In the foregoing papers, the authors have reported the decrease of sulfate meta bolism in vitamin D-deficient chicks (1), the participation of vitamin D3 in active sulfate metabolism of chicks (2), the biogenesis of vitamin D sulfate in animal tissues (3) and the distribution and isolation of vitamin D selfokinase in animal tissues (3). Further researches were made on the physicochemical and biological properties of pure synthetic vitamin D2 sulfate.Its identification and estimation were also made by means of antimony trichloride reaction, thin-layer chromatography and spectrophotometry.The authors first tested the occurence of water-soluble vitamin D sulfate in fresh cow's milk and found to be always positive. Further, its presence in human milk was studied, and found that there is a remarkable amount of the componnd. EXPERIMENTAL AND RESULTS Tests for the Presence o f Vitamin D Sulfate in Fresh Cow's MilkOne part of fresh cow's milk was evaporated under reduced pressure to a one-third part.To one part of the latter was added three parts of ethanol and the precipitate chiefly consisting of crude protein was removed by filtration. The filtrate was tested for the presence of vitamin D sulfate.After removal of the solvent, the residue was again dissolved in butanol or chloroform , and the solution was tested for the presence of vitamin D sulfate . The analysis was carried out using Libermann's reaction , antimony trichloride reaction and thin-layer chromato graphy.In the parallel experiments , the same compound in commercial non-processed evaporated milk and powdered milk was estimated . The results were quite similar.
In the forgoing papers, the authors have reported the relation between vitamin D and active sulfate metabolism (1-4), and the isolation of vitamin D sulfate from mammalian milk (5). Further tests for the antirachitic potency of human milk (containing 780I.U. of total vitamin D in 2ml as determined by the authors) by prophyrachitic and curative methods using rats previously fed on a vitamin D -defficient diet showed the positive results both in X-ray and AOAC ash test .
Concerning the enzymatic role of water-soluble vitamins many investigations have been published, but few papers are available on the mechanism of the action influencing lipid-soluble vitamins.Our recent interest in the biochemistry of lipid -soluble vitamins is an outgrowth of a curiosity about the correlation between vit amin D and sulfate metabolism.It was early reported by the present authors on the bioassay of vitamin D with rats fed on sterol-free synthetic diets and it was confirmed that no influence of vitamin B12 on the degree of healing in vitamin ID-deficient rats was observed (1).Thereafter many papers have been published on metabolic function of vitamin D, e.g., relation to calcium and phosphorus metabolism, relation to citrate metab olism and other metabolic relationships.Sano (2) summarized his researches on the rickets in Japan, e.g., thiamine and pyruvate levels in rachitic children, ribo flavin levels in blood and organs of experimental rachitic rabbits, phosphorus metab olism in young normal and rachitic rats, serum citrate levels in rachitic children and healing effect of cortisone on rachitic children. Keane et al. (3) reported that vitamin D markedly increased the absorption and retention of Ca45 given orally to chicks but no effect of the intramuscularly injected Ca45 was observed.It was concluded that the effect of vitamin D on calcium metabolism was primarily due to the absorption from the intestinal tract. Gershoff and Hegsted (4) fed diets containing various Ca/P ratios to chiks and found that the ratios had no significant effect on the intestinal absorption of Ca45 if an adequate amount of vitamin D was present.Otherwise, the calcium absorption was inverse ly proportional to Ca/P ratios.Cramer and Steenbock (5) found that the rise in calcium intake in the presence of vitamin D changed the negative calcium balance induced in rats by a low-phosphorus diet to a positive one.The relationship between vitamin D and citrate metabolism at the enzymatic level were investigated by DeLuca et al . (6). Addition of the vitamin to rachito genic or nonrachitogenic diets reduced the oxidation of citrate by kidney homo genates or mitochondria, but no effect was found on the oxidation of other Krebs cycle intermediates . These findings were presumed by the authors to account for 121
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