α-Tocopherol Intake Decreases Phylloquinone Concentration in Bone but Does Not Affect Bone Metabolism in Rats.

Previous studies have shown that α-tocopherol intake lowers phylloquinone (PK) concentration in some extrahepatic tissues in rats. The study's aim was to clarify the effect of α-tocopherol intake on vitamin K concentration in bone, as well as the physiological action of vitamin K. Male Wistar rats were divided into 4 groups. Over a 3-mo period, the K-free group was fed a vitamin K-free diet with 50 mg RRR-α-tocopherol/kg, the E-free group was fed a diet containing 0.75 mg PK/kg without vitamin E, the control group was fed a diet containing 0.75 mg PK/kg with 50 mg RRR-α-tocopherol/kg, and the E-excess group was fed a diet containing 0.75 mg PK/kg with 500 mg RRR-α-tocopherol/kg. PK concentration in the liver was higher in E-excess rats than in E-free rats, was lower in the tibias of control rats than in those of E-free rats, and was lower in E-excess rats than in control rats. Menaquinone-4 (MK-4) concentration in the liver was higher in E-excess rats than in E-free and control rats. However, MK-4 concentrations in the tibias of E-free, control, and E-excess rats were almost the same. Blood coagulation activity was lower in K-free rats than in the other rats but was not affected by the level of α-tocopherol intake. Additionally, dietary intake of PK and α-tocopherol did not affect uncarboxylated-osteocalcin concentration in the serum, femur density, or expression of the genes related to bone resorption and formation in the femur. These results suggest that α-tocopherol intake decreases PK concentration in bone but does not affect bone metabolism in rats.

Plasma vitamin K concentrations depend on CYP4F2 polymorphism and influence on anticoagulation in Japanese patients with warfarin therapy.

INTRODUCTION: Warfarin is characterized by a large inter-individual variability in dosage requirement. This study aimed to analyze the contribution of the CYP4F2 genetic polymorphism and plasma vitamin K concentration on the warfarin pharmacodynamics in patients and to clarify the plasma vitamin K concentration affecting warfarin sensitivity index in rats. MATERIALS AND METHODS: Genetic analyses of selected genes were performed and plasma concentrations of warfarin, vitamin K1 (VK1) and menaquinone-4 (MK-4) were measured in 217 Japanese patients. We also assessed the association of plasma VK1 and MK-4 concentrations with the warfarin sensitivity index (INR/Cp) in rats. RESULTS: Patients with the CYP4F2 (rs2108622) TT genotype had significantly higher plasma VK1 and MK-4 concentrations than those with CC and CT genotypes. The multiple linear regression model including VKORC1, CYP4F2, and CYP2C9 genetic variants, age, and weight could explain 42% of the variability in warfarin dosage. The contribution of CYP4F2 polymorphism was estimated to be 2.2%. In contrast, plasma VK1 and MK-4 concentrations were not significantly associated with warfarin dosage in patients. Nevertheless, we were able to demonstrate that the warfarin sensitivity index was attenuated and negatively correlated with plasma VK1 concentration by the oral administration of VK1 in rats, as it resulted in a higher VK1 concentration than that in patients. CONCLUSIONS: The plasma VK1 and MK-4 concentrations are significantly influenced by CYP4F2 genetic polymorphism but not associated with warfarin therapy at the observed concentration in Japanese patients. The CYP4F2 polymorphism is poorly associated with inter-individual variability of warfarin dosage requirement.

Deuterium-labeled phylloquinone fed to α-tocopherol-injected rats demonstrates sensitivity of low phylloquinone-containing tissues to menaquinone-4 depletion.

SCOPE: The influence of excess α-tocopherol (α-T) on tissue depletion of phylloquinone (PK) and menaquinone-4 (MK-4) was evaluated. METHODS AND RESULTS: Rats (n = 5 per group) were fed deuterium-labeled PK (2 µmol/kg diet) for 17 days, thereby labeling the conversion from deuterium-labeled PK to d4-MK-4. Then they were injected subcutaneously daily for the last 7 days with saline, vehicle, or α-T (100 mg/kg body weight). α-T injections (i) increased α-T concentrations by tenfold in liver, doubled them in plasma and most tissues, but they were unchanged in brain; (ii) increased the α-T metabolite, carboxyethyl hydroxychromanol (α-CEHC) concentrations: >25-fold in liver and kidney, tenfold in plasma and lung, and 50-fold in heart; brain contained detectable α-CEHC (0.26 ± 0.03 nmol/g) only in α-T-injected animals; and (iii) depleted most tissues' vitamin K. Compared with vehicle-injected rats, brains from α-T rats contained half the total vitamin K (10.3 ± 0.5 versus 21 ± 2 pmol/g, p = 0.0002) and one-third the d4-MK-4 (5.8 ± 0.5 versus 14.6 ± 1.7 pmol/g, p = 0.0002). Tissues with high PK concentrations (liver, 21-30 pmol/g and heart, 28-50 pmol/g) were resistant to K depletion. CONCLUSION: We propose that α-T-dependent vitamin K depletion is likely mediated at an intermediate step in MK-4 production; thus, tissues with high PK are unaffected.

Excess α-tocopherol decreases extrahepatic phylloquinone in phylloquinone-fed rats but not menaquinone-4 in menaquinone-4-fed rats.

SCOPE: The effects of vitamin E on vitamin K metabolism were elucidated by comparing the effect of tocopherol intake on vitamin K concentrations in rats fed phylloquinone (PK) or menaquinone (MK)-4. METHODS AND RESULTS: Initially, the dietary effect of RRR-α-tocopherol, but not RRR-γ-tocopherol, in decreasing extrahepatic PK concentrations was confirmed. Subsequently, rats were fed a PK or MK-4-containing diet (0.75 mg/kg) with RRR-α-tocopherol (0, 10, 50, or 500 mg/kg) for 6 weeks. In rats fed PK, α-tocopherol consumption decreased PK in kidney, lung, heart, muscle, testis, and brain but not in serum and liver. However, in rats fed MK-4, α-tocopherol consumption did not decrease MK-4 in serum and tissues. Finally, vitamin K- and E-depleted rats were administered PK or MK-4 (0.2 mg) with RRR-α-tocopherol (0, 1, or 10 mg) by gavage. After PK administration, α-tocopherol was observed to decrease PK in kidney, adrenal gland, lung, testis, and brain but not in serum and liver, whereas, after MK-4 administration, α-tocopherol did not affect MK-4 in serum and tissues. CONCLUSION: Excess α-tocopherol decreased extrahepatic PK in rats fed PK but not MK-4 in rats fed MK-4.

Menaquinone-4 enhances testosterone production in rats and testis-derived tumor cells.

BACKGROUND: Vitamin K is essential for the posttranslational modification of various Gla proteins. Although it is widespread in several organs, including the testis, the function of vitamin K in these organs is not well characterized. In this study, we investigated the function of vitamin K in the testis and analyzed its role in steroidogenesis. METHODS: Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K2 vitamins present in the testis, for 5 weeks. In vivo testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and in vitro testosterone levels of testis-derived tumor cells (I-10 cells) maintained in Ham's F-10 medium with 10% fetal bovine serum were measured following treatment with MK-4 (0 to 100 µM) at several time points. Testosterone and cellular protein levels were analyzed with respect to their effects on steroidogenesis. RESULTS: Testosterone levels in the plasma and testes of MK-4-fed rats were significantly increased compared to those of control rats, with no obvious differences in plasma luteinizing hormone levels. Secreted testosterone levels from I-10 cells were elevated by MK-4, but not by vitamin K1, in a dose-dependent manner independent of cAMP treatment. Western blot analysis revealed that expression of CYP11A, the rate-limiting enzyme in steroidogenesis, and phosphorylation levels of protein kinase A (PKA) and the cAMP response element-binding protein were all stimulated by the presence of MK-4. Enhancement of testosterone production was inhibited by H89, a specific inhibitor of PKA, but not by warfarin, an inhibitor of γ-glutamylcarboxylation. CONCLUSIONS: MK-4 stimulates testosterone production in rats and testis-derived tumor cells via activation of PKA. MK-4 may be involved in steroidogenesis in the testis, and its supplementation could reverse the downregulation of testosterone production in elders.

Vitamin K1 supplementation to improve the stability of anticoagulation therapy with vitamin K antagonists: a dose-finding study.

BACKGROUND: Poor anticoagulant stability in patients using vitamin K antagonists is a risk factor for both bleeding and thrombosis. In previous studies supplementation with low dose vitamin K(1) was shown to improve the stability of anticoagulant control. We set up a study to confirm earlier reports and to determine the optimal daily dose of vitamin K(1) in preparation of a large study with clinical endpoints. DESIGN AND METHODS: Four hundred patients from two anticoagulation clinics starting with vitamin K antagonists, independently of a possible history of instable anticoagulation, were randomized to receive either placebo or 100, 150 or 200 µg of vitamin K(1) together with their treatment with vitamin K antagonists. The treatment was administered for 6 to 12 months. Anticoagulation stability, expressed as the percentage of time that the International Normalized Ratio was within the therapeutic range, was compared between the groups. RESULTS: After adjustment for age, sex, vitamin K antagonist used, anticoagulation clinic and interacting drugs as confounding factors the difference in percentage of time with the International Normalized Ratio within the therapeutic range between the placebo group and the vitamin K(1) groups was 2.1% (95% CI: -3.2% - 7.4%) for the group taking 100 µg, 2.7% (95% CI: -2.3% -7.6%) for the group taking 150 µg and 0.9% (95% CI: -4.5% - 6.3%) for the group taking 200 µg vitamin K(1) group, in favor of the vitamin K(1) groups. The patients from both the 100 µg group and the 150 µg group had a 2-fold higher chance of reaching at least 85% of time with the International Normalized Ratio within the therapeutic range. There were no differences in thromboembolic or hemorrhagic complications between the groups. CONCLUSIONS: In patients starting vitamin K antagonists, supplementation with low dose vitamin K(1) resulted in an improvement of time that anticoagulation was within the therapeutic range. Differences between doses were, however, small and the improvement is unlikely to be of clinical relevance. For future studies we recommend selecting only patients with instable anticoagulant control. (This study was registered at www.isrctn.org as ISRCTN37109430).

Brodifacoum intoxication with marijuana smoking.

We report the case of a 17-year-old boy with a significant history of drug and alcohol abuse, which included smoking marijuana mixed with brodifacoum. As a consequence, the patient developed a prolonged coagulopathy that persisted for more than 1 year. To our knowledge, this is the first case reported in the literature in which super-warfarin intoxication has been associated with marijuana smoking. This report should increase the awareness of pathologists and clinicians when examining a patient with a history of drug abuse who exhibits persistent vitamin K1-dependent coagulopathy.

[Effects on the bone metabolism of long-term treatment with antivitamins K1]. / Retentissement sur le métabolisme osseux des traitements au long cours par les antivitamines K1.

Oral anticoagulants (OC) prevent the activation by carboxylation of coagulation proteins. However this action also affects osteocalcin, or bone Gla-protein, a parameter of bone remodelling. Phosphorus and calcium metabolism, osteocalcin levels and bone mineral content were studied in twelve men aged under 60, and who had been taking OC for more than a year, in comparison with a paired group of nine controls with the same cardiovascular pathology but not taking anticoagulants. Osteocalcin levels were lower in the OC group (3.44 ng/ml) than in the control group (5.88 ng/ml) (p = 0.01). There was no significant difference in other phosphorus/calcium balance parameters nor in bone density between the two groups. No evidence was found of any osteopathy in the OC group, but the decrease in serum osteocalcin could result either from inhibition of its secretion or of its carboxylation, or from an action on the osteoblast.

Sulfaquinoxaline inhibition of vitamin K epoxide and quinone reductase.

Sulfaquinoxaline (N1-(2-quinoxalinyl)sulfanilamide) has been shown to be a potent (Ki = 1 microM) freely reversible inhibitor of the dithiothreitol-dependent reduction of both vitamin K epoxide and vitamin K quinone by rat liver microsomes in vitro. This observation provides an explanation for the hemorrhagic syndrome occasionally seen in poultry on medicated feed and the efficacy of sulfaquinoxaline in anticoagulant based rodenticides. Sulfaquinoxaline inhibition resembled inhibition by coumarin anticoagulants (e.g., warfarin) and hydroxynaphthoquinones (e.g., lapachol). Inhibition was observed in assays using microsomes from control strain rats, but the enzyme was resistant to sulfaquinoxaline in microsomes from warfarin-resistant rats. Steady-state kinetics inhibition patterns were nearly competitive versus dithiothreitol and nearly uncompetitive versus vitamin K epoxide as is observed for warfarin and lapachol. These results suggest that this inhibitor binds to the oxidized form of vitamin K epoxide reductase in the same way as suggested for the coumarins and hydroxyquinones. Of 10 other sulfa drugs tested, none were inhibitors, and of fragments and related compounds tested, only 2-aminoquinoxaline benzenesulfonamide was active. These results provide a probably orientation in the binding site in relation to that for warfarin and lapachol.

A new warfarin metabolite: structure and function.

The metabolism of the clinically utilized, anticoagulant warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one] by rat liver microsomes has been investigated. The structure of a new warfarin metabolite [4-hydroxy-3-(3-oxo-1-phenyl-1-butenyl)-2H-1-benzopyran-2-one] (dehydrowarfarin) has been determined by mass spectral comparison with the chemically synthesized compound. The formation of dehydrowarfarin is catalyzed by cytochrome P-450 and is unusual in that the final product is effectively dehydrogenated warfarin.

Vitamin K and coumarin anticoagulants: dependence of anticoagulant effect on inhibition of vitamin K transport.

Coumarin anticoagulants inhibit the release of plasma clotting factor VII by vitamin K in liver slices from vitamin K-deficient animals without inhibition of protein synthesis. When the ratio of vitamin K to coumarin anticoagulant is kept constant, but the concentrations are increased, the inhibition disappears. This suggests that the pharmacological action of coumarin anticoagulants depends on irreversible inhibition of normal vitamin K transport to its site of action. At higher concentrations of vitamin K the inhibition can be surmounted, because vitamin K can enter the cell by an alternate route that is not inhibited by coumarin anticoagulants.