n-3 Canola oil effectively replaces fish oil as a new safe dietary source of DHA in feed for juvenile Atlantic salmon.

Limited availability of fish oils (FO), rich in n-3 long-chain (≥C20) PUFA, is a major constraint for further growth of the aquaculture industry. Long-chain n-3 rich oils from crops GM with algal genes are promising new sources for the industry. This project studied the use of a newly developed n-3 canola oil (DHA-CA) in diets of Atlantic salmon fingerlings in freshwater. The DHA-CA oil has high proportions of the n-3 fatty acids (FA) 18 : 3n-3 and DHA and lower proportions of n-6 FA than conventional plant oils. Levels of phytosterols, vitamin E and minerals in the DHA-CA were within the natural variation of commercial canola oils. Pesticides, mycotoxins, polyaromatic hydrocarbons and heavy metals were below lowest qualifiable concentration. Two feeding trials were conducted to evaluate effects of two dietary levels of DHA-CA compared with two dietary levels of FO at two water temperatures. Fish increased their weight approximately 20-fold at 16°C and 12-fold at 12°C during the experimental periods, with equal growth in salmon fed the FO diets compared with DHA-CA diets. Salmon fed DHA-CA diets had approximately the same EPA+DHA content in whole body as salmon fed FO diets. Gene expression, lipid composition and oxidative stress-related enzyme activities showed only minor differences between the dietary groups, and the effects were mostly a result of dietary oil level, rather than the oil source. The results demonstrated that DHA-CA is a safe and effective replacement for FO in diets of Atlantic salmon during the sensitive fingerling life-stage.

Biomimetic hydroxyapatite/poly xylitol sebacic adibate/vitamin K nanocomposite for enhancing bone regeneration.

Hydroxyapatite (HAP) is a significant bone mineral that establishes bone strength. HAP composites in combination with biodegradable and bioactive polymer poly xylitol sebacic adipate (PXSA) would result in a constant release at target sites. Numerous studies have shown that vitamin K (VK) might possess a vital function in bone metabolism. The purpose of the present study was to inspect the synthesized composite HAP/PXSA/VK in developing polymeric biomaterials composite for the application of bone tissue regeneration. FTIR, X-ray diffraction, SEM and TEM techniques were applied to characterize the prepared composites. The release of VK from the HAP/PXSA/VK composite was evidenced through UV-Vis spectroscopy. In vitro studies proved that the HAP/PXSA/VK composite is appropriate for mesenchymal stem cell culture. Compared to pure HAP prepared following the same method, HAP/PXSA/VK composite provided favourable microstructures and good biodegradation distinctiveness for the application of tissue engineering, as well as tissue in-growth characteristics and improved scaffold cell penetration. This work reveals that the HAP/PXSA/VK composites have the potential for applications in bone tissue engineering.

Combination of Double-Mediator System with Large-Scale Integration-Based Amperometric Devices for Detecting NAD(P)H:quinone Oxidoreductase 1 Activity of Cancer Cell Aggregates.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme providing cytoprotection from quinone species. In addition, it is expressed at high levels in many human tumors, such as breast cancer. Therefore, it is considered to be a potential target in cancer treatment. In order to detect intracellular NQO1 activity in MCF-7 aggregates as a cancer model, we present, in this study, a double-mediator system combined with large-scale integration (LSI)-based amperometric devices. This LSI device contained 20 × 20 Pt working electrodes with a 250 µm pitch for electrochemical imaging. In the detection system, menadione (MD) and [Fe(CN)6]3- were used. Since MD can diffuse into cells due to its hydrophobicity, it is reduced into menadiol by intracellular NQO1. The menadiol diffuses out of the cells and reduces [Fe(CN)6]3- of a hydrophilic mediator into [Fe(CN)6]4-. The accumulated [Fe(CN)6]4- outside the cells is electrochemically detected at 0.5 V in the LSI device. Using this strategy, the intracellular NQO1 activity of MCF-7 aggregates was successfully detected. The effect of rotenone, which is an inhibitor for Complex I, on NQO1 activity was also investigated. In addition, NQO1 and respiration activities were simultaneously imaged using the detection system that was further combined with electrochemicolor imaging. Thus, the double-mediator system was proven to be useful for evaluating intracellular redox activity of cell aggregates.

Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.

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.

The role of daily physical activity and nutritional status on bone turnover in cystic fibrosis: a cross-sectional study.

BACKGROUND: Nutritional status and daily physical activity (PA) may be an excellent tool for the maintenance of bone health in patients with cystic fibrosis (CF). OBJECTIVE: To evaluate the relationship between nutritional status, daily physical activity and bone turnover in cystic fibrosis patients. METHOD: A cross-sectional study of adolescent and adult patients diagnosed with clinically stable cystic fibrosis was conducted. Total body, femoral neck, and lumbar spine bone mineral density (BMD) were determined by dual energy X-ray absorptiometry and bone metabolism markers ALP, P1NP, PICP, and ß-CrossLaps. PA monitoring was assessed for 5 consecutive days using a portable device. Exercise capacity was also determined. Serum 25-hydroxyvitamin D and vitamin K were also determined in all participants. RESULTS: Fifty patients (median age: 24.4 years; range: 16-46) were included. BMI had positive correlation with all BMD parameters, with Spearman's coefficients ranging from 0.31 to 0.47. Total hip bone mineral density and femoral neck BMD had positive correlation with the daily time spent on moderate PA (>4.8 metabolic equivalent-minutes/day; r=0.74, p<0.001 and r=0.72 p<0.001 respectively), daily time spent on vigorous PA (>7.2 metabolic equivalent-minutes/day; r=0.45 p<0.001), body mass index (r=0.44, p=0.001), and muscle mass in limbs (r=0.41, p=0.004). Levels of carboxy-terminal propeptide of type 1 collagen were positively associated with the daily time spent on moderate (r=0.33 p=0.023) and vigorous PA (r=0.53, p<0.001). CONCLUSIONS: BMI and the daily time spent on moderate PA were found to be correlated with femoral neck BMD in CF patients. The association between daily PA and biochemical markers of bone formation suggests that the level of daily PA may be linked to bone health in this patient group. Further research is needed to confirm these findings.

A Mixed Micelle Formulation for Oral Delivery of Vitamin K.

PURPOSE: To develop a stable micellar formulation of vitamin K for oral delivery, because the commercial and clinically used formulation of vitamin K (Konakion® MM) destabilizes at gastric pH resulting in low bioavailability of this vitamin in neonates with cholestasis. METHODS: Mixed micelles composed of EPC, DSPE-PEG 2000 and glycocholic acid, with and without vitamin K, were prepared by a film hydration method. The influence of pH on the stability of the micelles was analyzed by dynamic light scattering (DLS). The critical micelle concentration (CMC) was determined by fluorescence spectroscopy using pyrene and the morphology was evaluated by transmission electron microscopy . Caco-2 cells were used to study the cytocompatibilty. RESULTS: Mixed micelles with mean diameters from 7.1 to 11.0 nm and a narrow size distribution (PDI < 0.2) were obtained after 3 membrane extrusion cycles. Konakion® MM formed aggregated particles at gastric pH, which was avoided through steric stabilization by introducing PEG. TEM showed that mixed micelles had a spherical size (diameter of around 10 nm) with a narrow size distribution in agreement with the DLS results. The loading capacities for vitamin K of mixed micelles with varying molar fractions of DSPE-PEG and EPC (from 0/100 to 50/50 (mol/mol)) were 10.8-5.0 w%, respectively. The mixed micelles showed good cytocompatibility at concentrations of glycocholic acid between 0.12 and 1.20 mM. CONCLUSIONS: Mixed micelles with superior stability to Konakion® MM at low pH were obtained by introducing DSPE-PEG 2000. These are therefore attractive oral formulations for vitamin K.

The role of daily physical activity and nutritional status on bone turnover in cystic fibrosis: a cross-sectional study

ABSTRACT Background Nutritional status and daily physical activity (PA) may be an excellent tool for the maintenance of bone health in patients with cystic fibrosis (CF). Objective To evaluate the relationship between nutritional status, daily physical activity and bone turnover in cystic fibrosis patients. Method A cross-sectional study of adolescent and adult patients diagnosed with clinically stable cystic fibrosis was conducted. Total body, femoral neck, and lumbar spine bone mineral density (BMD) were determined by dual energy X-ray absorptiometry and bone metabolism markers ALP, P1NP, PICP, and ß-CrossLaps. PA monitoring was assessed for 5 consecutive days using a portable device. Exercise capacity was also determined. Serum 25-hydroxyvitamin D and vitamin K were also determined in all participants. Results Fifty patients (median age: 24.4 years; range: 16-46) were included. BMI had positive correlation with all BMD parameters, with Spearman’s coefficients ranging from 0.31 to 0.47. Total hip bone mineral density and femoral neck BMD had positive correlation with the daily time spent on moderate PA (>4.8 metabolic equivalent-minutes/day; r=0.74, p<0.001 and r=0.72 p<0.001 respectively), daily time spent on vigorous PA (>7.2 metabolic equivalent-minutes/day; r=0.45 p<0.001), body mass index (r=0.44, p=0.001), and muscle mass in limbs (r=0.41, p=0.004). Levels of carboxy-terminal propeptide of type 1 collagen were positively associated with the daily time spent on moderate (r=0.33 p=0.023) and vigorous PA (r=0.53, p<0.001). Conclusions BMI and the daily time spent on moderate PA were found to be correlated with femoral neck BMD in CF patients. The association between daily PA and biochemical markers of bone formation suggests that the level of daily PA may be linked to bone health in this patient group. Further research is needed to confirm these findings.

Antivitamins for Medicinal Applications.

Antivitamins represent a broad class of compounds that counteract the essential effects of vitamins. The symptoms triggered by such antinutritional factors resemble those of vitamin deficiencies, but can be successfully reversed by treating patients with the intact vitamin. Despite being undesirable for healthy organisms, the toxicities of these compounds present considerable interest for biological and medicinal purposes. Indeed, antivitamins played fundamental roles in the development of pioneering antibiotic and antiproliferative drugs, such as prontosil and aminopterin. Their development and optimisation were made possible by the study, throughout the 20th century, of the vitamins' and antivitamins' functions in metabolic processes. However, even with this thorough knowledge, commercialised antivitamin-based drugs are still nowadays limited to antagonists of vitamins B9 and K. The antivitamin field thus still needs to be explored more intensely, in view of the outstanding therapeutic success exhibited by several antivitamin-based medicines. Here we summarise historical achievements and discuss critically recent developments, opportunities and potential limitations of the antivitamin approach, with a special focus on antivitamins K, B9 and B12 .

Inhibition of repair-related DNA polymerases by vitamin Ks, their related quinone derivatives and associated inflammatory activity (Review).

Vitamin Ks (VKs) are fat-soluble quinone compounds known to have various bioactivities. This review describes the inflammatory effects of VKs and their related quinone derivatives based on DNA polymerase (pol) inhibition. VK3, but not VK1 or VK2 (=MK-4), inhibited the activity of human pol γ, which is the DNA replicative pol in mitochondria. Of the intermediate compounds between VK2 and VK3 (namely MK-3, MK-2 and MK-1), MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B-, Y- and X-families of pols, respectively. Among the VK3 based quinone derivatives, such as 1,4-naphthoquinone (NQ), 2-dimethyl-1,4-naphthoquinone (1,2-dimethyl-NQ), 1,4-benzoquinone (BQ), 9,10-anthraquinone (AQ) and 5,12-naphthacenequinone (NCQ), NQ was the strongest inhibitor of mammalian pols α and λ, in particular, DNA repair-related pol λ. Among the all compounds tested, NQ displayed the strongest suppression of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in a cell culture system using RAW264.7 mouse macrophages. NQ also suppressed the expression of pol λ protein in these cells, after LPS-treated RAW264.7 cells were stimulated to induce pol λ expression. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of NQ into mice suppressed TNF-α production in peritoneal macrophages and serum. In an in vivo colitis mouse model induced using dextran sulfate sodium (DSS), NQ markedly suppressed DSS-evoked colitis. The promising anti-inflammatory candidates based on the inhibition of DNA repair-related pols, such as pol λ, by VKs quinone derivatives, such as NQ, are discussed.

Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein.

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson's disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.

Structure-activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines.

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure-activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.

Recovery of solanesol from tobacco as a value-added byproduct for alternative applications.

Solanesol in the waste streams of a bioprocess designed for alternative applications of low-alkaloid tobacco was recovered using three different extraction methods. Compared to the conventional heat-reflux extraction (HRE) and ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) using 1:3 hexane:ethanol (v/v) as the solvent after saponification treatment of tobacco biomass was found the most effective in terms of solanesol yield, processing time, and volume of solvent consumed. Quantification of solanesol was achieved by optimizing the mobile phase at 60/40 acetonitrile-isopropanol and lowering the oven temperature to 22 degrees C using a standard reverse-phase high performance liquid chromatography (RP-HPLC). The total solanesol recovered from tobacco biomass and chloroplast accounted for 30% (w/w) of the total solanesol in the fresh leaves. Since solanesol is the precursor of metabolically active quinones such as coenzyme Q10 and vitamin K analogues, extraction of solanesol from tobacco bioprocess waste is a feasible operation and could leverage the overall profitability of biorefining tobacco for alternative, value-added uses.

Participation of low molecular weight electron carriers in oxidative protein folding.

Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding.

The influence of bile acids on the oral bioavailability of vitamin K encapsulated in polymeric micelles.

The purpose of this study was to assess the ability of polymeric micelles to enable gastrointestinal absorption of the extremely hydrophobic compound vitamin K, by comparison of its absorption in bile duct ligated and sham operated rats. Hereto, vitamin K was encapsulated in micelles composed of mPEG(5000)-b-p(HPMAm-lac(2)), a thermosensitive block copolymer. Vitamin K plasma levels rose significantly upon gastric administration of 1 mg vitamin K encapsulated in polymeric micelles in sham operated rats, but not after bile duct ligation (AUC 4543 and 1.64 ng/mL/h respectively, p<0.01). Duodenal administration of polymeric micelles together with bile acids in bile duct ligated rats fully restored absorption. Dynamic light scattering time series showed a significant and dose dependent rise in micellar size in the presence of bile acids in vitro, indicating the gradual formation of mixed micelles during the first 3 h of incubation. The highest bile acid amounts (11 mM deoxycholic acid and 41 mM taurocholic acid) eventually caused aggregation of the loaded micelles after the formation of mixed micelles. These data suggest that the gastrointestinal absorption of encapsulated vitamin K from polymeric micelles is mediated by free bile and that uptake of intact micelles through pinocytosis is insignificant.

Metabolism and cell biology of vitamin K.

Naturally occurring vitamin K compounds comprise a plant form, phylloquinone (vitamin K(1)) and a series of bacterial menaquinones (MKs) (vitamin K(2)). Structural differences in the isoprenoid side chain govern many facets of metabolism of K vitamins including the way they are transported, taken up by target tissues, and subsequently excreted. In the post-prandial state, phylloquinone is transported mainly by triglyceride-rich lipoproteins (TRL) and long-chain MKs mainly by low-density lipoproteins (LDL). TRL-borne phylloquinone uptake by osteoblasts is an apoE-mediated process with the LRP1 receptor playing a predominant role. One K(2) form, MK-4, has a highly specific tissue distribution suggestive of local synthesis from phylloquinone in which menadione is an intermediate. Both phylloquinone and MKs activate the steroid and xenobiotic receptor (SXR) that initiates their catabolism, but MK-4 specifically upregulates two genes suggesting a novel MK-4 signalling pathway. Many studies have shown specific clinical benefits of MK-4 at pharmacological doses for osteoporosis and cancer although the mechanism(s) are poorly understood. Other putative non-cofactor functions of vitamin K include the suppression of inflammation, prevention of brain oxidative damage and a role in sphingolipid synthesis. Anticoagulant drugs block vitamin K recycling and thereby the availability of reduced vitamin K. Under extreme blockade, vitamin K can bypass the inhibition of Gla synthesis in the liver but not in the bone and the vessel wall. In humans, MK-7 has a greater efficacy than phylloquinone in carboxylating both liver and bone Gla proteins. A daily supplement of phylloquinone has shown potential for improving anticoagulation control.

Age and dietary form of vitamin K affect menaquinone-4 concentrations in male Fischer 344 rats.

Phylloquinone, the primary dietary form of vitamin K, is converted to menaquinone-4 (MK-4) in certain tissues. MK-4 may have tissue-specific roles independent of those traditionally identified with vitamin K. Fischer 344 male rats of different ages (2, 12, and 24 mo, n = 20 per age group) were used to compare the conversion of phylloquinone to MK-4 with an equivalent dose of another dietary form of vitamin K, 2',3'-dihydrophylloquinone. Rats were age- and diet-group pair-fed phylloquinone (198 +/- 9.0 microg/kg diet) or dihydrophylloquinone (172 +/- 13.0 microg/kg diet) for 28 d. MK-4 was the primary form of vitamin K in serum, spleen, kidney, testes, bone marrow, and brain myelin fractions, regardless of age group. MK-4 concentrations were significantly lower in kidney, heart, testes, cortex (myelin), and striatum (myelin) in the dihydrophylloquinone diet group compared with the phylloquinone diet group (P < 0.05). The MK-4 concentrations in 2-mo-old rats were lower in liver, spleen, kidney, heart, and cortex (myelin) but higher in testes compared with 24-mo-old rats (P < 0.05). However, there were no age-specific differences in MK-4 concentrations among the rats fed the 2 diets. These data suggest that dihydrophylloquinone, which differs from phylloquinone in its side phytyl chain, is absorbed but its intake results in less MK-4 in certain tissues. Dihydrophylloquinone may be used in models for the study of tissue-specific vitamin K deficiency.

Comparative uptake, metabolism, and utilization of menaquinone-4 and phylloquinone in human cultured cell lines.

It is generally accepted that the availability of vitamin K in vivo depends on its homologues, the biological activities of which would differ among organs. To test this hypothesis, we examined the uptake, metabolism, and utilization of menaquinone-4 (MK-4) and phylloquinone (PK) using 18O-labeled compounds in two cultured human cell lines (HepG2 and MG-63). Lipid extracts were prepared from the cells and media after 1, 3, and 6h of incubation. The detection of the vitamin K analogues (18O-, 16O-quinone, and epoxide forms) was carried out with LC-APCI-MS/MS as previously reported. The 18O of vitamin K was replaced with atmospheric 16O2 during the formation of vitamin K epoxide with a carboxylative catalytic reaction. As a result, a significant difference was observed between MK-4 and PK in the amounts taken up into the cells. The 18O-labeled MK-4 was rapidly and remarkably well absorbed into the cells and metabolized to the epoxide form via a hydroquinone form as compared to the 18O-labeled PK. The difference in uptake of MK-4 and PK was not affected by treatment with warfarin although the metabolism of both compounds was markedly inhibited. This methodology should be utilized to clarify some of the actions of vitamin K in target cells and facilitate the development of new vitamin K drugs.

Vitamin K epoxide reductase complex subunit 1 (VKORC1): the key protein of the vitamin K cycle.

Vitamin K epoxide, a by-product of the carboxylation of blood coagulation factors, is reduced to vitamin K by an enzymatic system possessing vitamin K epoxide reductase (VKOR) activity. This system is the target of coumarin-derived drugs widely used in thrombosis therapy and prophylaxis. Recently, the key protein of the VKOR system has been identified. The human VKORC1 gene maps to chromosome 16 and consists of 3 exons encoding a 163-amino acid integral ER membrane protein with three or four predicted transmembrane alpha- helices. Expression of human VKORC1 in Spodoptera frugiperda (Sf9) cells and in Pichia pastoris results in enhanced VKOR activity over low endogenous constitutive levels. Sequence based search methods reveal that human VKORC1 belongs to a large family of homologous genes found in vertebrates, insects, plants, protists, archea, and bacteria. All orthologs share five completely conserved amino acids, including two cysteines found in a tetrapeptide motif presumably required for redox function. The recent discovery of the VKORC1 gene has initiated renewed interest in understanding VKOR activity. Analysis of VKORC1 protein structure and function will be crucial in understanding the VKOR catalytic mechanism, how anticoagulant drugs modulate VKOR activity, and the role of VKORC1 in downstream physiological and pathological pathways.

Engineering of a recombinant vitamin K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic acid forming capacity: evidence for a functional CXXC redox center in the system.

The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.