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We predict increased hepatic á-tocopherol metabolism will increase phylloquinone, decrease MK-4, and decrease vitamin K-dependent carboxylation of glutamate (Gla) residues on specific proteins, especially those involved in coagulation. <P>
Our objectives are: <OL> <LI> Determine alterations in hepatic vitamin K1 conversion to K2 resulting from increased hepatic á-tocopherol concentrations; <LI> Define the mechanism(s) by which vitamin E alters Gla-protein levels; <LI>Determine the cytochrome P450 enzymes responsible for ù-hydroxylation of phylloquinone. </ol> Our rationale is that these findings will be key to defining the mechanism(s) for vitamin E and K interactions, a pivotal event in defining the potentially untoward effects of vitamin E on blood clotting. Currently, the mechanism for vitamin K1 conversion to K2 is unknown, the mechanism for regulating vitamin K metabolism to urinary excretion products is unknown, and why vitamin E supplements have such a dramatic effect, causing bleeding in some individuals and not in others, remains a mystery.
NON-TECHNICAL SUMMARY: Our project addresses the USDA's Nutrition, Food Safety and Quality Program, 31.0 Bioactive Food Components for Optimal Health program priority #3: "Novel studies of the functions and mechanisms of regulation of vitamins and minerals." Vitamin E and K interactions have been recognized for more than 50 years. However, the mechanisms for these interactions are unknown. The interactions are important because they alter blood clotting. When humans take vitamin E supplements, their risk of dying from venous blood clots is decreased. This information suggests vitamin E decreases the tendency for blood to clot. Vitamin K is important in regulating factors for blood clotting. However, vitamin E supplements in humans decrease factors involved in blood clotting. This information suggests that vitamin E is decreasing the activity of vitamin K. In rats, feeding diets extremely high in vitamin E increased bleeding. The bleeding could be prevented by giving the rats more vitamin K. We anticipate that our studies will fill gaps in our basic knowledge concerning regulation of vitamin K activation and metabolism. We believe that vitamin E supplements may have marked effects on vitamin K that currently are not understood.
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APPROACH: <BR> Objective 1, we plan to determine the degree and mechanisms by which vitamin E excess causes vitamin K depletion. Objective 2, we plan to determine the physiological consequences of the vitamin K depletion caused by vitamin E. Objective 3, we plan to assess which CYPs are involved in vitamin K metabolism and whether these are the same ones involved in vitamin E metabolism or are up regulated by vitamin E. <BR> <BR> Objective 1 <BR> Rats will be fed diets containing either phylloquinone (vitamin K1) or menadione, then subcutaneously (SQ) inject rats with either RRR-á-tocopherol, or vehicle. We will then measure 1) á-tocopherol, vitamin K1, MK-4 and menadione in plasma and tissues, 2) vitamin K metabolite (5C-aglycone) and menadione in liver and in urine. Replacing vitamin K1 with menadione as the vitamin K source of will allow us to distinguish between proposed mechanisms of vitamin E and K interactions. <BR> <BR> Objective 2 <BR> Determine alterations in Gla protein status in rats from Objective 1. Vitamin K is required for the posttranslational conversion of glutamyl to ã-carboxyglutamyl residues (Gla) in the precursors to several clotting factors synthesized in the liver. Carboxylated and under-carboxylated forms of prothrombin (synthesized in the liver), osteocalcin (synthesized in the bone) and MGP (synthesized in several extrahepatic tissues) in will be measured. Elevated tissue á-tocopherol concentrations will decrease the availability of vitamin K for vitamin K-dependent ã-glutamylcarboxylation thus resulting in under-ã-carboxylation of vitamin K dependant proteins. By measuring plasma concentrations of á-tocopherol, phylloquinone and MK-4 in Objective 1, we anticipate that we will be able to determine whether MK-4 itself is necessary for vitamin K-dependent ã-glutamylcarboxylation, whether phylloquinone can also serve this function, and whether vitamin E itself has a direct role in the process, as was previously suggested for other prenylquinones. <BR> <BR> Objective 3 <BR> Determine the cytochrome P450 enzyme(s) responsible for omega -hydroxylation of vitamin K.Individual recombinant human P450 enzymes expressed in insect microsomes will be used to determine the metabolic activity of the individual enzymes towards vitamin K1 or MK-4. Media plus microsomes will be extracted and vitamin K1 and its omega-hydroxylation metabolite will be determined by LC-MS. To determine the ability of á-tocopherol to alter the omega-hydroxylation of vitamin K, these insect microsomes will be incubated with or without á-tocopherol. Liver microsomes will be prepared (rats from Objective 1),then protein and gene expression of the CYP(s) responsible for omega-hydroxylation of phylloquinone will be determined. The CYP(s) responsible for phylloquinone omega-hydroxylation may be the first step in vitamin K1 conversion to MK-4, or in vitamin K metabolism, and a likely site for vitamin E and K interactions. Identification of the CYP(s) involved, vitamin E's effect on this CYP, and isolation of microsomes from animals studied in Objective 1 will provide significant new information on vitamin E and K interactions.