High-performance liquid chromatography (HPLC) was applied to the determination of ascorbic acid (AsA) and erythorbic acid (ErA). The apparatus was a Shimadzu model LC-2P Liquid Chromatograph equipped with a UV detector set at 254nm. The separation was achieved on a LiChrosorb-NH2 column which was pre-treated with 0.1M ammonium monophosphate solution using a mixture of acetonitrile, acetic acid and water (87:2:11, v/v) as an eluant. The HPLC method has the following advantages: AsA and ErA are quantitated after being distinctly separated, analysis time per one sample is short, and AsA or ErA levels as low as 1.0×10-2μg are detectable. Recovery experiments with dehydro-AsA and dehydro-ErA, involving reduction with H2S, gave satisfactory results.
A method for the separation and determination of ascorbic acid (AsA) and erythorbic acid (ErA) in animal tissues is described. It employs high-performance liquid chromatography of a LiChrosorb-NH2 column in conjunction with a mixture of acetonitrile, acetic acid and water as an eluant. Application of the method, which is sensitive, rapid and simple, to the analyses of AsA and ErA in animal tissues such as liver, adrenals, spleen, kidneys and heart gave satisfactory results. Dehydro AsA and dehydro-ErA in rat liver could be determined after reduction with H2S. The method was shown to be useful for the routine analyses.
When oxidized corn oil (100 nmol in terms of malondialdehyde/day/rat) was administered to a riboflavin-deficient rat, the body weight gain was markedly suppressed. However, when 20, ug of riboflavin/day/rat was administered with the oxidized corn oil, reasonable growth and normal flavin levels in the liver, kidney and heart could be attained, though they were somewhat less than those of the animals fed on the normal diet containing non-oxidized corn oil. It was noted that the elevation of lipid peroxide level in blood plasma of animals administered with the oxidized oil was effectively prevented by riboflavin. These results indicate the protective effect of riboflavin on suppression of growth caused by the oxidized oil.
The effect of nicotinic acid on the synthesis of cerebrosides in the brain was studied during brain development. The concentration of cerebrosides in the brain was significantly lower in nicotinic acid-deficient animals than in those receiving a nicotinic acid-supplemented diet. The total lipid concentration in the brain of nicotinic acid-deficient rats was slightly lower than that of rats fed on the nicotinic acid-supplemented diet. Therefore, the ratio of cerebrosides to total lipids of nicotinic aciddeficient rats was markedly lower than that of nicotinic acid-supplemented rats. However, this low cerebroside level in nicotinic acid-deficient rats was restored by the administration of the nicotinic acid-supplemented diet. Synthesis of cerebrosides was followed in the brain of developing rats after intracerebral injection of L-[U-14C]serine. The total amount of radioactivity incorporated into the cerebroside fraction of nicotinic aciddeficient rat was smaller than that of nicotinic acid supplemented rats. These observations suggest that nicotinic acid affects cerebroside synthesis in the brain of rats.
Effect of vitamin A compounds on the covalent binding of benzo (a) pyrene [B (a) P] to subnuclear components was studied using rat liver nuclei in vitro. The retinol-induced inhibition of the covalent binding of B (a) P depended both on the dose and the time of addition. Retinal and retinyl acetate were both potent inhibitors, but retinoic acid was less effective. Retinol induced marked inhibition of B (a) P binding to DNA and protein of the nuclear matrix but had little or no effect on the labeling of bulk DNA and protein in chromatin fractions.
A method for simultaneous determination of 25-hy droxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) in human plasma has been developed by using two steps of high-performance liquid chromatography (HPLC). Lipids extracted from 0.5ml of human plasma were first subjected to the preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) and a 25-OH-D fraction containing 25-OH D2 and 25-OH-D3 was separated. The separated fraction was subsequently subjected to the analytical HPLC using a Zorbax SIL column (straight phase type). Since the peaks corresponding to 25-OH-D2 and 25-OH-D3 were clearly separated from one another on the chromatogram of the analytical HPLC, the metabolites could be simultaneously determined by estimating the respective peak heights. When the fractions corresponding to the respective peaks were separately collected by repeatedly applying rather large quantities of human plasma and were subjected to gas chromatography-mass spectrometry (GC-MS), they were identified as containing 25-OH-D2 and 25-OH-D3, respectively. The proposed method was applied to plasma samples of human adults taking 400 I.U./day of vitamin D2 for 8 weeks and the values were 22.5±8.1 ng/ml for 25-OH-D3 and 11.5±1.8ng/ml for 25-OH-D2 (mean±S. D.), respectively.
A high-performance liquid chromatographic method for simultaneous assay of 25-hydroxyvitamin D2 (25-OH-D2) and 25 hydroxyvitamin D3 (25-OH-D3) proposed in a previous paper (1) was applied to determine the plasma levels of the metabolites in perinatal and postnatal periods. The plasma samples of maternal, cord and neonatal blood were collected in summer and winter seasons. 25-Hydroxyvitamin D3 was detected in all the samples. The plasma levels of the metabolite in mothers, cords and newborn infants (at life within 24hr) in summer were 33.9±12.5, 18.9±8.4 and 16.6±6.4 (mean±S. D.) ng/ml, respectively, while those in winter were 15.8±6.6, 8.8±3.4 and 7.7±3.2 ng/ml, respectively. The data in summer were significantly higher than the respective data in winter and there were high significant correlations between mothers and cords and between mothers and newborns. In both seasons, the plasma levels of mothers were about two times higher than the respective dataa of cords and newborns which were nearly identical with one another. The similar tendency was always observed in the individual data of mothers-cords-newborns pair samples. In this study, many plasma samples from mothers, cords and newborns were examined, but 25-OH-D2 was detected in only few samples (6/41 for mothers, 3/36 for cords and 2/34 for newborns). However, the metabolite began to appear in all the samples during nursing with vitamin D2-fortified dry milk to show 4.6±1.3 and 4.8±1.2 ng/ml in the summer and winter samples of neonates at life of 5-6 days, respectively. When the variation of plasma 25-OH-D2 and 25-OH-D3 levels was examined in postnatal periods until one month, the levels of exogenous 25-OH-D2 was increased while those of endogenous 25-OH-D3 was decreased. The sum of vitamin D2 intake from fortified dry milk was highly significantly correlated with the plasma levels of 25-OH-D2, which indicates that daily intake of exogenous vitamin D2 is very important in nutrition during postnatal periods of bottle-fed infants.