title: The potent antioxidant activity of the vitamin K cycle in microsomal lipid peroxidation.
abstract: In the vitamin K cycle, vitamin K-hydroquinone, the active cofactor for gamma-glutamylcarboxylase, is continuously regenerated. The successive pathways contain oxidation of the hydroquinone to the epoxide, followed by reduction to the quinone and reduction to the hydroquinone. Vitamin K-hydroquinone is a potent radical scavenging species (Mukai et al., J Biol Chem 267: 22277-22281, 1992). We tested the potential antioxidant activity of the vitamin K cycle in lipid peroxidation reactions (thiobarbituric acid reactive substances, TBARS) in rat liver microsomes. As prooxidant we used Fe2+/ascorbate, NADPH-Fe3+/ATP, and NADPH/CCl4. Vitamin K (< or = 50 microM) on its own did not influence the formation of TBARS. In combination with 1 mM dithiothreitol (DTT), the reductive cofactor for the microsomal enzyme vitamin K epoxide reductase, vitamin K suppressed lipid peroxidation with a concentration that blocked the maximal response by 50% (IC50) of ca. 0.2 microM. Vitamin K1 (phylloquinone) and vitamin K2 (menaquinone-4) were equally active. Warfarin (5 microM) and chloro-vitamin K (50 microM), inhibitors of vitamin K epoxide reductase and gamma-glutamylcarboxylase, respectively, were able to completely abolish the antioxidant effect. Lipid peroxidation was inversely related to the amount of vitamin K hydroquinone in the reaction. Vitamin K epoxide reductase seemed sensitive to lipid peroxidation, with half of the activity being lost within 10 min during oxidation with NADPH/CCl4. The inactivation could be attenuated by antioxidants such as vitamin E, reduced glutathione, and menadione and also by a K vitamin in combination with DTT, but not by superoxide dismutase and catalase. The results show that the vitamin K cycle could act as a potent antioxidant, that the active species in all probability is vitamin K-hydroquinone, and that the primary reaction product is the semiquinone. The results also show that the reaction product is processed in the vitamin K cycle to regenerate vitamin K-hydroquinone.
authors.affiliation: Department of Pharmacology, Cardiovascular Research Institute, University of Maastricht, The Netherlands.
authors.forename: L M
authors.initials: LM
authors.lastname: Vervoort
authors.forename: J E
authors.initials: JE
authors.lastname: Ronden
authors.forename: H H
authors.initials: HH
authors.lastname: Thijssen
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D002451:Cell Compartmentation; D004229:Dithiothreitol / Q000378:metabolism; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D014812:Vitamin K / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases
chemical_list: D000975:Antioxidants; D006873:Hydroquinones; D008054:Lipid Peroxides; D014812:Vitamin K; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D004229:Dithiothreitol