James H. Skala

Simultaneous Determination of Ascorbic Acid, Isoascorbic Acid (Erythorbic Acid) and Uric Acid in Human Plasma by High-Performance Liquid Chromatography with Amperometric Detection

Abstract A procedure is presented for the direct and simultaneous determination of ascorbic acid (AA), isoascorbic acid (IA), and uric acid (UA) in human plasma by paired-ion reversed-phase high-performance liquid chromatography. An Ultrasphere ODS (C18) column is used with a pH 5.25 mobile phase containing 0.04M sodium acetate, 0.005M tetrabutylammonium phosphate, and 0.2 mg/mL disodium EDTA. Plasma samples preserved with an equal volume of 10% metaphosphoric acid are diluted 10-fold with mobile phase and filtered through 0.2 micron filters. The injection volume is 10 uL. Detection of AA, IA, and UA is by amperometry using a glassy carbon electrode and Ag/AgCl reference electrode. The applied potential is +0.6 volt and the sensitivity setting is 100 nAmps. As little as 0.25 ng of each component can be detected at this setting and the electrode response is linear over the AA, IA, and UA ranges encountered in human plasma.

Experimental vitamin C depletion and supplementation in young men. Nutrient interactions and dental health effects.

Biochemical indices of AA clearly showed that the young men in this study were brought into various states of AA depletion and repletion according to their dietary AA intakes. While previous studies have postulated that supplemental intakes of AA may adversely affect body status of vitamins B6 and B12, we found no changes in the B vitamin status of the young men receiving varying AA intakes. Moderate AA supplementation (605 mg/day) showed no antagonistic effect on markers of vitamins B6 and B12. Blood markers of fat-soluble vitamins A and E and iron status were not affected by AA intakes. The propensity of the gingiva to become inflamed or bleed on probing was reduced after normal (65 mg/day) AA intakes as compared to deficient (5 mg/day) intakes and upon supplementary (605 mg/day) AA intakes as compared to normal intakes. The results suggest that AA status may influence early stages of gingival inflammation and crevicular bleeding, and warrant further study of the relationship between AA and periodontal health.

Effect of various levels of pyridoxine on erythrocyte aminotransferase activities in the rat

Abstract A study was designed to evaluate the performance of erythrocyte alanine (ALT) and aspartate (AST) aminotransferases in the insufficient, marginal, adequate and elevated dietary vitamin B-6 intake ranges of the rat. Female Long-Evans rats, 12 weeks old, were 4-hour meal-fed an AIN 76A diet devoid of pyridoxine (PN) for 3 weeks. Rats were then blocked by weight and randomly assigned within block to one of six dietary treatments (n=12). Four diets were formulated to contain 0.25, 0.5, 1.0, or 7.0 mg PN.HCl/kg. The rats receiving these diets were pair-fed to the 0.25 group; all were 4-hour meal-fed. Two additional diet treatments of 7.0 and 1400 mg PN.HCl/kg were fed ad libitum. Blood samples were collected by cardiac puncture at the end of the devoid period and at 2, 4, and 6 weeks of repletion. ALT and AST endogenous and stimulated activities were determined by an automated procedure (recently published by the senior author). Aminotransferase activities differed significantly between devoid and control groups at the end of the devoid period. After repletion at various levels for six weeks a significant dose-response was reached among the pair-fed groups. There was no difference in response between ad libitum and pair-fed groups at the 7.0 mg PN.HCl/kg level. While there was a significant increase in the alanine aminotransferase levels at nominal 1400 mg PN.HCl/kg, it was not suggestive of a dramatic change.