This proposal is part of the long term objectives to study the regulation of vitamin B6 metabolism and determine the alterations of this metabolism in vitamin B6 deficienty caused by various diseases. Vitamin B6 deficiency frequently associated with chronic alcohol abuse is attributed in part to a defect in vitamin B6 metabolism. Acetaldehyde may accelerate the degradation of the coenzyme form, pyridoxal-P. Human erythrocytes convert vitamin B6 to pyridoxal-P which is then either bound to protein (mainly hemoglobin) or hydrolyzed to pyridoxal by the action of phosphatases. Erythrobyte B6 phosphatase activity is assayed using (3H)pyridoxal-P as substrate; there is an alkaline B6 phosphatase associated with the stroma and a soluble neutral B6 phosphatase. The overall objective of this proposed research is to determine that there is an enhanced rate of degradation of pyridoxal-P in the erythrocytes of chronic alcohol abuse patients and to determine whether the enhanced rate of degradation is due to greater B6 phosphatase activity or to less protection of pyridoxal-P by binding to proteins. Blood will be drawn from chronic alcohol abuse and control Caucasian male subjects. The erythrocyte activities of alkaline and neutral B6 phosphatase, pyridoxal kinase and pycidoxine-P oxidase in alcoholic and control subjects will be analyzed and will be compared with the individual plasma and erythrocyte pyridoxal-P concentrations. To compare overall synthesis and degradation of pyridoxal-P in the erythrocytes, the kinetics of uptake and metabolism of (3H)pyridoxine by erythrocytes in whole blood taken from alcoholic and control subjects will be studied. The tritiated metabolites will be separated by ion exchange HPLC. To determine the importance of protein binding in maintaining normal pyridoxal-P concentrations, the relative amounts of pyridoxal-P bound to protein in erythrocytes and that degraded to pyridoxal and excreted into the plasma will be determined using blood from alcoholic and control subjects. The B6 phosphatases and the binding of pyridoxal-P to proteins will be characterized. The neutral and alkaline B6 phosphatases will be purified from erythrocytes; substrate specificity, kinetics, effect of divalent cations and anions, and inhibition will be studies. Equilibrium dialysis will be used to study the binding of pyridoxal-P under various conditions to erythrocyte proteins, plasma proteins, and purified hemoglobin and serum albumin. The effect of ethanol and acetaldehyde on B6 phosphatase activity and on pyridoxal and pyridoxal-P binding to erythrocyte and plasma proteins will be evaluated.
| Gao, G J; Fonda, M L (1994) Kinetic analysis and chemical modification of vitamin B6 phosphatase from human erythrocytes. J Biol Chem 269:7163-8 |
| Fonda, M L (1993) Vitamin B6 metabolism and binding to proteins in the blood of alcoholic and nonalcoholic men. Alcohol Clin Exp Res 17:1171-8 |
| Fonda, M L (1992) Purification and characterization of vitamin B6-phosphate phosphatase from human erythrocytes. J Biol Chem 267:15978-83 |
| Fonda, M L; Trauss, C; Guempel, U M (1991) The binding of pyridoxal 5'-phosphate to human serum albumin. Arch Biochem Biophys 288:79-86 |
| Fonda, M L; Brown, S G; Pendleton, M W (1989) Concentration of vitamin B6 and activities of enzymes of B6 metabolism in the blood of alcoholic and nonalcoholic men. Alcohol Clin Exp Res 13:804-9 |
