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Vitamin K epoxide reductases (VKORs) are a large family of integral membrane enzymes found from bacteria to humans. Human VKOR, specific target of warfarin, has both the epoxide and quinone reductase activity to maintain the vitamin K cycle. Bacterial VKOR homologs, however, are insensitive to warfarin inhibition and are quinone reductases incapable of epoxide reduction. What affords the epoxide reductase activity in human VKOR remains unknown. Here, we show that a representative bacterial VKOR homolog can be converted to an epoxide reductase that is also inhibitable by warfarin. To generate this new activity, we first substituted several regions surrounding the active site of bacterial VKOR by those from human VKOR based on comparison of their crystal structures. Subsequent systematic substitutions narrowed down to merely eight residues, with the addition of a membrane anchor domain, that are responsible for the epoxide reductase activity. Substitutions corresponding to N80 and Y139 in human VKOR provide strong hydrogen bonding interactions to facilitate the epoxide reduction. The rest of six substitutions increase the size and change the shape of the substrate-binding pocket, and the membrane anchor domain stabilizes this pocket while allowing certain flexibility for optimal binding of the epoxide substrate. Overall, our study reveals the structural features of the epoxide reductase activity carried out by a subset of VKOR family in the membrane environment., (© 2022 Federation of European Biochemical Societies.)

Background: Respiratory failure and thromboembolism are frequent in severe acute respiratory syndrome coronavirus 2-infected patients. Vitamin K activates both hepatic coagulation factors and extrahepatic endothelial anticoagulant protein S, required for thrombosis prevention. In times of vitamin K insufficiency, hepatic procoagulant factors are preferentially activated over extrahepatic proteins. Vitamin K also activates matrix Gla protein (MGP), which protects against pulmonary and vascular elastic fiber damage. We hypothesized that vitamin K may be implicated in coronavirus disease 2019 (COVID-19), linking pulmonary and thromboembolic disease., Methods: A total of 135 hospitalized COVID-19 patients were compared with 184 historic controls. Inactive vitamin K-dependent MGP (desphospho-uncarboxylated [dp-uc] MGP) and prothrombin (PIVKA-II) were measured inversely related to extrahepatic and hepatic vitamin K status, respectively. Desmosine was measured to quantify the rate of elastic fiber degradation. Arterial calcification severity was assessed using computed tomography., Results: dp-ucMGP was elevated in COVID-19 patients compared with controls (P < .001), with even higher dp-ucMGP in patients with poor outcomes (P < .001). PIVKA-II was normal in 82.1% of patients. dp-ucMGP was correlated with desmosine (P < .001) and with coronary artery (P = .002) and thoracic aortic (P < .001) calcification scores., Conclusions: dp-ucMGP was severely increased in COVID-19 patients, indicating extrahepatic vitamin K insufficiency, which was related to poor outcome; hepatic procoagulant factor II remained unaffected. These data suggest pneumonia-induced extrahepatic vitamin K depletion leading to accelerated elastic fiber damage and thrombosis in severe COVID-19 due to impaired activation of MGP and endothelial protein S, respectively., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

No epidemiologic studies have been conducted to assess the association of intake of dietary vitamin K with the risk of pancreatic cancer. We used prospective data from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial between 1993 and 2009 to fill this gap. A total of 101,695 subjects were identified. Dietary intakes of phylloquinone (vitamin K1), menaquinones (vitamin K2), and dihydrophylloquinone (dihydrovitamin K1) were assessed using a food frequency questionnaire. Cox regression was applied to calculate hazard ratios and 95% confidence intervals. During a mean follow-up of 8.86 years (900,744.57 person-years), 361 cases of pancreatic cancer were documented. In the fully adjusted model, dietary intakes of phylloquinone (for quartile 4 vs. quartile 1, hazard ratio (HR) = 0.57, 95% confidence interval (CI): 0.39, 0.83; P for trend = 0.002) and dihydrophylloquinone (for quartile 4 vs. quartile 1, HR = 0.59; 95% CI: 0.41, 0.85; P for trend = 0.006), but not menaquinones (for quartile 4 vs. quartile 1, HR = 0.93; 95% CI: 0.65, 1.33; P for trend = 0.816), were found to be inversely associated with the risk of pancreatic cancer in a nonlinear dose-response manner (all P values for nonlinearity < 0.05), and this was not modified by predefined stratification factors and remained in sensitivity analyses. In conclusion, dietary intakes of phylloquinone and dihydrophylloquinone, but not menaquinones, confer a lower risk of pancreatic cancer. Future studies should confirm our findings., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the vitamin K 1 sensitizing potential, the oxidized-isoform of vitamin K 1 (vitamin K 1 oxide, VKO), has been recently used for treating laser-induced purpura and hyperpigmentation in cosmetics. The objective of this study was to formulate VKO in nanoliposomes by using Box-Behnken experimental design to obtain an optimized formula with higher efficiency. The ratio of phospholipid to cholesterol (PC/CHO ratio), VKO concentration and sonication time in low, medium, and high levels were independent variables, while the percent of VKO entrapment efficiency (EE%) and vesicle size were selected as dependent variables. Optimum desirability was identified and an optimized formulation was prepared, characterized, and selected for in vitro VKO release and ex vivo skin permeation. The PC/CHO ratio showed the greatest effect on both responses (P < 0.0001). This effect was positive on EE%, while a negative effect was shown on vesicle size. The optimized formulation showed controlled drug release of 79.2% through a silicon membrane, and achieved flux of 327.36 ± 22.1 μg/cm 2 through human skin after 24 h. So, nanoliposomes were proven as a suitable drug delivery system for topical delivery of VKO., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Warfarin exerts its anticoagulation activity by blocking the vitamin K-epoxide cycle. A quantitative understanding of how warfarin and related genes interact with the vitamin K-epoxide cycle and the associated change of coagulation activity in the human body may help study the pharmacodynamics of warfarin. The plasma concentration of vitamin K 1 (VK 1 ) and vitamin K 1 2,3-epoxide (VK 1 O) could reflect the status of vitamin K-epoxide cycle. However, their determination is a challenging task due to their extremely low concentrations in human plasma and the severe interferences caused by co-extracted lipids. In this study, we developed an LC-APCI-MS/MS method for the simultaneous determination of VK 1 and VK 1 O in human plasma using stable deuterium-labeled vitamin K 1 (vitamin K 1 -d7) as the internal standard (IS). Plasma samples were prepared through protein denaturation followed by one-step liquid extraction with cyclohexane. Chromatographic separation of analytes from isobaric interferences and endogenous ion suppressor was performed on a Synergi Hydro-RP column (150 mm × 4.6 mm, 4 μm) under the reversed-phase condition with isocratic elution. The selective reaction monitoring (SRM) transitions were chosen as m/z = 451.5→187.3 for VK 1 , m/z = 467.5→161.2 for VK 1 O, and m/z = 458.6→194.3 for IS in APCI positive mode. The assay was linear in the range of 100-10,000 pg/mL for the two analytes and achieved considerable extraction recoveries (87.8-93.3%, 91.0-96.9%, and 92.0% for VK 1 , VK 1 O, and IS, respectively), negligible matrix effects (93.6-96.0%, 96.3-100.1%, and 95.5%), and high selectivity with a small sample volume requirement (0.2 mL) and short run time (15 min). The validated method was successfully applied in a clinical pharmacodynamic study of warfarin, and the clotting activity was found to be negatively correlated with the plasma concentration ratio of VK 1 O to VK 1 ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH 2 ) required for the carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. VKORC1 produces KH 2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH 2 Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH 2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH 2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1., (© 2018 by The American Society of Hematology.)

Background: Vitamin K is necessary for the synthesis of coagulation factors. Term infants, especially those who are exclusively breast fed, are deficient in vitamin K and consequently may have vitamin K deficiency bleeding (VKDB). Preterm infants are potentially at greater risk for VKDB because of delayed feeding and subsequent delay in the colonization of their gastrointestinal system with vitamin K producing microflora, as well as immature hepatic and hemostatic function.  OBJECTIVES: To determine the effect of vitamin K prophylaxis in the prevention of vitamin K deficiency bleeding (VKDB) in preterm infants., Search Methods: We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials (CENTRAL 2016, Issue 11), MEDLINE via PubMed (1966 to 5 December 2016), Embase (1980 to 5 December 2016), and CINAHL (1982 to 5 December 2016). We also searched clinical trials databases, conference proceedings, and the reference lists of retrieved articles., Selection Criteria: Randomized controlled trials (RCTs) or quasi-RCTs of any preparation of vitamin K given to preterm infants., Data Collection and Analysis: We evaluated potential studies and extracted data in accordance with the recommendations of Cochrane Neonatal., Main Results: We did not identify any eligible studies that compared vitamin K to no treatment.One study compared intravenous (IV) to intramuscular (IM) administration of vitamin K and compared various dosages of vitamin K. Three different prophylactic regimes of vitamin K (0.5 mg IM, 0.2 mg vitamin K 1 , or 0.2 mg IV) were given to infants less than 32 weeks' gestation. Given that only one small study met the inclusion criteria, we assessed the quality of the evidence for the outcomes evaluated as low.Intramuscular versus intravenousThere was no statistically significant difference in vitamin K levels in the 0.2 mg IV group when compared to the infants that received either 0.2 or 0.5 mg vitamin K IM (control) on day 5. By day 25, vitamin K 1 levels had declined in all of the groups, but infants who received 0.5 mg vitamin K IM had higher levels of vitamin K 1 than either the 0.2 mg IV group or the 0.2 mg IM group.Vitamin K 1 2,3-epoxide (vitamin K 1 O) levels in the infants that received 0.2 mg IV were not statistically different from those in the control group on day 5 or 25 of the study. All of the infants had normal or supraphysiologic levels of vitamin K 1 concentrations and either no detectable or insignificant amounts of prothrombin induced by vitamin K absence-II (PIVKA II).Dosage comparisonsDay 5 vitamin K 1 levels and vitamin K 1 O levels were significantly lower in the 0.2 mg IM group when compared to the 0.5 mg IM group. On day 25, vitamin K 1 O levels and vitamin K 1 levels in the 0.2 mg IM group and the 0.5 mg IM group were not significantly different. Presence of PIVKA II proteins in the 0.2 mg IM group versus the 0.5 mg IM group was not significantly different at day 5 or 25 of the study., Authors' Conclusions: Preterm infants have low levels of vitamin K and develop detectable PIVKA proteins during the first week of life. Despite being at risk for VKDB, there are no studies comparing vitamin K versus non-treatment and few studies that address potential dosing strategies for effective treatment. Dosage studies suggest that we are currently giving doses of vitamin K to preterm infants that lead to supraphysiologic levels. Because of current uncertainty, clinicians will have to extrapolate data from term infants to preterm infants. Since there is no available evidence that vitamin K is harmful or ineffective and since vitamin K is an inexpensive drug, it seems prudent to follow the recommendations of expert bodies and give vitamin K to preterm infants. However, further research on appropriate dose and route of administration is warranted.

A unique electron-accepting analog of vitamin K 1 found in photosystem I in several species of oxygenic photosynthetic microorganisms was confirmed to be 5'-hydroxyphylloquinone (1) through stereo-uncontrolled synthesis. Furthermore, the stereochemistry of 1 obtained from Synechococcus sp. PCC 7942 was assigned to be 5'S using proline-catalyzed stereocontrolled reactions.

Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH 2 ) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH 2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle., (© 2017 Elsevier Inc. All rights reserved.)

Phylloquinone (PhQ), or vitamin K 1 , is an essential electron carrier (A 1 ) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A 1 site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone-deficient mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome., (© 2016 The Authors.The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Vitamin K epoxide reductase (VKOR) catalyzes the reduction of vitamin K quinone and vitamin K 2,3-epoxide, a process essential to sustain γ-carboxylation of vitamin K-dependent proteins. VKOR is also a therapeutic target of warfarin, a treatment for thrombotic disorders. However, the structural and functional basis of vitamin K reduction and the antagonism of warfarin inhibition remain elusive. Here, we identified putative binding sites of both K vitamers and warfarin on human VKOR. The predicted warfarin-binding site was verified by shifted dose-response curves of specified mutated residues. We used CRISPR-Cas9-engineered HEK 293T cells to assess the vitamin K quinone and vitamin K 2,3-epoxide reductase activities of VKOR variants to characterize the vitamin K naphthoquinone head- and isoprenoid side chain-binding regions. Our results challenge the prevailing concept of noncompetitive warfarin inhibition because K vitamers and warfarin share binding sites on VKOR that include Phe55, a key residue binding either the substrate or inhibitor.

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we conducted structure simulations, which revealed a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.

Phylloquinone is a redox active naphthoquinone involved in electron transport in plants. The function of this reduced form remains unclear due to its instability, which has precluded detection. Herein, a simple method that permits the stabilization of the reduced form of phylloquinone by di-O-methylation and HPLC detection is described., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K(1)) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K(2) family and metabolite of K(1) in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K(1), K(1) 2,3-epoxide (K(1)O), and MK-4 in human serum and plasma at concentrations in the ng/L-μg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal "endogenous suppressors" and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760μg/L. MK-4 was not detected in either of the two groups., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.

Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 µM. The Vmax values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The Km values of resistant rats (47 ± 32 µM) were similar to those of sensitive rats (59 ± 18 µM). Warfarin-sensitive rats exhibited enzyme efficiencies (Vmax/Km) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

Δ⁹-tetrahydrocannabinol (THC), through its action on cannabinoid type-1 receptor (CB₁R), is known to activate dopamine (DA) neurotransmission. Functional evidence of a direct antagonistic interaction between CB₁R and DA D₂-receptors (D₂R) suggests that D₂R may be an important target for the modulation of DA neurotransmission by THC. The current study evaluated, in rodents, the effects of chronic exposure to THC (1 mg/kg/day; 21 days) on D₂R and D₃R availabilities using the D₂R-prefering antagonist and the D₃R-preferring agonist radiotracers [¹⁸F]fallypride and [³H]-(+)-PHNO, respectively. At 24 h after the last THC dose, D₂R and D₃R densities were significantly increased in midbrain. In caudate/putamen (CPu), THC exposure was associated with increased densities of D₂R with no change in D₂R mRNA expression, whereas in nucleus accumbens (NAcc) both D₃R binding and mRNA levels were upregulated. These receptor changes, which were completely reversed in CPu but only partially reversed in NAcc and midbrain at 1 week after THC cessation, correlated with an increased functionality of D₂/₃R in vivo, based on findings of increased locomotor suppressive effect of a presynaptic dose and enhanced locomotor activation produced by a postsynaptic dose of quinpirole. Concomitantly, the observations of a decreased gene expression of tyrosine hydroxylase in midbrain together with a blunted psychomotor response to amphetamine concurred to indicate a diminished presynaptic DA function following THC. These findings indicate that the early period following THC treatment cessation is associated with altered presynaptic D₂/₃R controlling DA synthesis and release in midbrain, with the concurrent development of postsynaptic D₂/₃R supersensitivity in NAcc and CPu. Such D₂/₃R neuroadaptations may contribute to the reinforcing and habit-forming properties of THC.

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Thylakoid membranes contain two types of quinones, benzoquinone (plastoquinone) and naphthoquinone, which are involved in photosynthetic electron transfer. Unlike the benzoquinone, the chemical species of naphthoquinone present (phylloquinone, menaquinone-4 and 5'-monohydroxyphylloquinone) varies depending on the oxygenic photosynthetic organisms. The green alga Chlamydomonas reinhardtii has been used as a model organism to study the function of the naphthoquinone bound to PSI. However, the level of phylloquinone and the presence of other naphthoquinones in this organism remain unknown. In the present study, we found that 5'-monohydroxyphylloquinone is the predominant naphthoquinone in cell and thylakoid extracts based on the retention time during reverse phase HPLC, absorption and mass spectrometry measurements. It was shown that 5'-monohydroxyphylloquinone is enriched 2.5-fold in the PSI complex as compared with thylakoid membranes but that it is absent from PSI-deficient mutant cells. We also found a small amount of phylloquinone in the cells and in the PSI complex and estimated that accumulated 5'-monohydroxyphylloquinone and phylloquinone account for approximately 90 and 10%, respectively, of the total naphthoquinone content. The ratio of these two naphthoquinones remained nearly constant in the cells and in the PSI complexes from logarithmic and stationary cell growth stages. We conclude that both 5'-monohydroxyphylloquinone and phylloquinone stably co-exist as major and minor naphthoquinones in Chlamydomonas PSI.

The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein environments but possess distinct thermodynamic and kinetic properties. Although the determinants responsible for the different properties of the phylloquinones are not completely understood, the strength and geometry of hydrogen bond interactions are significant factors in tuning and control of function. This study focuses on characterizing the hydrogen-bonding interactions of the phylloquinone acceptor, A(1A), by (1)H and (14)N HYSCORE spectroscopy. Photoaccumulation of PS I complexes at pH 8.0 results in the trapping of the phyllosemiquinone anion, A(1A)(-), on the A-branch of cofactors. The experiments described here indicate that A(1A)(-) forms a single H-bond. Using a simple point dipole approximation, we estimate its length to be 1.6 ± 0.1 Å. The value of the (1)H isotropic hyperfine coupling constant suggests that the H-bond has significant out-of-plane character. The (14)N HYSCORE spectroscopy experiments support the assignment of a H-bond wherein, the (14)N quadrupolar coupling constant is consistent with a backbone amide nitrogen as the hydrogen bond donor.

We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH₂) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH₂, and (4) the primary function of VKOR is the reduction of KO to vitamin K.

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.

Dysregulation of myelin sulfatides is a risk factor for cognitive decline with age. Vitamin K is present in high concentrations in the brain and has been implicated in the regulation of sulfatide metabolism. Our objective was to investigate the age-related interrelation between dietary vitamin K and sulfatides in myelin fractions isolated from the brain regions of Fischer 344 male rats fed one of two dietary forms of vitamin K: phylloquinone or its hydrogenated form, 2',3'-dihydrophylloquinone (dK), for 28 days. Both dietary forms of vitamin K were converted to menaquinone-4 (MK-4) in the brain. The efficiency of dietary dK conversion to MK-4 compared to dietary phylloquinone was lower in the striatum and cortex, and was similar to that in the hippocampus. There were significant positive correlations between sulfatides and MK-4 in the hippocampus (phylloquinone-supplemented diet, 12 and 24 months; dK-supplemented diet, 12 months) and cortex (phylloquinone-supplemented diet, 12 and 24 months). No significant correlations were observed in the striatum. Furthermore, sulfatides in the hippocampus were significantly positively correlated with MK-4 in serum. This is the first attempt to establish and characterize a novel animal model that exploits the inability of dietary dK to convert to brain MK-4 to study the dietary effects of vitamin K on brain sulfatide in brain regions controlling motor and cognitive functions. Our findings suggest that this animal model may be useful for investigation of the effect of the dietary vitamin K on sulfatide metabolism, myelin structure and behavior functions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In a recent human [(11)C]-(+)-PHNO positron emission tomography study, olanzapine, clozapine, and risperidone occupied D2 receptors in striatum (STR), but, despite their similar in vitro D2 and D3 affinities, failed to occupy D3 receptors in globus pallidus. This study had two aims: (1) to characterize the regional D2/D3 pharmacology of in vitro and ex vivo [(3)H]-(+)-PHNO binding sites in rat brain and (2) to compare, using [(3)H]-(+)-PHNO autoradiography, the ex vivo and in vitro pharmacology of olanzapine, clozapine, risperidone, and haloperidol. Using the D3-selective drug SB277011, we found that ex vivo and in vitro [(3)H]-(+)-PHNO binding in STR is exclusively due to D2, whereas that in cerebellar lobes 9 and 10 is exclusively due to D3. Surprisingly, the D3 contribution to [(3)H]-(+)-PHNO binding in the islands of Calleja, ventral pallidum, substantia nigra, and nucleus accumbens was greater ex vivo than in vitro. Ex vivo, systemically administered olanzapine, risperidone, and haloperidol, at doses occupying approximately 80% D2, did not occupy D3 receptors. Clozapine, which also occupied approximately 80% of D2 receptors ex vivo, occupied a smaller percentage of D3 receptors than predicted by its in vitro pharmacology. Across brain regions, ex vivo occupancy by antipsychotics was inversely related to the D3 contribution to [(3)H]-(+)-PHNO binding. In contrast, in vitro occupancy was similar across brain regions, independent of the regional D3 contribution. These data indicate that at clinically relevant doses, olanzapine, clozapine, risperidone, and haloperidol are D2-selective ex vivo. This unforeseen finding suggests that their clinical effects cannot be attributed to D3 receptor blockade.

Ethnic differences in warfarin maintenance doses have been documented amongst the three major Asian ethnic groups (Chinese, Malay and Indian) in Singapore. Studies have shown that cytochrome P450 2C9 (CYP2C9) polymorphisms alone did not entirely account for these differences. Recent reports suggest that VKORC1 (subunit of vitamin K epoxide reductase) haplotypes are more predictive of warfarin response. Population pharmacokinetic/pharmacodynamic (PK/PD) modelling techniques were employed to characterise the PK and PD of warfarin in a healthy volunteer study of 16 Chinese and Indian subjects following a single 25 mg dose of warfarin. To further investigate the underlying differences in warfarin response, a semi-mechanistic modelling approach (using an indirect response model for PCA activity) incorporating the vitamin K cycle was attempted using population methods with Bayesian inference. All eight Indian subjects had H7H7 VKORC1 haplotypes and three had either *2/wt or *3/wt CYP2C9 genotypes. Six Chinese subjects had H1H1 VKORC1 haplotypes and one had H1H7. All Chinese subjects were homozygous wt/wt for CYP2C9. Simulations to steady state were performed to examine warfarin response in subjects with different CYP2C9 and VKORC1 polymorphisms. The presence of a single *2 or *3 CYP2C9 allele reduced mean [SE (standard error)] S-warfarin clearance by 35% from 0.276 (0.04) to 0.180 (0.11) l/h. Subjects with VKORC1 haplotype groups of H7H7 had increased mean (SE) C (50,S) (concentration of S-warfarin required to achieve 50% of maximum effect) of 479 (7.3) compared to 206 (6.7) ng/ml in subjects with the H1H1 groups. For subjects with the H1H7 haplotype, mean (SE) C (50,S) increased 1.4 times to 288 (1.3) ng/ml compared to subjects with H1H1 haplotypes. Steady state simulations showed that whilst CYP2C9 polymorphisms affect the PK of warfarin, VKORC1 haplotypes may be better predictors of warfarin response. Since 90% of Chinese subjects had the VKORC1 H1 haplotype and 100% of Indian subjects the H7 haplotype in this study, ethnic differences in warfarin response in this study appear to be linked to differences in VKORC1 haplotypes.

Background and Objective: Facial purpura is a frequent barrier to patient acceptance and satisfaction with the results of various cosmetic procedures. Methods to shorten the duration of purpura after such procedures are often sought by patients. This study evaluated the efficacy and safety of a topical gel containing vitamin K oxide in the resolution of laser induced purpura., Methods: In this randomized, double-blinded, placebo-controlled split-face study, 20 subjects with bilateral facial telangiectasia were treated with a pulsed dye laser (PDL) device at purpuric settings. The test articles, a gel containing vitamin K oxide and placebo (vehicle), were each randomly assigned to one side of the subject's face. Subjects applied the test articles twice a day for the following 9 +/- 1 days. Improvement in both focal and general field purpura on each side of the face was assessed by the investigator using photographs. A scale of -100% (worsening) to 100% (improving) was used to rate photos against a baseline photograph obtained 15-30 minutes after treatment with the PDL device., Results: Resolution of the field of purpura was consistently greater with the vitamin K oxide gel after the second day of treatment. The greatest difference between the vitamin K oxide gel and placebo scores occurred on the fourth day after treatment. Although differences in active versus placebo scores did not reach statistical significance during the nine-day study period, a trend toward faster resolution of purpura with the active product was seen. Treatment-related adverse effects were not observed in any subject., Conclusion: Vitamin K oxide gel appears to hasten the resolution of pulsed dye laser-induced purpura in subjects being treated for bilateral facial telangiectasia, and may well be useful in accelerating resolution of facial bruising from other cosmetic procedures such as fillers used for soft-tissue augmentation as well as other types of cutaneous surgical procedures.

The glutamate agonist LY404,039 has been used to treat schizophrenia. Because all currently used antipsychotics act on dopamine receptors, it was decided to examine whether this glutamate agonist also had an affinity for dopamine D2 receptors in vitro. The present data show that LY404,039 inhibited the binding of [3H]domperidone and [3H]+PHNO by 15.5 +/- 1.5% to the high-affinity state, D2(High), of cloned dopamine D2(Long) receptors and rat striatal tissue with dissociation constants of between 8.2 and 12.6 nM. This high-affinity component of LY404,039 on the binding of [3H]domperidone was inhibited by the presence of guanine nucleotide, indicating an agonist action of the drug at D2(High). LY404,039 also stimulated the incorporation of [35S]GTP-gamma-S into D2(Long) receptors (EC50% = 80 +/- 15 nM) over the same range of concentrations as occurred for the inhibition of [3H]domperidone by LY404,039 at D2(High) (IC50%(High) = 50 +/- 10 nM). A possible clinical antipsychotic action of LY404,039 may depend on the combined stimulation of glutamate receptors and a partial dopamine agonist action that would interfere with neurotransmission at D2(High) receptors., (Copyright 2009 Wiley-Liss, Inc.)

Dopaminergic stabilizers are recognized as compounds that can either enhance or antagonize dopamine (DA)-dependent behaviors depending on the prevailing dopaminergic tone. The dopaminergic stabilizer ASP2314 is being tested clinically and has been reported to have antipsychotic effects in a clinical trial as an add on medication. To elucidate the mechanisms of action of this dopaminergic stabilizer, its potency on the functional dopamine D2(High) receptors was examined. In competition with D2 receptors selectively labeled by [3H]domperidone, ASP2314 had a dissociation constant, Ki(High), of 1.62 microM for D2(High) in human cloned D2Long receptors and 0.83 muM for rat homogenized striata. Using the D2 agonist ligand [3H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO), ASP2314 had a high-affinity Ki of 32 nM for D2(High) for rat homogenized striata. ASP2314 stimulated the incorporation of [35S]GTP-gamma-S into rat striata by 50% at 43 nM, and into the cloned D2Long membranes by 50% at 3.2 microM (compared to 100% stimulation by 10 microM dopamine). With similar concentrations of ASP2314 inhibiting the binding of ligands at D2(High) and stimulating [35S]GTP-gamma-S incorporation, the data indicate that the dopaminergic stabilizing action of ASP2314 may be related to the selectivity for the D2(high) state of the D2 receptor., (Copyright 2009 Wiley-Liss, Inc.)

Although the glutamate agonist LY 404,039 has been used to treat schizophrenia, its closest congener LY 379,268 has an affinity for both glutamate and dopamine (DA) D2(High) receptors. Considering that all antipsychotics act on dopamine receptors, and considering that another laboratory reported that LY 379,268 did not have any affinity for the D2(High) receptor, it was necessary to examine whether such glutamate agonists have an affinity for D2 and D3 dopamine receptors in vitro. The present data show that 50-200 nM LY 379,268 inhibited the binding of [(3)H]domperidone and [(3)H](+)PHNO to cloned dopamine D2 receptors consistently and reproducibly by 16% with dissociation constants of 2.1 and 2.5 nM at D2(High), respectively. In addition, LY 379,268 inhibited the binding of [(3)H]domperidone and [(3)H](+)PHNO to cloned dopamine D3 receptors with dissociation constants of 130 and 10 nM, respectively. LY 379,268 also inhibited the binding of [(3)H]domperidone to rat striata with a dissociation constant of 22 nM, predicting a clinical antipsychotic dose of 80-100 mg/day. LY 379,268 appears to act as an agonist at D2(High) and as an antagonist at D3, because guanine nucleotide eliminated the competition at D2(High) but had no effect on the competition at D3. The findings indicate that this type of glutamate agonist, LY 379,268, has a significant affinity for D2(High) and D3 receptors.

Genetic polymorphisms in VKORC1 and CYP2C9, genes controlling vitamin K(1) (VK1) epoxide reduction and (S)-warfarin metabolism, respectively, are major contributors to interindividual variability in warfarin dose. The V433M polymorphism (rs2108622) in CYP4F2 has also been associated with warfarin dose and speculatively linked to altered VK1 metabolism. Therefore, the purpose of the present study was to determine the role of CYP4F2 and the V433M polymorphism in the metabolism of VK1 by human liver. In vitro metabolic experiments with accompanying liquid chromatography-tandem mass spectrometry analysis demonstrated that recombinant CYP4F2 (Supersomes) and human liver microsomes supplemented with NADPH converted VK1 to a single product. A screen of all commercially available P450 Supersomes showed that only CYP4F2 was capable of metabolizing VK1 to this product. Steady-state kinetic analysis with recombinant CYP4F2 and with human liver microsomes revealed a substrate K(m) of 8 to 10 microM. Moreover, anti-CYP4F2 IgG, as well as several CYP4F2-selective chemical inhibitors, substantially attenuated the microsomal reaction. Finally, human liver microsomes genotyped for rs2108622 demonstrated reduced vitamin K(1) oxidation and lower CYP4F2 protein concentrations in carriers of the 433M minor allele. These data demonstrate that CYP4F2 is a vitamin K(1) oxidase and that carriers of the CYP4F2 V433M allele have a reduced capacity to metabolize VK1, secondary to an rs2108622-dependent decrease in steady-state hepatic concentrations of the enzyme. Therefore, patients with the rs2108622 polymorphism are likely to have elevated hepatic levels of VK1, necessitating a higher warfarin dose to elicit the same anticoagulant response.

The finding that the mGlu2/3 metabotropic glutamate receptor agonist, LY404039, improves clinical symptoms in schizophrenia warrants a search for a possible interaction between mGlu2/3 receptors and dopamine D2 receptors. Here, this topic is examined in striatal tissue of mice lacking either mGlu2 or mGlu3 receptor. Such mice are known to be behaviorally supersensitive to dopamine receptor agonists. Therefore, to determine the basis of this dopamine supersensitivity, the proportion of dopamine D2(High) receptors was measured in the striata of mGlu2 and mGlu3 receptor knockout mice. The proportion of D2(High) receptors was found to be elevated by 220% in the striata of both knockouts. To measure the functional dopamine supersensitivity, the D2 agonist (+)PHNO was used to stimulate the incorporation of GTP-gamma-S in the striatal homogenates in the presence of drugs that blocked the dopamine D1, D3, and D5 receptors. Compared with control striata, the mGlu2 receptor knockout tissues were 67-fold more sensitive to (+)PHNO, while the mGlu3 receptor knockout tissues were 17-fold more sensitive. These data suggest that group II mGlu receptors-mGlu2 receptors in particular-may normally regulate D2 receptors by reducing the proportion of high-affinity D2 receptors in membranes. Such regulation may contribute to the antipsychotic action of mGlu2/3 receptor agonists., ((c) 2008 Wiley-Liss, Inc.)

We studied vitamin K(1)(20), vitamin K(1)(25), and vitamin K(1) epoxide in n-hexane and ethanol solutions by gas chromatography/mass spectrometry (GC/MS) utilizing a DB-5 MS fused-silica capillary column. In ethanol solutions of K(1), we observed an extra peak eluting from the GC column with somewhat longer retention time than K(1)(20). A similar peak following K(1)(25) was also found. These peaks were not found in n-hexane solutions of K(1). A close examination of the mass spectra of these peaks indicated that they were vitamin K(1) variants containing a base peak at m/z 225 characteristic of the methylnaphthoquinone ring with a four-carbon side chain. In addition, they contained the molecular ions of K(1)(20) and K(1)(25), respectively. We conclude that K(1)(20) and K(1)(25), but not K(1) epoxide, might undergo rearrangements in ethanol involving an intramolecular proton transfer and a shift of the beta,gamma-double bond on the phytyl side chain toward the ring. The conjugation of the phytyl double bond with the quinone ring is probably the driving force of the rearrangement. We emphasize, however, that our conclusion is based only on mass spectral analysis and would require further investigation by other spectroscopic methods.

A sensitive HPLC-APCI-MS method for the determination of vitamin K(1) (VK-1) in human plasma was established. Target ions at [M+H](+)m/z 451.5 for VK-1 and [M+H](+)m/z 331.4 for the I.S. (teprenone). Calibration curve was linear over the range of 0.3-1,000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 8% and 15%, respectively. The C(max) was 210.1+/-86.7 ng/ml while the elimination half-life (t(1/2)) was 8.8+/-1.7h and time to the C(max) was 5.5+/-0.8h after administration of soft capsule containing 10mg VK-1.

Vitamin K epoxide reductase (VKOR) is an integral membrane protein that catalyzes the reduction of vitamin K 2,3-epoxide and vitamin K to vitamin K hydroquinone, a cofactor required for the gamma-glutamyl carboxylation reaction. VKOR is highly sensitive to inhibition by warfarin, the most commonly prescribed oral anticoagulant. Warfarin inhibition of VKOR decreases the concentration of reduced vitamin K, which reduces the rate of vitamin K-dependent carboxylation and leads to under-carboxylated, inactive vitamin K-dependent proteins. It is proposed that an active site disulfide needs to be reduced for the enzyme to be active. VKOR uses two sulfhydryl groups for the catalytic reaction and these two sulfhydryl groups are oxidized back to a disulfide bond during each catalytic cycle. The recent identification of the gene encoding VKOR allows us to study its structure and function relationship at the molecular level. The membrane topology model shows that VKOR spans the endoplasmic reticulum membrane three times with its amino-terminus residing in the lumen and the carboxyl-terminus residing in the cytoplasm. Both the active site (cysteines 132 and 135) and the proposed warfarin binding site (tyrosine 139) reside in the third transmembrane helix. VKOR is made at high levels in insect cells and is relatively easily purified. This should allow the determination of its three-dimensional structure. A detailed mechanism has been published and the purified enzyme should allow the testing of this mechanism. A major unanswered question is the physiological reductant of VKOR.

Vitamin K-dependent (VKD) protein carboxylation uses vitamin K epoxidation to convert Glus to carboxylated Glus (Glas), rendering VKD proteins active in physiologies that include hemostasis, apoptosis, bone mineralization, calcium homeostasis, growth control, and signal transduction. Clusters of Glus are modified by a processive carboxylase, generating a calcium-binding module that allows binding to either hydroxyapatite in the extracellular matrices or cell surfaces where anionic phospholipids become exposed, for example, during apoptosis or cell activation. Naturally occurring carboxylase mutations have been informative for function and are associated with bleeding complications and, surprisingly, a pseudoxanthoma elasticum (PXE)-like phenotype. A major advance in defining carboxylase function is the identification of the base that initiates carboxylation, which raises interesting possibilities for how vitamin K epoxidation is regulated by Glu substrate and carboxylase membrane topology. Vitamin K oxidoreductase (VKOR), the target of warfarin, generates the reduced vitamin K cofactor used by the carboxylase. Oxidation of active site thiols during vitamin K reduction inactivates VKOR, and activity is regenerated by an unknown reductase. The amounts of reduced vitamin K limit the capacity for carboxylation in cells, and overexpression of VKOR, but not carboxylase, improves carboxylation. However, the effect of VKOR overexpression is small, possibly because the reductase that regenerates VKOR activity is saturated. The review discusses these advances, as well as the potential impact of secretory components on carboxylation, which occurs during VKD protein secretion. Also discussed is the role of the carboxylase in mammals and lower organisms, including the bacterial pathogen Leptospira interrogans that has acquired a VKD carboxylase by horizontal transfer.

Background: Poor diet may affect bone status by displacing nutrients involved in bone health. Dihydrophylloquinone, a form of vitamin K present in foods made with partially hydrogenated fat, is a potential marker of a low-quality dietary pattern., Objective: Our objective was to examine the cross-sectional associations between dihydrophylloquinone intake and bone mineral density (BMD) of the hip and spine in men and women., Design: Dihydrophylloquinone intake was estimated with a food-frequency questionnaire, and BMD (in g/cm(2)) was measured by dual-energy X-ray absorptiometry in 2544 men and women (mean age: 58.5 y) who had participated in the Framingham Offspring Study. General linear models were used to examine the associations between dihydrophylloquinone intake (in tertiles: <15.5, 15.5-29.5, and >29.5 microg/d) and hip and spine BMD after adjustment for age, body mass index, energy intake, calcium intake, vitamin D intake, smoking status, physical activity score, and, for women, menopause status and estrogen use., Results: Higher dihydrophylloquinone intakes were associated with lower mean BMD at the femoral neck [lowest-to-highest tertiles (95% CI): 0.934 (0.925, 0.942), 0.927 (0.919, 0.935), and 0.917 (0.908, 0.926), P for trend = 0.02], the trochanter [lowest-to-highest tertiles (95% CI): 0.811 (0.802, 0.820), 0.805 (0.797, 0.813), and 0.795 (0.786, 0.804), P for trend = 0.02], and the spine [lowest-to-highest tertiles (95% CI): 1.250 (1.236, 1.264), 1.243 (1.242, 1.229), and 1.227 (1.213, 1.242), P for trend = 0.03] in men and women after adjustment for the covariates. Further adjustment for markers of healthy and low-quality dietary patterns did not affect the observed associations., Conclusions: Higher dihydrophylloquinone intakes are associated with lower BMD in men and women. This association remains significant after adjustment for other markers of diet quality.

The physiological function and putative health roles of vitamin K-dependent proteins now extend beyond their classical role in hemostasis and include bone mineralization, arterial calcification, apoptosis, phagocytosis, growth control, chemotaxis, and signal transduction. Current assessments of vitamin K status do not reflect the variety of molecular forms of vitamin K. We assessed whether urinary excretion of 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone) and 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone), vitamin K metabolites common to both phylloquinone and the menaquinone series, reflect dietary vitamin K intake. In a randomized crossover study, 9 adults resided in a metabolic unit for two 30-d periods separated by a free-living period of > or = 4 wk. During each residency, subjects consumed 3 sequential diets: a control diet (93 microg phylloquinone/d) for 5 d, a phylloquinone-restricted diet (11 microg/d) for 15 d, followed by a randomly assigned repletion diet for 10 d with either phylloquinone (206 microg/d) or dihydrophylloquinone (240 microg/d). During the second residency, the alternative repletion diet was assigned. Urinary excretion of the 5C- and 7C-aglycones was measured in sequential 24-h collections. The 5C-aglycone accounted for approximately 75% of total excretion and declined in response to phylloquinone restriction (P = 0.001) to approximately 30% of that during the control diet period. Repletion with phylloquinone and dihydrophylloquinone doubled the excretion rate of the major 5C-aglycone by 24 h (P < 0.001), and tripled excretion by 4 d. There was a linear relationship between the logarithm of total urinary excretion and dietary vitamin K intake (r = 0.699, P < 0.001). We conclude that the urinary excretion of vitamin K metabolites reflects dietary phylloquinone intake and offers the first candidate marker of global vitamin K status.

Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.

More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is approximately 21 kDa, as expected; fully active; and > 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

A phylloquinone molecule (2-methyl-3-phytyl-1,4-naphthoquinone) occupies the A1 binding site in photosystem I. Previously, we have obtained A1(-)/A1 FTIR difference spectra using labeled and unlabeled photosystem I particles and proposed assignments for many of the bands in the spectra [Sivakumar, V., Wang, R., and Hastings, G. (2005) Biochemistry 44, 1880-1893]. In particular, we suggested that a negative/positive band at 1654/1495 cm(-1) in A1(-)/A1 FTIR DS is due to a C=O/C-:O mode of the neutral/anionic phylloquinone, respectively. To test this hypothesis, we have obtained A1(-)/A1 FTIR DS for menG mutant PS I particles. In menG mutant PS I, phylloquinone in the A1 binding site is replaced with an analogue in which the methyl group at position 2 of the quinone ring is replaced with a hydrogen atom (2-phytyl-1,4-naphthoquinone). In A1(-)/A1 FTIR DS obtained using menG mutant PS I particles, we find that the 1654/1495 cm(-1) bands are upshifted by approximately 6 cm(-1). To test if such upshifts are likely for C=O/C-:O modes of neutral/anionic phylloquinone, we have used density functional theory to calculate the "anion minus neutral" infrared difference spectra for both phylloquinone and its methyl-less analogue. We have also undertaken calculations in which the C4=O carbonyl group of phylloquinone and its methyl-less analogue are hydrogen bonded (to a water or leucine molecule). We find that, irrespective of the hydrogen bonding state of the C4=O group, the C=O/C-:O modes of neutral/reduced phylloquinone are indeed expected to be upshifted by at least 6 cm(-1) upon replacement of the methyl group at position 2 with hydrogen. The calculations also suggest that certain C=C/C-:C modes of neutral/reduced phylloquinone do not shift upon replacement of the methyl group. On the basis of these calculated results, we suggest which bands in the A1(-)/A1 FTIR DS may be associated with C=C/C-:C modes of neutral/reduced phylloquinone, respectively.

Phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. Neither the route nor the function of the conversion is known. One possible metabolic route might be the release of menadione from phylloquinone by catabolic activity. In the present study we explored the presence of menadione in urine and the effect of vitamin K intake on its excretion. Menadione in urine was analysed by HPLC assay with fluorescence detection. Urine from healthy male volunteers was collected before and after administration of a single dose of K vitamins. Basal menadione excretion in non-supplemented subjects (n 6) was 5.4 (sd 3.2) microg/d. Urinary menadione excretion increased greatly after oral intake of the K vitamins, phylloquinone and menaquinone-4 and -7. This effect was apparent within 1-2 h and peaked at about 3 h after intake. Amounts of menadione excreted in 24 h after vitamin K intake ranged, on a molar basis, from 1 to 5 % of the administered dose, indicating that about 5-25 % of the ingested K vitamins had been catabolized to menadione. Menadione excretion was not enhanced by phylloquinone administered subcutaneously or by 2',3'-dihydrophylloquinone administered orally. In archived samples from a depletion/repletion study (Booth et al. (2001) Am J Clin Nutr 74, 783-790), urinary menadione excretion mirrored dietary phylloquinone intake. The present study shows that menadione is a catabolic product of K vitamins formed after oral intake. The rapid appearance in urine after oral but not subcutaneous administration suggests that catabolism occurs during intestinal absorption. The observations make it likely that part of the menaquinone-4 in tissues results from uptake and prenylation of circulating menadione.

Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational gamma-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here,we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.

Commercial hydrogenation results in the formation of trans fatty acids. An unintended consequence of the hydrogenation process is conversion of phylloquinone (vitamin K1) to dihydrophylloquinone. Plasma dihydrophylloquinone concentrations have yet to be characterized in population-based studies. Dietary determinants of plasma dihydrophylloquinone were estimated using a semi-quantitative food frequency questionnaire in 803 men and 913 women in the Framingham Offspring Study. Geometric mean dihydrophylloquinone intake was 21.3 (95 % CI 20.4, 22.3) microg/d in men and 19.4 (95 % CI 18.5, 20.2) microg/d in women. Detectable (>0.05 nmol/l) plasma dihydrophylloquinone concentrations were measured in 41 % and 30 % of men and women, respectively. The multivariate odds ratio (OR) of detectable plasma dihydrophylloquinone from the lowest to the highest quartile category of dihydrophylloquinone intake were 1 (referent), 1.13 (95 % CI 0.83, 1.53), 1.66 (95 % CI 1.21, 2.26) and 1.84 (95 % CI 1.31, 2.58), P for trend <0.001, adjusted for sex, age, body mass index, triacylglycerols, season and energy intake. Higher trans fatty acid intake was associated with higher multivariate OR for detectable plasma dihydrophylloquinone (OR comparing extreme quartiles 2.41 (95 % CI 1.59, 3.64), P for trend <0.001). There were limitations in the use of plasma dihydrophylloquinone, evident in the high proportion of the population that had non-detectable dihydrophylloquinone concentrations. Despite this caveat, higher plasma dihydrophylloquinone was associated with higher dihydrophylloquinone intake and higher trans fatty acid intake.