The bioavailability of vitamin B-6 from four selected foods was investigated in five men, aged 22 to 25 years, who were receiving a pyridoxine supplement. The subjects received a constant diet containing 1.34 mg of vitamin B-6 throughout this five-week study, except on Saturdays and Sundays when they ate their self-chosen diets. Starting on day 6 of week 1, following a five-day adjustment period, the subjects received orally 5-mg crystalline pyridoxine supplement daily, except on Tuesday and Thursday of each week. On these two days, the subjects were given orally 0 mg or 2 mg of crystalline pyridoxine, or test doses of bananas, filberts, soybeans and beef which contained around 2 mg of vitamin B-6. Vitamin B-6 was determined by microbiological assay with Saccharomyces uvarum. Vitamin B-6 bioavailability in the test food was determined by comparing 24-hour urinary total vitamin B-6 in response to the test food doses to that excreted following a 2-mg crystalline pyridoxine dose in each subject. Compared to the 100 percent bioavailability of the 2-mg crystalline PN dose, the average vitamin B-6 bioavailability in bananas was 115 + 32% and that in filberts, soybeans and beef was 93 + 8%, 73 + 20% and 87 + 7%, respectively. The metabolism of vitamin B-6 in pyridoxine-supplemented subjects was also investigated by measuring changes in plasma total vitamin B-6 which increased and was stablized after three weeks of pyridoxine supplementation. It was concluded that urinary total vitamin B-6 in pyridoxine-supplemented subjects can be used as a measure of vitamin B-6 bioavailability from test food doses. / Graduation date: 1983

Pyridoxal phosphate is a coenzyme in about 50 known enzymatic reactions. A simple and accurate method for the determination of pyridoxal phosphate would be desirable because it could provide a means to assess the nutritional status of vitamin B₆ in the human. The cyanohydrin methods to determine pyridoxal phosphate appear to be simple and promising. Cyanohydrin methods have been devised by Bonavita and Scardi, and Bonavita, and applied to biological materials by Yamada et al. The cyanohydrin procedure of Yamada et al. was investigated. In this procedure, the pyridoxal phosphate and pyridoxal in a deproteinized sample are separated with the use of a column of SM-cellulose (1 gm., equilibrated with 0.01 N acetic acid). Pyridoxal phosphate is eluted from SM-cellulose with 0.01 N acetic acid, and pyridoxal is eluted with 0.1 M sodium phosphate buffer, pH 7.4. Pyridoxal phosphate and pyridoxal are converted to their respective cyanohydrin derivatives by reaction with potassium cyanide. These cyanohydrin derivatives are measured fluorometrically at their activation and fluorescence maxima. In preliminary studies on the procedure by Yamada et al., the activation and fluorescence spectra of the cyanohydrin derivatives of pyridoxal phosphate and pyridoxal were obtained to determine the appropriate activating and fluorescent wavelength settings to use for subsequent fluorometric analyses. Pyridoxal phosphate cyanohydrin at pH 3.8 in 0.2 M sodium phosphate buffer had an activation maximum at 325 mμ and a fluorescence maximum at 415 mμ; and pyridoxal cyanohydrin at pH 10 in 0.2 M sodium phosphate buffer had an activation maximum at 355 mμ and a fluorescence maximum at 435 mμ. To obtain maximum fluorescence of the cyanohydrin derivatives, pyridoxal phosphate had to be reacted with potassium cyanide at 50°C for 60 minutes, and pyridoxal had to be reacted for 150 minutes. Following these preliminary studies, the elution pattern of pyridoxal phosphate and pyridoxal from a column of SM-cellulose was investigated. Pyridoxal phosphate was eluted with 0.01 N acetic acid; and pyridoxal, with both 0.01 N acetic acid and 0.1 M sodium phosphate buffer, pH 7.4. The recovery of pyridoxal phosphate from SM-cellulose was 93.5% when pyridoxal phosphate alone was applied to the column, and that of pyridoxal was 108.8% when pyridoxal alone was applied. When a mixture of pyridoxal phosphate and pyridoxal was applied to SM-cellulose, the recovery of pyridoxal phosphate was 105.5% and that of pyridoxal was only 59.8%. When either standard alone was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 85.0%, and that of pyridoxal was only 29.1%. When a mixture of pyridoxal phosphate and pyridoxal was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 62.6%, and that of pyridoxal was 52.1%. This lower recovery of pyridoxal phosphate in blood was due mainly to the high readings of the blanks. This higher recovery of pyridoxal phosphate in blood may be explained by the low concentration of pyridoxal in the buffer fractions from a column of SM-cellulose to which a mixture of pyridoxal phosphate and pyridoxal had been applied that was used to calculate the recovery. Determining the recovery of standards added to the supernatant after the precipitation of the proteins in blood, rather than to the hemolyzed blood before precipitation, would indicate whether pyridoxal phosphate and pyridoxal were lost by adsorption on the protein precipitate. The modified procedure of Yamada et al. is not sensitive enough to determine the pyridoxal phosphate and pyridoxal content of human blood. / Graduation date: 1968

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Methods for assessing vitamin A status in the horse and rabbit were developed and evaluated using a modified Relative Dose Response Test (%RDR = A₄-A₀/A₄ X 100, where A₄ and A₀ represent four hours post-feeding and fasting serum total vitamin A levels, respectively) for horses and a Vitamin A Fractionation (VAF) Test monitoring serum levels of vitamin A palmitate, vitamin A acetate and retinol for horses and rabbits. In Experiment 1 (RDR test), 5 horses per treatment group were fed 0 (deficient), 10,000 (control) or 80,000 (excess) I.U. vitamin A palmitate daily, for 30 days. RDR Test was positive (>20%) for all horses receiving diets deficient in vitamin A and negative «20%) for all horses receiving control or excess diets. In Experiment 2 (VAF test), rabbits were fed varying dietary levels of vitamin A palmitate (ranging from 0 to 58000 I.U./kg feed) for up to 87 days. Percentages of retinol and vitamin A palmitate were reflective of vitamin A status. An approaching vita'min A deficiency or toxicity is indicated when percentages of vitamin A palmitate and retinol are more than 1 SD from the means observed for control rabbits (6.2±1.8 and 92.9±3.5, respectively). If a deficiency is approaching then percentage of vitamin A palmitate will be between 21% and 73% and percentage retinol between 8% and 21%. If toxicity is approaching then percentage vitamin A palmitate and retinol will be greater than 21% and less than 73%. Rabbit is normal if percentages are maintained within the ± SD of the mean. Experiment 3 (VAF test) was conducted using the same horses and conditions as in Experiment 1. After 30 days on treatment, percentages of retinol and vitamin A palmitate were significantly lower and higher (P<.05) than controls, for deficient and excess horses, respectively. The percentages of vitamin A palmitate and retinol in deficient horses were intermediate between values observed in horses from the other two treatment groups. If percentage retinol is between 45% and 65% and percentage vitamin A palmitate is between 31% and 45% the horse is approaching deficiency. If the percentage retinol is less than 45% and and vitamin A palmitate is greater than 45%, then the horse is probably approaching toxicity. Results suggests that both RDR and VAF tests can be used to determine vitamin A status before appearance of overt signs of deficiency occur, however only the VAF test is suitable for detecting toxicity.

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