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.

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.

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.

Vitamin K status in healthy volunteers.

Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for clotting factor synthesis, but substantial concentrations of undercarboxylated extra-hepatic Gla-proteins are found in the circulation of non-supplemented individuals. This suggests that vitamin K intake above the RDA is required for an optimal extra-hepatic vitamin K status. Circulating uncarboxylated osteocalcin (ucOC) and desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP) are considered markers of the vitamin K status in bone and the vasculature, respectively. We measured these markers in 896 samples of healthy volunteers and defined target groups for vitamin K supplementation based on increased levels indicative of tissue-specific vitamin K deficiency. We studied the response to vitamin K supplements at different states of vitamin K deficiency by measuring the circulating dp-ucMGP level in samples from two short-term trials on menaquinone-7 (MK-7, vitamin K2) supplementation in 42 children and 68 adults. Children had high ucOC levels (3.4-96.9 ng ml(-1)); other age groups had values in the range of 1.5-5.0 ng ml(-1). From the age of 40 years, dp-ucMGP levels gradually increased. Children and adults with more pronounced vitamin K deficiency gave the highest responses to MK-7 supplementation. Children and adults above 40 years showed the largest tissue-specific vitamin deficiency and accordingly may benefit from MK-7 supplementation to improve their extra-hepatic vitamin K status.

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.

Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women.

UNLABELLED: Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K(2) intake promotes bone mineral density and bone strength. Results showed that K(2) improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K(2) intake may contribute to preventing postmenopausal bone loss. INTRODUCTION: Vitamin K is involved in the synthesis of several proteins in bone. The importance of K vitamins for optimal bone health has been suggested by population-based studies, but intervention studies with DXA-BMD as a clinical endpoint have shown contradicting results. Unlike BMC, DXA-BMD does not take into account the geometry (size, thickness) of bone, which has an independent contribution to bone strength and fracture risk. Here we have tested whether BMC and femoral neck width are affected by high vitamin K intake. METHODS: A randomized clinical intervention study among 325 postmenopausal women receiving either placebo or 45 mg/day of vitamin K(2) (MK-4, menatetrenone) during three years. BMC and hip geometry were assessed by DXA. Bone strength indices were calculated from DXA-BMD, femoral neck width (FNW) and hip axis length (HAL). RESULTS: K(2) did not affect the DXA-BMD, but BMC and the FNW had increased relative to placebo. In the K(2)-treated group hip bone strength remained unchanged during the 3-year intervention period, whereas in the placebo group bone strength decreased significantly. CONCLUSIONS: Vitamin K(2) helps maintaining bone strength at the site of the femoral neck in postmenopausal women by improving BMC and FNW, whereas it has little effect on DXA-BMD.

Vitamin K1 supplementation retards bone loss in postmenopausal women between 50 and 60 years of age.

Although several observational studies have demonstrated an association between vitamin K status and bone mineral density (BMD) in postmenopausal women, no placebo-controlled intervention trials of the effect of vitamin K1 supplementation on bone loss have been reported thus far. In the trial presented here we have investigated the potential complementary effect of vitamin K1 (1 mg/day) and a mineral + vitamin D supplement (8 microg/day) on postmenopausal bone loss. The design of our study was a randomized, double-blind, placebo-controlled intervention study; 181 healthy postmenopausal women between 50 and 60 years old were recruited, 155 of whom completed the study. During the 3-year treatment period, participants received a daily supplement containing either placebo, or calcium, magnesium, zinc, and vitamin D (MD group), or the same formulation with additional vitamin K1 (MDK group). The main outcome was the change in BMD of the femoral neck and lumbar spine after 3 years, as measured by DXA. The group receiving the supplement containing additional vitamin K1 showed reduced bone loss of the femoral neck: after 3 years the difference between the MDK and the placebo group was 1.7% (95% Cl: 0.35-3.44) and that between the MDK and MD group was 1.3% (95% Cl: 0.10-3.41). No significant differences were observed among the three groups with respect to change of BMD at the site of the lumbar spine. If co-administered with minerals and vitamin D, vitamin K1 may substantially contribute to reducing postmenopausal bone loss at the site of the femoral neck.

Evaluation of a bead-based enzyme immunoassay for the rapid detection of osteocalcin in human serum.

BACKGROUND: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin. METHODS: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS((R)) EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH(2) terminus. RESULTS: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 microg/L and a linear dose-response curve between 4.5 and 100 microg/L. The intraassay imprecision (CV) was 5-8% (n = 21); the interassay variation was 6-9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays. CONCLUSIONS: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.

Effect of oral anticoagulant treatment on markers for calcium and bone metabolism.

Vitamin K-dependent proteins regulate blood coagulation as well as bone growth and calcification. Here, we have compared the effects of oral anticoagulants on circulating vitamin K-dependent proteins and on markers for calcium and bone metabolism. Patients with a clinical indication for antithrombotic therapy were randomized into three groups and treated with either aspirin, regular-intensity anticoagulation [target international normalized ratio (INR) values: 2.5-3.5] or low-intensity anticoagulation (target INR values: 1.1-1.6). At the start and after 1 year of treatment, various biochemical markers were assessed. Both the circulating levels and the degree of carboxylation of the various gamma-carboxyglutamate (Gla)-containing proteins were affected differently by oral anticoagulant treatment. Circulating osteocalcin was more sensitive to poor vitamin K status than other Gla proteins. From the fact that - except for osteocalcin - neither markers for osteoblast nor osteoclast function were affected by oral anticoagulant treatment, we conclude that bone turnover remained unaltered, which is indicative of an unchanged rate of bone loss. Whether the long-term production of undercarboxylated bone Gla proteins may have a negative effect on the quality of bone (e.g. bone strength) cannot be concluded from this study.

Improved bone metabolism in female elite athletes after vitamin K supplementation.

In female elite athletes strenuous exercise may result in hypoestrogenism and amenorrhoea. As a consequence a low peak bone mass and rapid bone loss are often seen in relatively young athletes. In postmenopausal women, increased intake of vitamin K may result in an increase of serum markers for bone formation, a decrease of urinary markers for bone resorption, and a decrease in urinary calcium loss. In the present paper we report an intervention study among eight female athletes, four of whom had been amenorrhoeic for more than one year, whereas the others had been using oral contraceptives. All participants received vitamin K supplementation (10 mg/day) during one month, and various bone markers were measured before and after treatment. At baseline the athletes not using oral contraceptives were biochemically vitamin K-deficient as deduced from the calcium binding capacity of the circulating bone protein osteocalcin. In all subjects increased vitamin K was associated with an increased calcium-binding capacity of osteocalcin. In the low-estrogen group vitamin K supplementation induced a 15-20% increase of bone formation markers and a parallel 20-25% decrease of bone resorption markers. This shift is suggestive for an improved balance between bone formation and resorption.

Correlation of serum osteocalcin fractions with bone mineral density in women during the first 10 years after menopause.

Serum immunoreactive osteocalcin (irOC) consists of two fractions that differ from each other by their affinity for hydroxyapatite. The high and low affinity fractions are referred to as irOCbound and irOCfree, respectively. To evaluate whether these fractions are determinants for different characteristics of bone or bone metabolism, we have performed a cross-sectional study among 212 apparently healthy women between 20 and 90 years of age. Bone mineral density (BMD) was determined at the lumbar spine, and the right femur neck, trochanter, and Ward's triangle using dual-energy X-ray absorptiometry (DXA). Biochemical markers for bone formation and resorption were determined in serum and in urine. After classification according to menopausal age, an inverse correlation was found in the 1-10 years postmenopausal women between irOCfree and BMD, notably of the Ward's triangle and femur neck. It is concluded that in 1-10 years postmenopausal women, irOCfree is an independent marker for BMD, but that in other age groups the association is less clear or is absent.

Bone markers during a 6-month space flight: effects of vitamin K supplementation.

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.

Strategies for developing human osteocalcin standards: a critical evaluation.

Osteocalcin is a small protein uniquely produced by osteoblasts and odontoblasts. Since about 30% of the de novo synthesized osteocalcin is set free in the blood stream, it is widely used as a marker for bone formation. However, circulating immunoreactive osteocalcin (irOC) consists of several fractions, which may differ from each other with respect to size and calcium binding properties. Whereas it is generally assumed that the fraction with high affinity for hydroxyapatite reflects bone formation, we have found a high correlation between bone mass and irOC with low affinity for hydroxyapatite. Since the antibodies used in various commercial test kits for osteocalcin have different affinities for the various irOC fractions, well-defined homogenous standards have to be prepared which may be used for standardization of the detection techniques used in various laboratories. In this paper we give a critical evaluation of the strategies which may be followed.

Osteocalcin detection in aging serum and whole blood: stability of different osteocalcin fractions.

Human serum osteocalcin is a well known bone formation marker. On the basis of their different affinities for hydroxyapatite, the total immunoreactive osteocalcin may be separated into two fractions. Six commercial test kits for osteocalcin were compared. All kits reacted with both osteocalcin fractions but the absolute amounts found in the same serum samples differed widely. During serum storage at room temperature, there was no significant loss of osteocalcin during the first 6 h. After longer storage periods, the recorded decrease of osteocalcin depended on the system used: with two kits, over 80% of the original immunoreactive antigen was left after 9 days. It is considered that the different osteocalcin fractions may become useful as markers for different metabolic bone processes. A more precise definition of the various circulating osteocalcin fractions, and the development of separate tests for each fraction, are requirements for the optimal use of osteocalcin as a diagnostic tool for metabolic bone disorders.

Effects of vitamin K on bone mass and bone metabolism.

Vitamin K is involved in blood coagulation and in bone metabolism via the carboxylation of glutamate residues in (hepatic) blood coagulation factors and (osteoblastic) bone proteins. The bioavailability of nutritional vitamin K depends on the type of food, the dietary fat content, the length of the aliphatic side chain in the K-vitamer and probably also the genetically determined polymorphism of apolipoprotein E. Although undercarboxylation of blood coagulation factors is very rare, undercarboxylated osteocalcin (bone Gla-protein) is frequently found in postmenopausal women. Supplementation of these women with extra vitamin K causes the markers for bone formation to increase. In parallel, a decrease of the markers for bone resorption is frequently seen. Insufficient data are available to conclude that the regular administration of vitamin K concentrates will reduce the loss of bone mass in white women at risk for developing postmenopausal osteoporosis.

Role of vitamin K in bone metabolism.

Vitamin K is a cofactor required for the formation of gamma-carboxyglutamate (Gla) residues in proteins. Osteoblasts produce at least three different Gla-containing proteins: osteocalcin, matrix Gla-protein, and protein S. After cellular secretion of these proteins, the main part of each remains bound to the hydroxyapatite matrix in bone, but their function remains unclear. Part of the newly synthesized osteocalcin is also set free into the bloodstream, where it may be used as a diagnostic marker for bone formation. Several studies have demonstrated that a poor vitamin K status is associated with an increased risk of osteoporotic bone fractures. Whether vitamin K supplementation will reduce the rate of bone loss in postmenopausal women remains a matter of debate.

Vitamin K-induced changes in markers for osteoblast activity and urinary calcium loss.

The objective of this study was to identify subjects in whom vitamin K has an effect on markers for calcium and bone metabolism and to detect hitherto-unnoticed correlations between vitamin K-induced changes in these markers. Participants in our studies were apparently healthy women, in whom we measured serum-immunoreactive osteocalcin (irOC) before and after adsorption to hydroxylapatite; total serum alkaline phosphatase (T-AP) and bone-specific alkaline phosphatase (B-AP); and fasting urinary calcium and creatinine. We describe a trial among 145 women who were treated with vitamin K (1 mg/day) for 2 weeks, and a prospective placebo-controlled trial among two groups each of 70 postmenopausal women with a treatment period of 3 months. It turned out that in elderly women vitamin K induced increased levels of serum irOC with a high affinity for hydroxylapatite (irOCbound), whereas that with low affinity (irOCfree) remained unaffected. In placebo-treated women the ratio irOCfree/irOCbound shifted from 0.38 to 0.65 around the 50th year of age. This shift was not found in vitamin K-treated women. After 3 months of treatment the vitamin K-induced changes in irOCbound were correlated with changes in B-AP, whereas irOCfree was correlated to urinary calcium excretion. In fast losers of urinary calcium vitamin K induced a 30% decrease of calcium excretion. The hypothesis is put forward that irOCbound may be a marker for bone formation, that serum irOCfree may be a marker for bone resorption, and that the serum irOCfree/irOCbound ratio may become a marker for skeletal remodeling.(ABSTRACT TRUNCATED AT 250 WORDS)