The association between vitamin K status and knee osteoarthritis features in older adults: the Health, Aging and Body Composition Study.

BACKGROUND: Vitamin K-dependent (VKD) proteins, including the mineralization inhibitor matrix-gla protein (MGP), are found in joint tissues including cartilage and bone. Previous studies suggest low vitamin K status is associated with higher osteoarthritis (OA) prevalence and incidence. OBJECTIVE: To clarify what joint tissues vitamin K is relevant to in OA, we investigated the cross-sectional and longitudinal association between vitamin K status and knee OA structural features measured using magnetic resonance imaging (MRI). METHODS: Plasma phylloquinone (PK, vitamin K1) and dephosphorylated-uncarboxylated MGP ((dp)ucMGP) were measured in 791 older community-dwelling adults who had bilateral knee MRIs (mean ± SD age = 74 ± 3 y; 67% female). The adjusted odds ratios (and 95% confidence intervals) [OR (95%CI)] for presence and progression of knee OA features according to vitamin K status were calculated using marginal models with generalized estimating equations (GEEs), adjusted for age, sex, body mass index (BMI), triglycerides and other pertinent confounders. RESULTS: Longitudinally, participants with very low plasma PK (<0.2 nM) were more likely to have articular cartilage and meniscus damage progression after 3 years [OR (95% CIs): 1.7(1.0-3.0), 2.6(1.3-5.2) respectively] compared to sufficient PK (≥ 1.0 nM). Higher plasma (dp)ucMGP (reflective of lower vitamin K status) was associated with higher odds of meniscus damage, osteophytes, bone marrow lesions, and subarticular cysts cross-sectionally [ORs (95% CIs) comparing highest to lowest quartile: 1.6(1.1-2.3); 1.7(1.1-2.5); 1.9(1.3-2.8); 1.5(1.0-2.1), respectively]. CONCLUSION: Community-dwelling men and women with very low plasma PK were more likely to have progression of articular cartilage and meniscus damage. Plasma (dp)ucMGP was associated with presence of knee OA features but not progression. Future studies are needed to clarify mechanisms underlying vitamin Ks role in OA.

Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women.

UNLABELLED: We have investigated whether low-dose vitamin K2 supplements (menaquinone-7, MK-7) could beneficially affect bone health. Next to an improved vitamin K status, MK-7 supplementation significantly decreased the age-related decline in bone mineral density and bone strength. Low-dose MK-7 supplements may therefore help postmenopausal women prevent bone loss. INTRODUCTION: Despite contradictory data on vitamin K supplementation and bone health, the European Food Safety Authorities (EFSA) accepted the health claim on vitamin K's role in maintenance of normal bone. In line with EFSA's opinion, we showed that 3-year high-dose vitamin K1 (phylloquinone) and K2 (short-chain menaquinone-4) supplementation improved bone health after menopause. Because of the longer half-life and greater potency of the long-chain MK-7, we have extended these investigations by measuring the effect of low-dose MK-7 supplementation on bone health. METHODS: Healthy postmenopausal women (n = 244) received for 3 years placebo or MK-7 (180 µg MK-7/day) capsules. Bone mineral density of lumbar spine, total hip, and femoral neck was measured by DXA; bone strength indices of the femoral neck were calculated. Vertebral fracture assessment was performed by DXA and used as measure for vertebral fractures. Circulating uncarboxylated osteocalcin (ucOC) and carboxylated OC (cOC) were measured; the ucOC/cOC ratio served as marker of vitamin K status. Measurements occurred at baseline and after 1, 2, and 3 years of treatment. RESULTS: MK-7 intake significantly improved vitamin K status and decreased the age-related decline in BMC and BMD at the lumbar spine and femoral neck, but not at the total hip. Bone strength was also favorably affected by MK-7. MK-7 significantly decreased the loss in vertebral height of the lower thoracic region at the mid-site of the vertebrae. CONCLUSIONS: MK-7 supplements may help postmenopausal women to prevent bone loss. Whether these results can be extrapolated to other populations, e.g., children and men, needs further investigation.

Sensitivity of Clarireedia spp. to benzimidazoles and dimethyl inhibitors fungicides and efficacy of biofungicides on dollar spot of warm season turfgrass.

Dollar spot caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa) is an economically destructive fungal disease of turfgrass that can significantly compromise turf quality, playability, and aesthetic value. Fungicides are frequently used to manage the disease but are costly and potentially unfavorable to the environment. Repeated use of some active ingredients has resulted in reduced efficacy on C. jacksonii causing dollar spot in cool-season turfgrasses in the US. Experiments were conducted to study fungicide sensitivity of Clarireedia spp. as well as to develop alternatives to fungicides against dollar spot on warm-season turfgrass in Georgia. First, 79 isolates of Clarireedia spp. collected across the state were tested on fungicide-amended agar plates for their sensitivity to thiophanate-methyl (benzimidazole) and propiconazole (dimethyl inhibitor). Seventy-seven isolates (97.5%) were sensitive (0.001 to 0.654 µg/mL) and two isolates (2.5%) were found resistant (>1000 µg/mL) to thiophanate-methyl. However, in the case of propiconazole, 27 isolates (34.2%) were sensitive (0.005 to 0.098 µg/mL) while 52 isolates (65.8%) were resistant (0.101 to 3.820 µg/mL). Next, the efficacy of three bio- and six synthetic fungicides and ten different combinations were tested in vitro against C. monteithiana. Seven bio- and synthetic fungicide spray programs comprising Bacillus subtilis QST713 and propiconazole were further tested, either alone or in a tank mix in a reduced rate, on dollar spot infected bermudagrass 'TifTuf' in growth chamber and field environments. These fungicides were selected as they were found to significantly reduce pathogen growth up to 100% on in vitro assays. The most effective spray program in growth chamber assays was 100% B. subtilis QST713 in rotation with 75% B. subtilis QST713 + 25% propiconazole tank mix applied every 14 days. However, the stand-alone application of the biofungicide B. subtilis QST713 every seven days was an effective alternative and equally efficacious as propiconazole, suppressing dollar spot severity and AUDPC up to 75%, while resulting in acceptable turf quality (>7.0) in field experiments. Our study suggests that increased resistance of Clarireedia spp. to benzimidazoles and dimethyl inhibitors warrants continuous surveillance and that biofungicides hold promise to complement synthetic fungicides in an efficacious and environmentally friendly disease management program.

Undercarboxylated matrix Gla protein is associated with indices of heart failure and mortality in symptomatic aortic stenosis.

OBJECTIVE: Matrix Gla protein (MGP) is a calcification inhibitor and alterations in circulating MGP have been observed in different populations characterized by vascular calcification. We hypothesized that patients with calcific valvular aortic stenosis (AS) would have dysregulated circulating MGP levels. DESIGN AND SUBJECTS: We examined plasma levels of nonphosphorylated carboxylated and undercarboxylated MGP (dp-cMGP and dp-ucMGP, respectively) in 147 patients with symptomatic severe AS and in matched healthy controls. MAIN OUTCOME MEASURES: We further investigated the relationship between MGP levels and aortic pressure gradients and valve area by echocardiography and measures of heart failure. Finally, we assessed the prognostic value of elevated plasma dp-ucMGP level in relation to all-cause mortality in patients with AS. RESULTS: We found markedly enhanced plasma levels of dp-cMGP and in particular of dp-ucMGP in patients with symptomatic AS. Although only weak correlations were found with the degree of AS, circulating dp-ucMGP was associated with cardiac function and long-term mortality in multivariate analysis. CONCLUSIONS: A dysregulated MGP system may have a role in the development of left ventricular dysfunction in patients with symptomatic AS.

Calcinosis in juvenile dermatomyositis: a possible role for the vitamin K-dependent protein matrix Gla protein.

OBJECTIVES: The aims of the present study were to investigate whether the calcification inhibitor matrix Gla protein (MGP) is expressed in muscle biopsies of patients with juvenile dermatomyositis (JDM), and whether different forms of MGP are differentially expressed in JDM patients with and without subcutaneous calcifications. METHODS: Muscle tissue from six JDM patients (three without calcinosis, two with calcinosis and one recently diagnosed patient), four patients with muscular dystrophy, three patients with IBM and five normal histological control subjects was used for immunohistochemistry staining using novel antibodies to different conformations of MGP. RESULTS: In the JDM patients, all forms of MGP [non-carboxylated MGP (ucMGP), carboxylated MGP (cMGP), non-phosphorylated MGP (serMGP) and phosphorylated MGP (pserMGP)] were more intensely stained in the perifascicular compared with the central muscle fibres. In addition, these MGP species were demonstrated in the pathological muscle fibres of IBM and dystrophy patients, but hardly in normal histological muscle tissue. In JDM patients with calcifications, only pserMGP was increased compared with those without calcifications. All forms of MGP were also found in various staining intensities in the microvasculature and macrophages of normal histological and disease biopsies. CONCLUSIONS: MGP was expressed at the site of muscle damage in JDM patients as well as in patients with muscular dystrophy and IBM. The difference in staining intensity of pserMGP appeared to distinguish between JDM patients with and without calcifications, whereas cMGP, the other functional form, was equally expressed.

Extremes in vitamin K status of bone are related to bone ultrasound properties in children with juvenile idiopathic arthritis.

OBJECTIVE: Osteopenia is a common complication of juvenile idiopathic arthritis (JIA). In adults, low bone density and increased fracture risk are associated with low vitamin K status of bone. The vitamin K-dependent protein osteocalcin plays an important role in bone metabolism. Its activity depends upon post-translational carboxylation in which vitamin K is an essential co-factor. Hence, vitamin K deficiency leads to under-carboxylated (i.e., inactive) osteocalcin (ucOC). Little is known about the vitamin K status and bone health in children with juvenile idiopathic arthritis (JIA). We studied the vitamin K status of bone and its association with bone mass properties in children with JIA compared to healthy children. METHODS: We performed a cross sectional study in 55 children with JIA and 54 healthy controls between 6-18 years of age. Bone markers, ultrasound bone mass properties and vitamin K status of bone were determined. RESULTS: Overall, no differences in vitamin K status of bone were found between the study groups. Among children with JIA, a high ratio of ucOC/cOC indicating low vitamin K status was associated with low bone ultrasound parameters, whereas children with a high vitamin K status had markedly higher bone properties. This association was independent of physical activity, age, gender and BMI. CONCLUSION: These results suggest that vitamin K may be one of multiple risk factors for low bone mass in children with JIA, in addition to other recognized determinants of bone mass. The question remains whether JIA patients would benefit from increased dietary vitamin K intake.

Vitamin K-induced effects on body fat and weight: results from a 3-year vitamin K2 intervention study.

BACKGROUND/OBJECTIVES: Vitamin K status has been linked to fat and glucose metabolism by several authors, but whether high vitamin K intake influences body weight or composition has remained unclear. Here we tested the hypothesis that increased vitamin K intake decreases body fat or fat distribution. SUBJECTS/METHODS: In a randomized placebo-controlled human intervention trial, 214 postmenopausal women, 55-65 years of age, received either 180 mcg/day of vitamin K2 (menaquinone-7, MK-7) or placebo for 3 years. Osteocalcin (OC) carboxylation was used as a marker for vitamin K status, and fat distribution was assessed by dual-energy X-ray absorptiometry total body scan. RESULTS: In the total cohort, MK-7 supplementation increased circulating carboxylated OC (cOC) but had no effect on body composition. In those with an above-median response in OC carboxylation ('good responders'), MK-7 treatment resulted in a significant increase in total and human molecular weight adiponectin and a decrease in abdominal fat mass and in the estimated visceral adipose tissue area compared with the placebo group and the poor responders. CONCLUSIONS: The fact that changes in body composition measures or markers for fat or glucose metabolism were not associated with changes in uncarboxylated OC (ucOC) does not support the assumption that ucOC stimulates fat metabolism in humans. Instead, high vitamin K2 intake may support reducing body weight, abdominal and visceral fat, notably in subjects showing a strong increase in cOC. A causal relation between the changes in cOC and body fat or distribution cannot be concluded from these data.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Reproducibility and relative validity of a food frequency questionnaire to estimate intake of dietary phylloquinone and menaquinones.

BACKGROUND/OBJECTIVES: This study aims to investigate the reproducibility and relative validity of the Dutch food frequency questionnaire (FFQ), to estimate intake of dietary phylloquinone and menaquinones compared with 24-h dietary recalls (24HDRs) and plasma markers of vitamin K status. SUBJECTS/METHODS: In a cross-sectional study among 63 men and 58 women, the FFQ was completed three times over a 1-year period and the reproducibility was calculated over these measurements. Twelve-monthly 24HDR were collected to estimate relative validity. In addition, the relative validity of the FFQ, compared with plasma phylloquinone and desphospho-uncarboxylated matrix Gla protein (dpucMGP), was assessed cross-sectionally among 507 postmenopausal women. RESULTS: Intraclass correlations showed a good reproducibility, with correlations ranging from 0.65 to 0.83. The relative validity for phylloquinone intake compared with 24HDR was lower for women (rs=0.28) than men (rs=0.40). The relative validity, compared with 24HDR, for intake of short-chain menaquinones were ranging between 0.30 and 0.34. Long-chain menaquinones showed good relative validity (rs=0.60-0.69). Plasma phylloquinone concentrations were weakly correlated with phylloquinone intake (rs=0.16 (0.07-0.24). Plasma dpucMGP was negatively but weakly correlated with phylloquinone intake (rs=-0.09 (-0.18; -0.01)) and long-chain menaquinones (rs=-0.13 (-0.21; -0.04)), but not with short-chain menaquinones (rs=-0.04 (-0.13; 0.05)). CONCLUSIONS: The FFQ is reproducible to rank subjects for phylloquinone and menaquinone intake.The relative validity of our FFQ, compared with 24HDR, to estimate intake of phylloquinone and short-chain menaquinones was low, but the relative validity for long-chain menaquinones was good. The relative validity of our FFQ, compared with plasma phylloquinone and dpucMGP, was relatively low for both phylloquinone and menaquinone intake.

Desphospho-uncarboxylated matrix Gla protein is associated with increased aortic stiffness in a general population.

Matrix Gla protein (MGP), a natural inhibitor of calcification, strongly correlates with the extent of coronary calcification. Vitamin K is the essential cofactor for the activation of MGP. The nonphosphorylated-uncarboxylated isoform of MGP (dp-ucMGP) reflects the status of this vitamin. We investigated whether there is an association between dp-ucMGP and stiffness of elastic and muscular-type large arteries in a random sample from the general population. In a cross-sectional design, we analyzed 1087 subjects from the Czech post-MONICA study. Aortic and femoro-popliteal pulse wave velocities (PWVs) were measured using a Sphygmocor device. Dp-ucMGP concentrations were assessed in freshly frozen samples by enzyme-linked immunosorbent assay methods using the InaKtif MGP iSYS pre-commercial kit developed by IDS and VitaK. Aortic PWV significantly (P<0.0001) increased across the dp-ucMGP quartiles. After adjustment for all potential confounders, aortic PWV independently correlated with dp-ucMGP (with beta coefficient (s.d.) 11.61 (5.38) and P-value=0.031). In a categorized manner, subjects in the top quartile of dp-ucMGP (⩾ 671 pmol l(-1)) had a higher risk of elevated aortic PWV, with corresponding adjusted odds ratio (95% confidence interval) 1.73 (1.17-2.5). In contrast, no relation between dp-ucMGP and femoro-popliteal PWV was found. In conclusion, increased dp-ucMGP, which is a circulating biomarker of vitamin K status and vascular calcification, is independently associated with aortic stiffness, but not with stiffness of distal muscular-type arteries.

Steady-state vitamin K2 (menaquinone-7) plasma concentrations after intake of dairy products and soft gel capsules.

BACKGROUND: In a previous human intervention study, we observed an improved vitamin K status after 8 weeks of intake of a yogurt that was fortified with vitamin K2 (as menaquinone-7, MK-7) and enriched with vitamins C and D3, magnesium and polyunsaturated fatty acids. It was hypothesized that the added nutrients contributed to this improvement. Here we report on a study in which we compared the fasting plasma concentrations of MK-7 from (a) yogurt enriched with MK-7, vitamins D3 and C, magnesium, n-3 poly unsaturated fatty acids (n-3 PUFA) and fish oil (yogurt Kplus), (b) yogurt fortified with MK-7 only (yogurt K) and (c) soft gel capsules containing only MK-7. SUBJECTS/METHODS: For 42 days, healthy men and postmenopausal women between 45 and 65 years of age daily consumed either yogurt K, yogurt Kplus or capsules. Circulating MK-7, 25-hydroxy vitamin D (25(OH)D) and markers for vitamin K status (uncarboxylated osteocalcin (ucOC) and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP)) were assessed. Plasma MK-7 was also measured during the washout period of 2 weeks. MK-7 and dp-ucMGP were measured in citrated plasma, and 25(OH)D3 and ucOC were measured in the serum. RESULTS: The increase in plasma MK-7 with the yogurt Kplus product was more pronounced than the increase in MK-7 with the capsules. Circulating dp-ucMGP and ucOC were significantly lowered after consumption of the yogurt products and the MK-7 capsules, reflecting vitamin K status improvement. No significant differences in fasting plasma concentrations of various biomarkers between the yogurts were found. CONCLUSIONS: Dairy matrix and nutrient composition may affect MK-7 delivery and improvement of vitamin K status. Yogurt fortified with MK-7 is a suitable matrix to improve the nutritional status of the fat-soluble vitamins.

Effect of Vitamin K on Vascular Health and Physical Function in Older People with Vascular Disease--A Randomised Controlled Trial.

BACKGROUND AND AIMS: Vitamin K insufficiency is common and linked to an increased risk of cardiovascular disease and osteoporotic fractures. The aim of this study was to examine whether daily supplementation with oral vitamin K could improve vascular health and physical function in older people with established vascular disease. METHODS AND RESULTS: A double blind, randomised, placebo-controlled trial. Participants aged ≤ 70 years with a history of vascular disease were randomised to receive 6 months of daily oral 100mcg vitamin K2 (MK7 subtype) or matching placebo with outcomes measured at 0, 3 and 6 months. The primary outcome was between-group difference in endothelial function assessed using flow-mediated dilatation of the brachial artery at 6 months. Secondary outcomes included carotid-radial pulse wave velocity, augmentation index, blood pressure, carotid intima-media thickness, C-reactive protein, B-type natriuretic peptide, cholesterol and desphospho-uncarboxylated matrix Gla protein levels. Handgrip strength and the Short Physical Performance Battery assessed physical function, while postural sway was measured using a 3-dimensional force platform. RESULTS: 80 participants were randomised, mean age 77 (SD 5) years; 44/80 were male. Vitamin K levels rose in the intervention arm compared to placebo (+48 pg/ml vs -6 pg/ml, p=0.03) at 6 months. Desphospho-uncarboxylated Matrix Gla protein levels fell in the intervention group compared to placebo at 6 months (-130 [SD 117] pmol/L vs +13 [SD 180] pmol/L, p<0.001). No change was seen in endothelial function (between group difference -0.3% [95%CI -1.3 to 0.8], p=0.62). A modest, non-significant improvement in pulse wave velocity was seen in the vitamin K group (-0.8m/s [95%CI -1.8 to 0.3], p=0.15) while all other vascular and physical function outcomes unchanged. CONCLUSIONS: Six months of vitamin K2 supplementation did not improve markers of vascular health or physical function in older patients with vascular disease.

In vitro prothrombin synthesis from a purified precursor protein. II. Partial purification of bovine carboxylase.

In this paper, we describe the isolation and partial purification of an enzyme system that converts bovine decarboxyfactor II (PIVKA-II) into prothrombin (factor II). It is shown that the increase in factor II activity occurs in parallel with 14CO2 incorporation into BaSO4 adsorbable proteins. The system is not strictly vitamin K-dependent because it is obtained from the livers of normal healthy cows. By preincubating the enzyme(s) with an excess of warfarin, an absolute vitamin K1-dependence can be obtained. The reaction is inhibited by its own product, factor II.

Tissue distribution of K-vitamers under different nutritional regimens in the rat.

Two forms of vitamin K [phylloquinone (K1) and menaquinone-4 (MK-4)] were added to vitamin K-deficient rat food in varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary requirements (MDR) to attain maximal prothrombin synthesis were determined to be 0.6 and 6-10 microg/g of food for K1 and MK-4, respectively. The difference between both vitamers could be explained by the limited hepatic accumulation of MK-4. Next, vitamin K was offered to rats at concentrations ranging between 0.6 and 3000 microg/g of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K1 and MK-4 was found in all tissues investigated, but both the absolute tissue concentration and the ratio between K1 and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no gamma-glutamylcarboxylase, the function of vitamin K in this tissue remains obscure. High tissue concentrations of MK-4 were also found in pancreas and testis after a diet containing K1 exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its mechanism are known.

Vitamin K-dependent carboxylation of poly-L-glutamate.

Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length.

Comparison of the vitamins K1, K2 and K3 as cofactors for the hepatic vitamin K-dependent carboxylase.

The vitamins phylloquinone (K1), menadione (K3) and various menaquinones (K2) were compared for their ability to serve as a cofactor for the hepatic vitamin K-dependent carboxylase. It was found that the cofactor activity of the menaquinones varied with the length of the aliphatic side-chain and showed an optimum at MK-3. Menadione was not active at all. The concentration required for half-maximal reaction velocity (K 1/2) was determined for the various menaquinones and decreased at increasing chain length. The K 1/2 value for MK-4 was 3-times lower than that for vitamin K1. Under our in vitro conditions both vitamin K1 and the K2 vitamins were rapidly metabolized into a mixture of the quinone, the hydroquinone and the epoxide form. The fact that at equilibrium the level of these three metabolites was independent of the starting material shows that the vitamin K cycle is operational for vitamin K1 as well as for K2.

The in vivo effects of acenocoumarol, phenprocoumon and warfarin on vitamin K epoxide reductase and vitamin K-dependent carboxylase in various tissues of the rat.

In rats the in vivo effects of a chronic low-dose treatment (+/- 60 micrograms/rat per day) with different coumarins (acenocoumarol, phenprocoumon and warfarin) on hepatic and non-hepatic vitamin K-dependent enzyme systems were compared. The plasma concentrations of the three coumarins differed largely but these differences were not reflected in the microsomal coumarin contents. The non-hepatic microsomes contained less than 20% of the coumarins found in liver microsomes. No substantial differences were observed between the following effects of the three anticoagulant treatments. The blood coagulation factor activities were about 10% of normal. The hepatic microsomal vitamin K epoxide reductase activity was diminished to about 35% of control values. The vitamin K epoxide reductase activities present in kidney, lung, spleen, testis and brain microsomes were less influenced by the coumarin treatments; activities ranged between 45 and 65% of normal. In the liver microsomes a 15-fold accumulation of non-carboxylated precursor proteins was found; in the non-hepatic microsomes this effect was less pronounced but still present. The hepatic vitamin K-dependent carboxylase activity was enhanced but the corresponding non-hepatic enzyme activities were slightly or not affected. In addition, the effects of a chronic low-dose warfarin treatment were compared with those after an acute high dose of the drug.

Nutritional vitamin K-intake and urinary gamma-carboxyglutamate excretion in the rat.

Using the rat as an experimental animal model we have found that prothrombin synthesis reaches its maximal level at a relatively low dietary vitamin K intake. At still higher vitamin K intakes, however, the urinary Gla-excretion was substantially increased, showing a different vitamin K requirement for liver and extrahepatic tissues. The increased urinary Gla-excretion was found for both phylloquinone and menaquinone-4, but not for menaquinone-8, which questions the bioavailability of higher menaquinones for extrahepatic tissues. A discrepancy was found between effects of nutritional vitamin K-deficiency and treatment with a vitamin K-antagonist (brodifacoum). With both regimens plasma prothrombin rapidly decreased to well below 10% of the starting values, but in case of K-deficiency urinary Gla had hardly decreased in 7 days, whereas after 3 days of brodifacoum treatment Gla-excretion had decreased to 17% of the starting values. An explanation for this observation is that prothrombin procoagulant activity does not decrease proportional to the prothrombin Gla-content, but that a wide range of undercarboxylated prothrombins have lost nearly all activity. During vitamin K-deficiency the remaining low levels of vitamin K would mainly give rise to undercarboxylated prothrombin, whereas during brodifacoum treatment only non-carboxylated prothrombin is formed. It seems plausible that in the latter case the urinary Gla originates from proteins with long half-life times, such as the bone Gla-proteins.

A comparison between vitamin K-dependent carboxylase from normal and warfarin-treated cows.

Detergent-solubilized microsomal preparations that catalyse the vitamin K-dependent gamma-carboxylation of glutamic acid residues in peptide and protein substrates, have been obtained from the livers of normal and warfarin-treated cows. The preparations from warfarin-treated animals contained more endogenous substrate than those from normal cows, but otherwise the two preparations were indistinguishable. The enzymes vitamin K reductase and gamma-glutamyl carboxylase, may function independently of each other in this system. They are, nevertheless, intimately linked in some way, so that the reduced vitamin K that is produced by the former enzyme can be used immediately by the latter.

Intestinal flora is not an intermediate in the phylloquinone-menaquinone-4 conversion in the rat.

To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinone-4 (MK-4) we investigated the tissue distribution of vitamin K in germ-free rats. The rats were made vitamin K deficient by feeding a vitamin K-free diet for 13 days. In a subsequent period of 6 days, phylloquinone and menadione were supplied via the drinking water in concentrations of 10 and 50 micromol l(-1). Menadione supplementation led to high levels of tissue MK-4, particularly in extrahepatic tissues like pancreas, aorta, fat and brain. Liver and serum were low in MK-4. Phylloquinone supplementation resulted in higher phylloquinone levels in all tissues when compared with vitamin K-deficient values. The main target organs were liver, heart and fat. Remarkably, tissue MK-4 levels were also higher after the phylloquinone supplementation. The MK-4 tissue distribution pattern after phylloquinone intake was comparable with that found after menadione intake. Our results demonstrate that the conversion of phylloquinone into MK-4 in extrahepatic tissues may occur in the absence of an intestinal bacterial population and is tissue specific. A specific function for extrahepatic MK-4 or a reason for this biochemical conversion of phylloquinone into MK-4 remains unclear thus far.