Biochimica et Biophysica Acta (BBA) - General Subjects
The biological activity and tissue distribution of 2′,3′-dihydrophylloquinone in rats
Introduction
The only known function of vitamin K in mammals is to act as a coenzyme for the endoplasmic enzyme γ-glutamylcarboxylase during the posttranslational conversion of glutamic acid residues of specific proteins to γ-carboxyglutamic acid (Gla) to form Gla-containing proteins [1]. A number of blood coagulation factors including coagulation factors II (prothrombin), VII, IX, and X are Gla-containing proteins, and are synthesized in the liver. Osteocalcin, a bone-specific protein synthesized by osteoblasts, is also a Gla-containing protein. The nutritional requirement of vitamin K for the complete γ-carboxylation of osteocalcin is thought to be higher than that for the maintenance of normal blood coagulation in humans [2].
There are two naturally occurring forms of vitamin K: vitamin K1 (phylloquinone, 2-methyl-3-phytyl-1,4-naphthoquinone), which is derived from plants, and vitamin K2 (menaquinones), a series of vitamers with multi-isoprene units at position 3. Vitamin K1 (K1) is present in large amounts in various green vegetables and plant oils, and is the primary dietary source of vitamin K [2]. The hydrogenation of plant oils, such as rapeseed oil, soybean oil and corn oil, which are widely used in processed foods, increases the oxidative stability of polyunsaturated oils and converts liquid oils to solid fats. During the course of the hydrogenation, K1 in plant oils is also hydrogenated and converted to 2′,3′-dihydrophylloquinone (dihydro-K1) [3], [4]. Appreciable amounts of dihydro-K1 exist in processed foods, but the property of this K-vitamer has not been well clarified. Recently, Booth et al. [5], [6] reported that dihydro-K1 has a low biological activity in hepatic vitamin K-dependent Gla-containing proteins and no biological activity in extrahepatic vitamin K-dependent Gla-containing proteins in humans.
Several reports showed that menaquinone-4 (MK-4) accumulates in the tissues of rats following the administration of K1 [7], [8], [9], [10]. MK-4 was detected at low levels in the plasma and liver, and at much higher levels in the extrahepatic tissues such as the tissues in the brain, kidney, pancreas, salivary gland and sternum. We also found the accumulation of MK-4 in the bones of rats fed diet with K1 [11]. The presence of the pathway that converts K1 to MK-4 and the accumulation of MK-4 in the extrahepatic tissues indicate that MK-4 may have specific but as yet unknown functions that are independent of the coenzyme function for γ-carboxylation of vitamin K-dependent proteins.
Warfarin inhibits the vitamin-K-dependent synthesis of Gla residues in proteins in the liver [12] and bone [13], [14]. Warfarin inhibits vitamin K epoxide reductase that is necessary for the regeneration of the hydroquinone of vitamin K needed for the carboxylation reaction [12]. It has been also demonstrated that warfarin decreases the osteocalcin de novo synthesis in a bone cell culture [15]. In this study, we examined whether dihydro-K1 affects blood coagulation, the serum total and undercarboxylated osteocalcin (ucOC) levels in warfarin-treated rats. Then, the tissue distribution of K1, dihydro-K1 and MK-4 was investigated in rats fed diets containing K1 or dihydro-K1.
Section snippets
Chemicals
K1 and MK-4 were purchased from Wako Pure Chemicals Industries (Osaka, Japan). Menaquinone-5 (MK-5) was purified from Bacillus subtilis [16]. Dihydro-K1 was prepared in our laboratory and the structure was confirmed by mass spectrometry. Hydroxyapatite and other chemicals from Wako were of reagent grade. Sodium warfarin was purchased from Sigma (St. Louis, MO).
Animals and diet
The study was conducted in accordance with the current legislation on animal experimentation in Japan. Male Sprague–Dawley rats (Charles
Counteracting effects of vitamin K1 and dihydro-K1 on vitamin K deficiency induced by sodium warfarin
Table 1 shows the changes in the blood coagulation status and serum osteocalcin level after the 1-week coadministration of sodium warfarin and K-vitamers in rats. In the low-K1+warfarin diet group, after 1 week, both PT and APTT were not measurable. On the other hand, PT and APTT were prolonged in both the high-K1+warfarin diet group and the dihydro-K1+warfarin diet group, but blood coagulation was protected as compared with the case of the low-K1+warfarin diet group, indicating that dihydro-K1
Discussion
This study was undertaken to reveal the property of dihydro-K1 as vitamin K using warfarin-treated rats. Rats were fed a diet containing sodium warfarin and either a low or high concentration of K1 or a high concentration of dihydro-K1. The coadministration of warfarin and a low K1 concentration induced hypoprothrombinaemia, whereas high concentrations of dihydro-K1 and K1 ameliorated the effect of warfarin. The counteracting effect of dihydro-K1 appeared to be slightly greater than that of K1.
Acknowledgments
This work was partly supported by a grant from the Japanese Research and Development Association for New Functional Foods.
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