The effects of riboflavin deficiency on serum proteins have been studied in rats following protein depletion and repletion with high-protein diet. Riboflavin deficiency causes a reduced serum total protein con-centration, reflecting changes in all fractions except β-globulin. The decrease in serum total protein level with changes in albumin and γ-globulin fractions following protein depletion is less pronounced in riboflavin deficiency. Although these changes are reversed upon repletion with 40% protein diet, the values of serum total protein and its different fractions with the exception of a₂- and β-globulins in ribo-flavin-deficient rats are still lower than those of control rats undergoing the same dietary treatment. It has been suggested that lowered serum protein level in riboflavin deficiency on 16% protein diet or on repletion with 40% protein diet following protein depletion may result from reduced synthesis by the liver as well as increased utilization by the peripheral tissues.

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Male albino rats were fed methylthiouracil (0.2%) and after 6 weeks their ascorbic acid, dehydroascorbic acid, and diketogulonic acid were determined in liver, kidney, and urine, while only total ascorbic acid was determined in blood. Activities of ascorbic acid synthesizing enzymes, D-glucurono-δ-lactone hydrolase, L-gulono-γ-lactone hydrolase, and L-gulono-γ-lactone oxidase were estimated in liver. The activities of degrading enzymes, dehydroascorbatase and 2, 3-diketoaldonate decarboxylase, were also studied in liver and kidney. In hypothyroid rats there was a significant decrease in the ascorbic acid level and dehydro-ascorbic acid content of liver. On the other hand there was a marked increase in the diketogulonic acid content of the liver. The content of ascorbic acid in urine of hypothyroid rats increased slightly, while there was no change in the dehydroascorbic acid and diketogulonic acid content. Total ascorbic acid in blood was found to be unaltered. There were no alterations in the activities of L-gulono-γ-lactone oxidase, D-glucurono-δ-lactone hydrolase and L-gulono-γ-lactone hydrolase. Appreciable increases in the activity of dehydroascorbatase and 2, 3-diketoaldonate decarboxylase, were observed in liver of hypothyroid rats.

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The determination of vitamin D₂ isomers in mixtures was investigated in order to use in kinetic experiments on their isomerization with acidic reagents. Vitamin D₂, pre-D₂, 5, 6-trans-D₂, iso-D₂, T₂ and iso-T₂, thought to exist in the reaction mixtures, were chosen as standard compounds of vitamin D₂ isomers. When a mixture of the standard compounds was ap-plied to the GLC using OV-17 as a stationary phase after trimethylsily-lation, the separation of vitamin D₂ from pre-D₂ and of T₂ from iso-T₂ were impossible while the two groups and the other compounds were separated one another (Both vitamin D₂ and pre-D₂ were quantitatively isomerized into the thermal cyclized compounds “pyro- and isopyro-D₂” in the same manner.). Therefore, it was possible to determine the compounds by the GLC when the coexistences of vitamin D₂ with pre-D₂ and of T₂ with iso-T₂ were simultaneously unidentified, whereas pre-treatment was thought necessary to separate them when their coexistence was identified. When a mixture of the standard compounds was applied to the TLC using Kieselgel GF₂₅₄ as an adsorbent and benzene-acetone (95:5) as a developing solvent, vitamin D₂ from pre-D₂ and T₂ from iso-T₂ were separated each other. From these results, a method based on the TLC and GLC was proposed for the determination of vitamin D₂ isomers in mixtures.

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Kinetic experiments on the isomerization of vitamin D₂, pre-D₂, 5, 6-trans-D₂ and T₂ with BF₃ were investigated by using the determination method as reported previously (1). The related components in the reaction mixtures were confirmed to be one of the starting compounds, iso-D₂ and iso-T₂ in all cases by applying the qualitative method using both TLC and GLC. Then, the components were individually determined by the proposed method according to lapse reaction times. The con-version rates of all the starting compounds followed apparent first-order kinetics and the rate constants of vitamin D₂, pre-D₂, 5, 6-trans-D₂ and T₂ were 0.277, 0.462, 0.693 and 0.330, respectively. When the yields of iso-D₂ and iso-T₂ to the concentrations of converted starting compounds were calculated on the basis of the corresponding reaction times, the initial rates of yields of iso-D₂ and iso-T₂ at zero conversions of the starting compounds were respectively 100 and 0% in all cases. The results induced the conclusions in all cases that iso-D₂ was originated as the first reaction product directly from the starting compounds while iso-T₂ was the second reaction product originated via iso-D₂. The conclusions are summarized as follows:
Vitamin D₂, pre-D₂, 5, 6-trans-D₂ or T₂ →BF₃ iso-D₂ →BF₃ iso-T₂
The reaction mechanism was discussed from the conclusions (Fig. 4).

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The coenzymatic activities of 2-nor-2-hydroxymethyl-pyridoxal 5'-phosphate (2'-hydroxy PLP) for various vitamin B₆-dependent enzymes were studied in order to obtain information on the role of the methyl group at position 2 of pyridoxal 5'-phosphate (PLP) for the binding to apoprotein and for the catalytic action of the holoenzyme.
Although it has been demonstrated that this coenzyme analog binds to the catalytic center and serves as a coenzyme for a variety of B₆ en-zymes, the mode of binding as well as the catalytic activity of the holo-enzyme reconstituted with the analog was different according to the kinds of enzyme and assay conditions.
In the ordinary assay system using Tris-HCl buffer, 2'-hydroxy PLP showed almost an equal affinity for pig heart muscle mitochondrial and cytoplasmic aspartate aminotransferase (GOT_s, and GOT_m) to that of PLP. The maximal velocity (V_max value) of the reaction mediated by GOT_s, or GOT_m reconstituted with 2'-hydroxy PLP was somewhat large as compared with that catalyzed by the native holoenzyme. How-ever, when phosphate buffer was used for the assay system, the affinity of the coenzyme analog decreased markedly and the V_max value catalyzed by the analog-bound GOT_s, or GOT_m became approximately 70% of that by the native enzyme. Thus as judged from the V_max value under ordinary assay conditions, 2'-hydroxy PLP is considered to be rather superior to PLP as the coenzyme for GOT. Phosphate anion inhibited the binding of PLP or 2'-hydroxy PLP to apoGOT in a competitive manner and further caused an appreciable decrease in the apparent V_max of the reaction catalyzed by the resulting holoenzyme. The K₁, values of phosphate anion against PLP and 2'-hydroxy PLP were 11.9 and 3.75, respectively.
On the other hand, in the case of E. coli tryptophanase, both the affinity for 2'-hydroxy PLP and the V_max value of the reaction mediated by the analog-bound enzyme were significantly smaller than those of the native coenzyme irrespective of the buffer used.
Of the other B₆ enzymes tested hitherto, E. intermedia tyrosine phenol-lyase and Pseudomonas dacunhae aspartate β-decarboxylase showed the same behaviors toward 2'-hydroxy PLP as that of tryptophanase. On the contrary, the coenzyme analog served as a more efficient co-enzyme than the native coenzyme for E. coli D-serine dehydrase in the presence of phosphate anion. These results indicate that there are at least three groups of B₆ enzymes exhibiting different interaction with the 2-methyl group of PLP.

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With a view to study the changes in collagen crosslinking in vitamin E deficiency, two groups of weanling albino rats were fed with control and vitamin E-deficient diets respectively for 16 weeks and the samples of skins and urine were analysed. The results showed compared to the controls, vitamin E-deficient group showed an elevation of labile col-lagen, a decrease in the aldehyde content of neutral salt soluble col-lagen, increased susceptibility of insoluble collagen to denaturants and pronase. There is an increase of a₁ and a₂ subunits and a decrease of β₁₁ and β₁₂ subunits while the urinary excretion of hydroxyproline is not altered. These results suggest an impairment of collagen cross-linking probably involving a reduction in the aldehydic crosslinking precursor of collagen. The changes observed do not seem to be due to increased catabolism.

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Pyridoxal 5'-phosphate (PLP), in high concentrations, was found to bind to lysine residues at non-catalytic sites of mammalian aspartate aminotransferase (GOT) and E. coil tryptophanase. This excess bind-ing of PLP caused significant decreases in the activities of these enzymes. The concentrations of PLP required for the 50% inhibition of GOT and tryptophanase were 2.5mM and 6.3mM, respectively. Pyridoxal (PL) also combined with these enzymes by forming SCHIFF's bases with lysine residues at the catalytic as well as non-catalytic sites. The concentration of PL necessary for the 50% inhibition of GOT activity was 5mM, indicating that the inhibitory action of PL is lower than that of PLP.

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Experiments were carried out to study the effect of acetoacetate admini-stration on pyridine nucleotides in liver and blood in male albino rats. Acetoacetate, when administered along with tryptophan (through diet) to tryptophan-niacin-deficient rats for twenty days, resulted in decreased pyridine nucleotide values as compared to the corresponding controls. Prolonged administration of acetoacetate to rats kept on normal diet decreased the pyridine nucleotide contents of liver and blood.

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This study was undertaken to determine the effects of the excess intake of organic calcium upon the absorption of other nutrients of foods taken at the same time.
Male albino rats of Wistar strain with initial body weight averaging 60g were used. Calcium lactate was fed to the rats as the calcium source. The rats fed with an excess calcium diet show a loss of weight, increased fecal weight and decreased absorption and retention of nitrogen and phosphorus when compared with the control group. High dietary calcium to phosphorus ratio enhanced the excretion of combined fatty acid in feces. Excess levels of calcium and phosphorus in diet caused a functional kidney obstruction.

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The relative turnover rate of protein components of the thin filament was measured by a continuous double isotope method. Both ³H-lysine and ¹⁴C-lysine were administered continuously in the diet to male adult rabbits for 7 days. On the 7th day, the ¹⁴C-lysine was discontinued while the ³H-lysine was continued for 14 additional days. The relative turnover rate of the protein components was estimated from the ³H/¹⁴C ratios of the isolated protein of the same animal. From the different ratios of ³H/¹⁴C of each protein fraction, asynchronous turnover of the thin fila-ment proteins (actin, tropomyosin and troponin) was concluded. The order of degree in relative turnover rate is as follows: troponin>tropomyosin>actin.

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