Naturally occurring UBIAD1 mutations differentially affect menaquinone biosynthesis and vitamin K-dependent carboxylation.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained ˜ 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu.

LRP1 polymorphisms associated with warfarin stable dose in Chinese patients: a stepwise conditional analysis.

Aim: The aim of this study was to investigate whether variability in warfarin stable dose (WSD) could be influenced by vitamin K-related polymorphisms in patients with heart valve replacement. Patients & methods: Twenty-nine vitamin K-related SNPs in 208 patients who initially took warfarin and achieved WSD were genotyped. Results: After conducting conditional analysis for both VKORC1 -1639G>A and CYP2C9*3, LRP1 rs1800139 and LRP1 rs1800154 were significantly associated with WSD (p = 0.007 and p = 0.015, respectively). Multivariate analysis showed that LRP1 rs1800139 accounted for 5.9% WSD variability. Conclusion: Our results suggest that a novel vitamin K-related gene, LRP1, exerts a relevant influence on WSD, independent of VKORC1 -1639G>A and CYP2C9*3.

The Role of Vitamin K and Its Related Compounds in Mendelian and Acquired Ectopic Mineralization Disorders.

Ectopic mineralization disorders comprise a broad spectrum of inherited or acquired diseases characterized by aberrant deposition of calcium crystals in multiple organs, such as the skin, eyes, kidneys, and blood vessels. Although the precise mechanisms leading to ectopic calcification are still incompletely known to date, various molecular targets leading to a disturbed balance between pro- and anti-mineralizing pathways have been identified in recent years. Vitamin K and its related compounds, mainly those post-translationally activated by vitamin K-dependent carboxylation, may play an important role in the pathogenesis of ectopic mineralization as has been demonstrated in studies on rare Mendelian diseases, but also on highly prevalent disorders, like vascular calcification. This narrative review compiles and summarizes the current knowledge regarding the role of vitamin K, its metabolism, and associated compounds in the pathophysiology of both monogenic ectopic mineralization disorders, like pseudoxanthoma elasticum or Keutel syndrome, as well as acquired multifactorial diseases, like chronic kidney disease. Clinical and molecular aspects of the various disorders are discussed according to the state-of-the-art, followed by a comprehensive literature review regarding the role of vitamin K in molecular pathophysiology and as a therapeutic target in both human and animal models of ectopic mineralization disorders.

Nuclear receptor gene polymorphisms and warfarin dose requirements in the Quebec Warfarin Cohort.

Warfarin is primarily metabolized by cytochrome 2C9, encoded by gene CYP2C9. Here, we investigated whether variants in nuclear receptor genes which regulate the expression of CYP2C9 are associated with warfarin response. We used data from 906 warfarin users from the Quebec Warfarin Cohort (QWC) and tested the association of warfarin dose requirement at 3 months following the initiation of therapy in nine nuclear receptor genes: NR1I3, NR1I2, NR3C1, ESR1, GATA4, RXRA, VDR, CEBPA, and HNF4A. Three correlated SNPs in the VDR gene (rs4760658, rs11168292, and rs11168293) were associated with dose requirements of warfarin (P = 2.68 × 10-5, P = 5.81 × 10-4, and P = 5.94 × 10-4, respectively). Required doses of warfarin were the highest for homozygotes of the minor allele at the VDR variants (P < 0.0026). Variants in the VDR gene were associated with the variability in response to warfarin, emphasizing the possible clinical relevance of nuclear receptor gene variants on the inter-individual variability in drug metabolism.

Vitamin K-Dependent Coagulation Factors That May be Responsible for Both Bleeding and Thrombosis (FII, FVII, and FIX).

Vitamin K-dependent clotting factors are commonly divided into prohemorrhagic (FII, FVII, FIX, and FX) and antithrombotic (protein C and protein S). Furthermore, another protein (protein Z) does not seem strictly correlated with blood clotting. As a consequence of this assumption, vitamin K-dependent defects were considered as hemorrhagic or thrombotic disorders. Recent clinical observations, and especially, recent advances in molecular biology investigations, have demonstrated that this was incorrect. In 2009, it was demonstrated that the mutation Arg338Leu in exon 8 of FIX was associated with the appearance of a thrombophilic state and venous thrombosis. The defect was characterized by a 10-fold increased activity in FIX activity, while FIX antigen was only slightly increased (FIX Padua). On the other hand, it was noted on clinical grounds that the thrombosis, mainly venous, was present in about 2% to 3% of patients with FVII deficiency. It was subsequently demonstrated that 2 mutations in FVII, namely, Arg304Gln and Ala294Val, were particularly affected. Both these mutations are type 2 defects, namely, they show low activity but normal or near-normal FVII antigen. More recently, in 2011-2012, it was noted that prothrombin defects due to mutations of Arg596 to Leu, Gln, or Trp in exon 15 cause the appearance of a dysprothrombinemia that shows no bleeding tendency but instead a prothrombotic state with venous thrombosis. On the contrary, no abnormality of protein C or protein S has been shown to be associated with bleeding rather than with thrombosis. These studies have considerably widened the spectrum and significance of blood coagulation studies.

Missense mutation of VKORC1 leads to medial arterial calcification in rats.

Vitamin K plays a crucial role in the regulation of vascular calcifications by allowing activation of matrix Gla protein. The dietary requirement for vitamin K is low because of an efficient recycling of vitamin K by vitamin K epoxide reductase (VKORC1). However, decreased VKORC1 activity may result in vascular calcification. More than 30 coding mutations of VKORC1 have been described. While these mutations have been suspected of causing anticoagulant resistance, their association with an increase in the risk of vascular calcification has never been considered. We thus investigated functional cardiovascular characteristics in a rat model mutated in VKORC1. This study revealed that limited intake in vitamin K in mutated rat induced massive calcified areas in the media of arteries of lung, aortic arch, kidneys and testis. Development of calcifications could be inhibited by vitamin K supplementation. In calcified areas, inactive Matrix Gla protein expression increased, while corresponding mRNA expression was not modified. Mutation in VKORC1 associated with a limited vitamin K intake is thus a major risk for cardiovascular disease. Our model is the first non-invasive rat model that shows spontaneous medial calcifications and would be useful for studying physiological function of vitamin K.

Effect of propeptide amino acid substitution in γ-carboxylation, activity and expression of recombinant human coagulation factor IX.

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre-propeptide sequences. The propeptide is connected to γ-carboxylase enzyme through the γ-carboxylase recognition site for the direct γ-carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ-carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ-carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT-hFIX-M14 expression cassette, containing cDNA of hFIX with substituted -14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4-fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD-14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated 2-fold enhanced recovery in the S2-expressing hFIXD-14A relative to that expressed native hFIX. These results show that changing -14 residues leads to a decrease in the binding affinity to substrate, increase in γ-carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515-520, 2018.

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI.

The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

The Arg98Trp mutation in human VKORC1 causing VKCFD2 disrupts a di-arginine-based ER retention motif.

Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of all vitamin K-dependent (VKD) proteins. Until now, only 1 human VKORC1 mutation, p.Arg98Trp, is known to cause combined deficiency of VKD clotting factors type 2 (VKCFD2), a disease phenotype reported in 3 unrelated families. VKCFD2 patients suffer from spontaneous bleeding episodes because of decreased levels of γ-carboxylated VKD clotting factors. Daily supraphysiological vitamin K supplementation restores clotting for VKCFD2 patients and results in high serum levels of vitamin K 2,3-epoxide, suggesting that supplemented vitamin K is reduced in vivo. Although the p.Arg98Trp mutation results in reduced vitamin K 2,3-epoxide reductase activity, the molecular mechanism underlying this pathophysiology is unknown. Using a combination of in silico analysis and confocal microscopy, we demonstrate for the first time that VKORC1:p.Arg98Trp disrupts a di-arginine ER retention motif resulting in 20% ER colocalization only. As a consequence, VKORC1 exits the ER membrane by cellular quality control systems and results in the observed VKCFD2 phenotype.

A model predicting fluindione dose requirement in elderly inpatients including genotypes, body weight, and amiodarone.

Indandione VKAs have been widely used for decades, especially in Eastern Europe and France. Contrary to coumarin VKAs, the relative contribution of individual factors to the indandione-VKA response is poorly known. In the present multicentre study, we sought to develop and validate a model including genetic and non-genetic factors to predict the daily fluindione dose requirement in elderly patients in whom VKA dosing is challenging. We prospectively recorded clinical and therapeutic data in 230 Caucasian inpatients mean aged 85 ± 6 years, who had reached international normalized ratio stabilisation (range 2.0-3.0) on fluindione. In the derivation cohort (n=156), we analysed 13 polymorphisms in seven genes potentially involved in the pharmacological effect or vitamin-K cycle (VKORC1, CYP4F2, EPHX1) and fluindione metabolism/transport (CYP2C9, CYP2C19, CYP3A5, ABCB1). We built a regression model incorporating non-genetic and genetic data and evaluated the model performances in a separate cohort (n=74).Body-weight, amiodarone intake, VKORC1, CYP4F2, ABCB1 genotypes were retained in the final model, accounting for 31.5% of dose variability. None influence of CYP2C9 was observed. Our final model showed good performances: in 83.3% of the validation cohort patients, the dose was accurately predicted within 5 mg, i.e.the usual step used for adjusting fluindione dosage. In conclusion, in addition to body-weight and amiodarone-intake, pharmacogenetic factors (VKORC1, CYP4F2, ABCB1) related to the pharmacodynamic effect and transport of fluindione significantly influenced the dose requirement in elderly patients while CYP2C9 did not. Studies are required to know whether fluindione could be an alternative VKA in carriers of polymorphic CYP2C9 alleles, hypersensitive to coumarins.

The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.

Clinical significance of AFP and PIVKA-II responses for monitoring treatment outcomes and predicting prognosis in patients with hepatocellular carcinoma.

AIM: Recently, the utility of tumor markers in the hepatocellular carcinoma (HCC) field has received a good deal of attention. Here, we review and summarize the results of studies on the roles played by the α -fetoprotein (AFP) and prothrombin induced by the absence of vitamin K or antagonist-II (PIVKA-II) responses in terms of the monitoring of outcomes and prediction of prognosis after various HCC treatments. METHODS: Studies lodged in PUBMED and that satisfied our inclusion criteria were reviewed. RESULTS: We reviewed 12 studies measuring both AFP and PIVKA-II responses in HCC patients treated in various ways. The results are presented by treatment modality. CONCLUSION: Measurement of AFP and PIVKA II marker levels before and after HCC treatment is clinically useful in monitoring of treatment outcomes and prognosis and in predicting recurrence and survival.

[Structure-activity relationship of novel vitamin K analogues as steroid and xenobiotic receptor (SXR) agonists].

Vitamin K2 is a ligand for a nuclear receptor, steroid and xenobiotic receptor (SXR), that induces the gene expressions of CYP3A4. We synthesized new vitamin K analogues with the same isoprene side chains symmetrically introduced at the 2 and 3 positions of 1,4-naphthoquinone and vitamin K2 analogues with hydroxyl or phenyl groups at the ω-terminal of the side chain. The upregulation of SXR-mediated transcription of the target gene by the analogues was dependent on the length of the side chain and the hydrophobicity of the ω-terminal residues. Phenyl analogue menaquinone-3 was as active as the known SXR ligand rifampicin.

Retrospective evidence for clinical validity of expanded genetic model in warfarin dose optimization in a South Indian population.

AIM: To optimize warfarin dose in patients at risk for thrombotic events, we have recently developed a pharmacogenomic algorithm, which explained 44.9% of the variability in warfarin dose requirements using age, gender, BMI, vitamin K intake, CYP2C9 (*2 and *3) and VKORC1 (*3, *4 and -1639 G>A) as predictors. The aim of the current study is to develop an expanded genetic model that can explain greater percentage of warfarin variability and that has clinical validity. PATIENTS & METHODS: CYP2C9*8, CYP4F2 V433M, GGCX G8016A and thyroid status were added to an expanded genetic model (n = 243). RESULTS: The expanded genetic model explained 61% of the variability in warfarin dose requirements, has a prediction accuracy of ±11 mg/week and can differentiate warfarin sensitive and warfarin resistant groups efficiently (areas under receiver operating characteristic curves: 0.93 and 0.998, respectively; p < 0.0001). Higher percentage of International Normalized Ratios in therapeutic range (52.68 ± 4.21 vs 43.80 ± 2.27; p = 0.04) and prolonged time in therapeutic range (61.74 ± 3.18 vs 47.75 ± 5.77; p = 0.03) were observed in subjects with a prediction accuracy of <1 mg/day compared with subjects with prediction accuracy >1 mg/day. In the warfarin-resistant group, primary hypothyroidism was found to induce more resistance while in the warfarin-sensitive group, hyperthyroidism was found to increase sensitivity. CONCLUSION: The expanded genetic model explains greater variability in warfarin dose requirements and it prolongs time in therapeutic range and minimizes out-of-range International Normalized Ratios. Thyroid status also influences warfarin dose adjustments.

Current challenges in personalizing warfarin therapy.

In an exciting era where novel oral anticoagulants, such as the factor Xa and direct thrombin inhibitors, are beginning to emerge as therapeutic options, the vitamin K antagonists (VKAs) such as warfarin, which have been in clinical use for over half a century, will remain an important part of the therapeutic landscape for the foreseeable future. The optimal effectiveness and safety of the VKAs is limited by significant inter- and intra- patient variability in dose response. As such, routine laboratory monitoring with subsequent dose adjustment to achieve and maintain an international normalized ratio (INR) that falls within a narrow therapeutic range is necessary; even with frequent INR monitoring, time in therapeutic range of VKAs is generally <60% in usual care settings. Yet, personalized approaches to warfarin therapy, such as the routine incorporation of pharmacogenetic data into dose selection and adjustment, the selective use of prescribed doses of vitamin K for those patients with unstable INRs, and integration of patient self-testing /self-management, has the potential to improve the safety, efficacy and ease of use of warfarin. To date, no randomized trials have proven the benefits of routine pharmacogenetic testing for warfarin initiation; however, pivotal trials are ongoing. Through further investigative work, allowing these personalized strategies to realize their full potential, warfarin may remain a preferred therapeutic oral anticoagulant for years to come.

ABCC6 as a target in pseudoxanthoma elasticum.

The ABCC6 gene encodes an organic anion transporter protein, ABCC6/MRP6. Mutations in the gene cause a rare, recessive genetic disease, pseudoxanthoma elasticum, while the loss of one ABCC6 allele is a genetic risk factor in coronary artery disease. We review here the information available on gene structure, evolution as well as the present knowledge on its transcriptional regulation. We give a detailed description of the characteristics of the protein, and analyze the relationship between the distributions of missense disease-causing mutations in the predicted three-dimensional structure of the transporter, which suggests functional importance of the domain-domain interactions. Though neither the physiological function of the protein nor its role in the pathobiology of the diseases are known, a current hypothesis that ABCC6 may be involved in the efflux of one form of Vitamin K from the liver is discussed. Finally, we analyze potential strategies how the gene can be targeted on the transcriptional level to increase protein expression in order to compensate for reduced activity. In addition, pharmacologic correction of trafficking-defect mutants or suppression of stop codon mutations as potential future therapeutic interventions are also reviewed.

Vitamin K-dependent coagulation factors deficiency.

All vitamin K-dependent coagulation factors require normal function of gamma-glutamyl carboxylase and vitamin K epoxide reductase enzyme complex (VKORC1). Heritable dysfunction of gamma-glutamyl carboxylase or of the VKORC1 complex results in the secretion of poorly carboxylated vitamin K-dependent proteins that play a role in coagulation. The following review will summarize the clinical manifestations of vitamin K-dependent coagulation factors deficiency I and II and will provide a detailed explanation about the gene and protein structure, the function of the protein, and an analysis of the previously reported mutations. Laboratory assays used for diagnosis will be discussed, and treatment for various clinical settings will be recommended.

Genetic and non-genetic correlates of vitamins K and D.

OBJECTIVE: To assess the genetic and nongenetic correlates of circulating measures of vitamins K and D status in a community-based sample of men and women. SUBJECTS/METHODS: A cross-sectional study of 1762 participants of the Framingham Offspring Study (919 women; mean age 59 years). Vitamin K status was measured as plasma phylloquinone and serum percent undercarboxylated osteocalcin (ucOC), and vitamin D was measured using plasma 25-hydroxyvitamin D (25(OH)D). Associations between vitamin K status and vitamin D status with biologically plausible nongenetic factors were assessed using stepwise regression. Heritability and linkage were determined using Sequential Oligogenic Linkage Analysis Routines (SOLAR). RESULTS: Nongenetic factors accounted for 20.1 and 12.3% of the variability in plasma phylloquinone in men and women respectively, with triglycerides and phylloquinone intake being the primary correlates. In men 12.2% and in women 14.6% of the variability in %ucOC was explained by nongenetic factors in our models. Heritability estimates for these vitamin K status biomarkers were nonsignificant. Season, vitamin D intake, high-density lipoprotein (HDL) cholesterol and waist circumference explained 24.7% (men) and 24.2% (women) of the variability in plasma 25(OH)D. Of the three vitamins examined, only 25(OH)D was significantly heritable (heritability estimate=28.8%, P<0.01), but linkage analysis of 25(OH)D did not achieve genome-wide significance. CONCLUSIONS: Variability in biomarkers of vitamin K status was attributed to nongenetic factors, whereas plasma 25(OH)D was found to be significantly heritable. Further studies are warranted to investigate genetic loci influencing vitamin D status.

Determinants of vitamin K status in humans.

To understand the role of vitamin K in human health, it is important to identify determinants of vitamin K status throughout the life cycle. Our current understanding of vitamin K physiology and metabolism only partially explains why there is wide interindividual variation in vitamin K status, as measured by various biochemical measures. Dietary intake of vitamin K is one of the primary determinants of vitamin K status, and intakes vary widely among age groups and population subgroups. How dietary sources of vitamin K are absorbed and transported varies with the form and food source of vitamin K. Likewise, the role of plasma lipids as a determinant of vitamin K status varies with the form of vitamin K ingested. There is also some evidence that other fat-soluble vitamins antagonize vitamin K under certain physiological conditions. Infants are at the greatest risk of vitamin K deficiency because of a poor maternal-fetal transfer across the placenta and low vitamin K concentrations in breast milk. During adulthood, there may be subtle age-related changes in vitamin K status but these are inconsistent and may be primarily related to dietary intake and lifestyle differences among different age groups. However, there is some suggestion that absence of estrogen among postmenopausal women may be a determinant of vitamin K, status. Genetics may explain some of the observed interindividual variability in vitamin K, but to date, there are few studies that have systematically explored the associations between individual genetic polymorphisms and biochemical measures of vitamin K status.

Nutritional effects of gamma-glutamyl carboxylase gene polymorphism on the correlation between the vitamin K status and gamma-carboxylation of osteocalcin in young males.

Vitamin K is a cofactor for gamma-glutamyl carboxylase (GGCX), which is an essential enzyme for the gamma-carboxylation of vitamin K-dependent proteins such as osteocalcin (OC). Associations among dietary vitamin K intake, vitamin K status, and bone metabolism have not been thoroughly investigated. Recently, it has been reported that single nucleotide polymorphisms of GGCX (R325Q, 974G>A) were associated with age-related bone loss and the kinetic affinity for the substrate. In the present study, we investigated the associations among dietary vitamin K intake, the level of serum vitamin K, and the ratio of undercarboxylated OC (ucOC) to intact OC. The subjects were 60 healthy young male volunteers (mean age, 22.6 y; standard deviation, 1.6). Dietary nutrient intake was assessed by consecutive individual 3-d food records taken before the day of blood examinations. Serum concentrations of vitamin K (phylloquinone: PK, menaquinone 4: MK-4, and menaquinone 7: MK-7), ucOC, and intact OC were measured. All subjects were genotyped for polymorphism (R325Q) presence. Dietary vitamin K intake from vegetables was significantly correlated with the level of serum PK, and vitamin K intake from fermented beans, natto, was also significantly correlated with the level of serum MK-7. The ratio of ucOC to intact OC showed a negative association with the total vitamin K intake (r=-0.331, p=0.010) and serum MK-7 (r=-0.394, p=0.002). Interestingly, grouped by the GGCX genotype, a significant interaction between the ratio of ucOC to intact OC with serum MK-7 was observed in 325R homozygotes (r=-0.572, p=0.003), but not in heterozygotes, nor in 325Q homozygotes. This is the first report to suggest the effects of the single nucleotide polymorphism R325Q in the GGCX gene on the correlation between the level of serum MK-7 and gamma-carboxylation of serum OC.