Study of the Vitamin K Cyclic Metabolism in absence and presence of coumarin anticoagulants by Liquid Chromatography- Linear Ion Trap Mass Spectrometry

In mammalians the only known function of vitamin K is that of γ-glutamylcarboxylase cofactor, enzyme catalyzing the posttranslational carboxylation of the so-called vitamin K-depending proteins. Well-known for the activation of the coagulation proteins, vitamin K has also been shown to be required by extra-hepatic proteins involved in bone metabolism, vascular calcification, and apoptosis. This vitamin is characterized by a cyclic metabolism: it is its stable quinonic form to be absorbed and transported in blood, but it is the hydroquinone one to act as enzymatic cofactor and to be transformed into vitamin K 2,3-epoxide. The latter is then recycled to quinone and hydroquinone in successive reactions catalyzed by vitamin K reductases, dithiol-depending enzymes inhibited by coumarin drugs. A second NAD(P)H-dependent quinone reductase is relatively insensitive to these anticoagulants and operates at high concentrations of vitamin K. Probably because of its efficient recycle system, very low levels of vitamin K circulate in plasma. Moreover, its Recommended Daily Allowance has currently been set at 1 μg/kg/day; this value is surely suitable for its hepatic function, but an extra demand might be required for guaranteeing the bone and vessel health, especially in subjects under anticoagulant therapy. For these reasons, an accurate determination of the vitamin K and its metabolites is a real analytical challenge. This work was just addressed to overcome the above-mentioned difficulties making use of an advanced analytical technique such as liquid chromatography-linear ion trap mass spectrometry. After its development and validation, the method was applied for refining the status of phylloquinone (vitamin K1) and for defining that of vitamin K1 2,3-epoxide in a significant cohort of healthy subjects and of patients under anticoagulant therapy. An accumulation of both forms was verified in individuals taking long-term coumarin drugs.