Gas chromatography for the identification of vitamin D₂, 5, 6-trans vitamin D₂, isocalciferol (isovitamin D₂), isotachysterol₂, tachysterol₂, lumisterol₂ and ergosterol was investigated, applying the following conditions: apparatus, a Shimazu GC-1B Gas Chromatograph with hydrogen flame ionization detector; column, a U-shaped stainless steel tube (0.4×75cm) packed with silianized 60- to 80-mesh Gas-Chrom W coated with 1.5% SE-30; column temperature, 220°; vaporizer temperature, 250° to 260°; detector temperature, 220°; nitrogen gas flow, 90 to 120ml/min. Lumisterol₂ from thermal cyclization products of vitamin D₂ (pyro- and isopyrocalciferol), formed during the gas chromatography, 5, 6-trans vitamin D₂ from isocalciferol and isotachysterol₂ from tachysterol₂ were not separated. Therefore, the identification of these compounds was impossible by applying gas chromatography alone.

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Two methods of alumina column chromatography, method I and II, for the identification of vitamin D₂, 5, 6-trans vitamin D₂, isocalciferol, isotachysterol₂, tachystrol₂, lumisterol₂ and ergosterol were investigated, and the following results were obtained.
1. Vitamin D₂ from tachysterol₂, 5, 6-trans vitamin D₂ from isocalciferol, and isotachysterol₂ from lumisterol₂ were not separated by the column chromatography according to method I.
2. However, the separation of vitamin D₂ from tachysterol₂, and isotachysterol₂ from lumisterol₂ were possible by the gas chromatography reported previously, so the identifications of these compounds were possible by the application of gas chromatography for the eluates obtained by the alumina column chromatography according to method I. This is a new identification method for the compounds except 5, 6-trans vitamin D₂ and isocalciferol.
3. 5, 6-trans Vitamin D₂ and isocalciferol were separated from other compounds by the column chromatography according to method I, but the identification between them were impossible by the method mentioned above.
4. The separative identification of 5, 6-trans vitamin D₂ from isocalciferol was possible by the column chromatography according to method II.
5. It was confirmed that these identification methods were applicable to the further investigations on the chemical isomerization of vitamin D₂.

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Thin layer chromatography for the identification of vitamin D₂, 5, 6-trans vitamin D₂, isocalciferol, isotachysterol₂, tachysterol₂, lumisterol₂ and ergosterol was investigated, and the following results were obtained.
1. When Kieselgel G as an adsorbent and n-hexane-methyl ethyl ketone (10:2) as a developing solvent were used, the most desirable separation was obtained.
2. However, the separation of vitamin D₂ from tachysterol₂, 5, 6-trans vitamin D₂ from isocalciferol, and isotachysterol₂ from lumisterol₂ was not possible even under the most desirable condition.
3. These results were identical with those obtained by the alumina column chromatography according to method I described in the previous report (2).
4. There are some difficulties to apply other identification methods after thin layer chromatography, because the application of large quantities of the compounds to the chromatography is generally impossible. Based on these observations, it was concluded that thin layer chromatography might be unsuitable for the identification of vitamin D₂ isomers.

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The chemical isomerization course of vitamin D₂ to isotachysterol₂ by acetyl chloride was investigated by using the identification method for vitamin D₂ isomers described in the previous papers, and it was confirmed that the isomerization went on through the course: vitamin D₂→isocalciferol→isotachysterol₂.

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1. The hepatic CoA value determined by Wakil's sobyl-CoA method was about a half of the value obtained by Kaplan-Lipmann's sulfanilamide method which is sensitive to phosphopantetheine and dephospho-CoA. In addition, the former corresponded to about a half of the total PaA value. From this fact it is suggested that a fair amount of bound pantothenic acid exists in the liver of rats as forms other than CoA.
2. The high value of pantothenic acid was obtained in the blood of cord and newborn infant and the value fell as the age advanced. The proportion of the free to the total pantothenic acid was greater in the lower age group. The amount of urinary pantothenic acid excretion showed a reverse relationship and the younger age group tended to show the lower excretion.
3. The total hepatic CoA in rats was markedly increased with the growth and the amount of CoA per unit of weight was also increased until 30 days. Thereafter, the value reached a constant level. The total amount of kidney CoA was increased with the growth but the value per unit of weight reached a constant level 10 days after birth. In the brain, both the total amount and the value of CoA per unit of weight reached a constant level 20 days after birth.
4. The changes of total pantothenic acid in liver, kidney and brain with the growth of rats showed the same tendencies with those of CoA. The kidney showed a presence of relatively high proportion of free PaA as compared with other organs.

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We observed the influences of dietary protein and amino acid compositions on hepatic CoA contents of young albino rats.
By low protein diet feeding, the body weight, liver weight and hepatic hitrogen content were all lower than in the standard diet group, and the total hepatic CoA was remarkably reduced.
CoA per unit of liver weight was higher in the standard diet group during the first one to two weeks of feeding, and the amount of CoA per unit of hepatic weight or nitrogen content was increased in the low protein diet group as the days of feeding elapsed.
Methionine, lysine, threonine or tryptophan deficient group did not show any difference in the gain of body weight but the amount of hepatic CoA was greatly reduced in methionine deficiency, slightly decreased in lysine and tryptophan deficiencies and unaltered in threonine deficiency.

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1. The in vitro biosynthesis from 4′-phosphopantetheine to CoA was investigated and if was applied for determination of hepatic CoA precursors, such as 4′-phosphopantetheine and dephospho CoA, in albino rats. By this method, changes in CoA and its precursors in the liver with the days after birth were determined. In the liver a fairly large amount of precursors were found to exist as bound PaA along with CoA. This finding does not agree with the report of Novelli who found the majority of bound form of PaA to be CoA. These CoA and precursors were gradually increased as the days after birth elapsed until 30 days but their proportions were not particularly changed with the number of days.
2. Hepatic CoA value was lowered by administration of pantothenic acid deficient diet and the values of CoA precursors were further markedly reduced to replace the majority of bound form of pantothenic acid with CoA.
3. When various amounts of pantothenic acid and pantethine were added to a pantothenic acid deficient diet, the maximum hepatic CoA value was found in the group fed on the diet added with 9.2 μmoles per 100g of the diet. Further administration resulted in a tendency of mild decrease.
4. The group added with 2.3 to 23 μmoles showed an increase of CoA precursors, similarly to the CoA level. Addition of as large as 230 μmoles resulted in a decrease of precursors but the other bound form of pantothenic acid was reserved in a large amount.
5. Comparing the pantothenic acid added group and pantethine added group, the amount of hepatic CoA did not show any difference between the two groups. However, the proportion occupied by CoA in the total pantothenic acid amount was always high in the pantethine added group.

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Through the study of fundamental conditions for colorimetric determination of homopantothenic acid (HOPA) in presence of sodium naphthoquinone sulfonate solution, a standard quantitative analysis and a quantitative separation of HOPA from the decomposition product were established. The physicochemical properties were investigated simultaneously. The general conclusion drawn from the experi ments is as follows.
1. The principle in the quantitative determination of calcium homopantothenate was based on the colorimetry of an orange yellow-colored solution obtainable in the reaction of sodium-1, 2-naphthoquinone-4-sulfonate with γ-aminobutyric acid (GABA). It is the alkaline hydrolyzate of HOPA in buffer solutions of pH 8.8-9.6.
2. There was no significant variation of analytical values in the use of Clark-Lubs buffer solutions having different pH values between 8.8 and 9.6.
3. Colorization was stable and its maximum absorption was present at 465 mμ. The concentrations of HOPA solutions between 200 and 1000μg/25ml were directly proportional to the absorbances.
4. The quantitative separation of HOPA from the decomposition product, GABA, was successfully conducted by the removal of GABA by the use of cationicion exchange resine Amberlite CG-120.
5. The recovery rate of HOPA by resinous treatment was over 98%.
6. The solubility of HOPA in water was approximately 70%, while it was insoluble in organic solvents though the solubility in methanol was 1.3%.
7. HOPA was found to be an optically active compound of which specific rotation [α]_D²⁰ was+23.8°.
8. The apparent dissociation constant, pKa, of HOPA was 4.52 at 25° when calculated from its potentiometric curve.
9. One spot was detected on a thin-layer chromatogram using a solvent composed of n-butanol, acetic acid and water.
10. Four functional groups, OH, NH, CONH and COO were identified by the infrared spectrophotometry of HOPA.
11. The ultraviolet spectrum of HOPA indicated no presence of the peak at the wavelengths over 200mμ.
12. Kinetic observation of the stability of HOPA disclosed that it was most stable in pH values ranging from 5 to 6.

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The influence of high dose of choline and riboflavin on the fecal excretion of cholesterol and bile acids of albino rats kept on a high cholesterol diet was studied. Choline increased the fecal excretion of bile acid without showing any appreciable increase in the excretion of cholesterol in feces. Riboflavin did not show any appreciable increase or decrease in the excretion of cholesterol and bile acid in the feces of the animals fed a high cholesterol diet. Both choline and riboflavin exerted a cholesterol-lowering effect in the liver and blood of the animals fed a high cholesterol diet.

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The interaction of PAL-P with ADH in vitro was observed, and electrophoretic mobility and absorption spectrum also changed. The molar ratios (PAL-P:ADH) of PAL-P-ADH complex was about 1:9.

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Oxidation of S-alkylthiamines and S-alkoxycarbonylthiamines, which are difficult to convert to thiamine by sulfhydryl compounds, was investigated. S-alkylthiamines were oxidized with hydrogen peroxide and perbenzoic acid in glacial acetic acid to afford the corresponding sulfoxides and sulfones in accordance with the amount of the peroxide. α-Acyloxysulfide-type thiamine, which was presumed to be produced via the Pummere's reaction, obtained by the reaction of sulfoxide with acid anhydride.
O-Benzoyl-S-ethoxycarbonylthiamine, one of S-alkoxycarbonylthiamines, was oxidized with hydrogen peroxide to give O-benzoylthiaminic acid.

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The subcellular distribution of ³H-vitamin D₃ in the intestinal mucosa was observed using the cellular fractionation techniques and the electronmicroscopic autoradiography. As a result, it was demonstrated that vitamin D was bound to the membrane of mitochondria and endoplasmic reticula, however, consisted inside nuclei.

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