins are useful for elimination of cyclic-FMN from FAD preparations, since cyclic-FMN has been known to have similar properties as FMN.

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Elimination of cyclic-FMN from FAD preparations was tried by using several anion exchange resins. A satisfactory result was obtained when adsorption on Dowex-2 (Cl) and gradient elution with 0.1N NaCl were carried out.

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Elimination of salts from FAD solution eluted from anion exchange resins was tried by the use of active carbon. The procedure was simultaneously used for concentrating a dilute solution of FAD. After a dilute solution of FAD (25μg/cc) containing 0.1N NaCl was subjected to a thin layer of active carbon, the salt was washed away from the layer with water, and then the layer was washed again with acetone. The second washing with acetone made the following elution of FAD easy. FAD solution of 40 fold concentration was obtained with the recovery of 89% by the procedure mentioned above using a mixture of acetone and p-cresol saturated water (3 : 7) as the solvent for elution.

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The existence of an enzyme which synthesizes FAD from FMN and ATP was proved in cells of Eremothecium ashbyii. The enzyme required Mg^<++> or Mn^<++> for its full activity. The optimum pH was found to be 8.0. When riboflavin and ATP were used for the substrates, a very slight amount of FAD was formed. Therefore, it seemed that the synthesis of FAD by Eremothecium ashbyii might be carried out through the initial phosphorylation of riboflavin, followed by the interaction of FMN and ATP.

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To study the effect of stress on the metabolism of riboflavin, the timely change of riboflavin content of various organs was investigated after the administration of ATCH or adrenalin in rats. In a group administered with ATCH, it was observed that the riboflavin contents of blood, liver and brain decreased at first and then increased. In a group given by adrenalin, the riboflavin content in blood slightly increased and then remarkably decreased, and in liver and brain it decreased at first and then increased. From the above results, it is supposed that the riboflavin metabolism under the stress is influenced by the anteriorpituitary adreno-cortical system or sympathico-adrenal system.

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Determination of vitamin A in blood serum and serum protein of healthy human subjects showed that about 90% of the vitamin in serum was present in protein fractions. Fractional determination by salting-out with varying sodium sulfite concentrations revealed that about 70% of the vitamin in protein fractions occurred in globulin and 30% in albumin. Determination of vitamin A in serum, serum protein and serum globulin by salting-out of the serum taken 3 hours after orally administering 1 to 12 mg vitamin A palmitate to healthy adult subjects, when the serum vitamin A levels reached the maximum values, showed that the increase of the vitamin in total body was calculated to be about 11 to 50% of the amount administered, the percentage recoveries being the less, the more the vitamin was administered. Determination of vitamin A in serum protein fractions of normal healthy subjects by zone electrophosesis showed that about 1/4 of the total protein vitamin A was albumin vitamin A and 3/4 globulin vitamin A, of which α_1-vitamin A was greatest and α_2-vitamin A smallest. The ratios of vitamin A alcohol and vitamin A ester in each protein fraction were about the same. The serum protein vitamin A values in patients with kidney disturbances were about twice those of the normal subjects. The values of patients with liver disturbances were about the same as those of normal subjects. The ratios of the vitamin values of each fraction were somewhat different in kidney disturbances. Incubation of 3 ml serum with varying amounts (5 to 100 μg) of vitamin A palmitate resulted in the increase of the vitamin of each protein fraction by 1 to 8%, whereby the recoveries were the less, the more the amounts added. When vitamin A palmitate equivalent to 20 μg of vitamin A alcohol was added to 3 ml of dialyzed serum incubated for varying periods, and the vitamin contents in protein fractions were determined, the values rose gradually with time, reaching the maximum in two hours. The ratios of the vitamin values of each protein fraction varied somewhat, but it remained roughly constant after two hours. After adding vitamin A alcohol, vitamin A aldehyde, β-carotene, vitamin D_2,D_3,α-tocopherol, vitamin K_3,respectively, to dialyzed serum and incubated at 37℃ for varying periods, the vitamin values of the protein fractions became maximum after one hour. The ratios of the vitamin in each protein fraction varied somewhat according to the kinds of fat-soluble vitamins, whereby about 1 to 12% of the vitamin added was recovered. Following incubation of riboflavin, FMN, FAD, respectively, to dialyzed serum, about 10% of the vitamin added was detected in protein fraction. Each vitamin added to the serum became associated with each protein fraction without showing any special affinity to a certain protein fraction.

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