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 -γ-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 (⩽50 μM) 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 μM. Vitamin K₁ (phylloquinone) and vitamin K₂ (menaquinone-4) were equally active. Warfarin (5 μM) and chloro-vitamin K (50 μM), inhibitors of vitamin K epoxide reductase and γ-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/CC1₄. 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.