Cees Vermeer - Academia.edu (original) (raw)

Papers by Cees Vermeer

BioMed Research International, 2014

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1985

Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of war... more Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of warfarintreated cows. Their enzymatic activities were compared with substrate-free carboxylase in microsomes from normal cows and substrate-bound carboxylase in microsomes from warfarin-treated cows. A number of exogenous substrates for carboxylase have been purified and tested. We found that large substrates, such as descarboxyprothrombin, are carboxylated only by substrate-free carboxylase and not by the substrate-bound enzyme. No differences in apparent K m values between solid-phase carboxylases II and X were observed.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2001

In the present study, we examined the expression of regulators of bone formation and osteoclastog... more In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification. (Arterioscler Thromb Vasc Biol.

Nephrology Dialysis Transplantation, 2013

Haemodialysis patients suffer from accelerated vascular calcification. The vitamin K-dependent ma... more Haemodialysis patients suffer from accelerated vascular calcification. The vitamin K-dependent matrix Gla protein (MGP) is one of the most powerful inhibitors of vascular calcification. Haemodialysis patients have high levels of the inactive form of MGP (desphosphorylated-uncarboxylated-MGP, dp-uc-MGP) and may benefit from pharmacological doses of vitamin K2 (menaquinone) to improve the calcification inhibitory activity of MGP. To determine the optimal dose of menaquinone-7 (MK-7) for MGP activation, 200 chronic haemodialysis patients were recruited to randomly receive 360, 720 or 1080 µg of MK-7 thrice weekly for 8 weeks. Dp-uc-MGP was measured at baseline and after 8 weeks. Dietary intake of vitamin K1 (phylloquinone) and menaquinone was estimated based on a detailed questionnaire. At baseline, dp-uc-MGP was not associated with phylloquinone intake (P = 0.92), but correlated inversely with menaquinone intake (P = 0.023). MK-7 supplementation dose dependently reduced dp-uc-MGP. The levels decreased by 17, 33 and 46% in the respective groups. Drop-outs were mainly due to gastrointestinal side-effects related to the unpleasant smell of the tablets. Chronic haemodialysis patients have high levels of inactive MGP, possibly related to a low dietary vitamin K intake. Pharmacological doses of MK-7 dose-dependently reduce dp-uc-MGP. Menaquinone supplementation may be a novel approach to prevent vascular calcifications in chronic haemodialysis patients.

Meat Science, 2014

Meat is a natural source of vitamin K, a vitamin associated with reduced bone loss and prevention... more Meat is a natural source of vitamin K, a vitamin associated with reduced bone loss and prevention of osteoporosis. Whether vitamin K content varies between breeds and muscles in cattle is not known. In the present study, contents of vitamin K1 (phylloquinone) and K2 (menaquinone, MK) were analysed in three different muscles from steers of two different breeds, Norwegian Red and Jersey, respectively. Results showed that MK4 was the most dominant of the vitamin K2 analogues, while only traces were found of MK6 and MK7. Both breeds had higher levels of MK4 in M. biceps femoris (BF) and M. longissimus dorsi (LD) compared to M. psoas major (PM). The results also showed significantly higher MK4 levels in muscles from Jersey compared to Norwegian Red. Furthermore, MK4 was not associated with intramuscular fat, suggesting a physiological role for MK4 in skeletal muscle cells. There were no association between vitamin K content and tenderness.

Hypertension, Jan 18, 2015

Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor ... more Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor of mortality. Matrix Gla-protein (MGP) is a vascular calcification inhibitor that needs vitamin K to be activated. Inactive MGP, known as desphospho-uncarboxylated MGP (dp-ucMGP), can be measured in plasma and has been associated with various cardiovascular markers, cardiovascular outcomes, and mortality. In this study, we hypothesized that high levels of dp-ucMGP are associated with increased PWV. We recruited participants via a multicenter family-based cross-sectional study in Switzerland. Dp-ucMGP was quantified in plasma by sandwich ELISA. Aortic PWV was determined by applanation tonometry using carotid and femoral pulse waveforms. Multiple regression analysis was performed to estimate associations between PWV and dp-ucMGP adjusting for age, renal function, and other cardiovascular risk factors. We included 1001 participants in our analyses (475 men and 526 women). Mean values were 7...

Hypertension, 2015

Matrix Gla-protein is a vitamin K-dependent protein that strongly inhibits arterial calcification... more Matrix Gla-protein is a vitamin K-dependent protein that strongly inhibits arterial calcification. Vitamin K deficiency leads to production of inactive nonphosphorylated and uncarboxylated matrix Gla protein (dp-ucMGP). The risk associated with dp-ucMGP in the population is unknown. In a Flemish population study, we measured circulating dp-ucMGP at baseline (1996-2011), genotyped MGP, recorded adverse health outcomes until December 31, 2012, and assessed the multivariable-adjusted associations of adverse health outcomes with dp-ucMGP. We applied a Mendelian randomization analysis using MGP genotypes as instrumental variables. Among 2318 participants, baseline dp-ucMGP averaged 3.61 μg/L. Over 14.1 years (median), 197 deaths occurred, 58 from cancer and 70 from cardiovascular disease; 85 participants experienced a coronary event. The risk of death and non-cancer mortality curvilinearly increased (P≤0.008) by 15.0% (95% confidence interval, 6.9-25.3) and by 21.5% (11.1-32.9) for a dou...

PLOS ONE, 2015

Vascular calcification is a common, serious and elusive complication of end-stage renal disease (... more Vascular calcification is a common, serious and elusive complication of end-stage renal disease (ESRD). As a pro-calcifying risk factor, non-thyroidal illness may promote vascular calcification through a systemic lowering of vascular calcification inhibitors such as matrix-gla protein (MGP) and Klotho. In 97 ESRD patients eligible for living donor kidney transplantation, blood levels of thyroid hormones (fT3, fT4 and TSH), total uncarboxylated MGP (t-ucMGP), desphospho-uncarboxylated MGP (dp-ucMGP), descarboxyprothrombin (PIVKA-II), and soluble Klotho (sKlotho) were measured. The degree of coronary calcification and arterial stiffness were assessed by means of cardiac CT-scans and applanation tonometry, respectively. fT3 levels were inversely associated with coronary artery calcification (CAC) scores and measures of arterial stiffness, and positively with dp-ucMGP and sKlotho concentrations. Subfractions of MGP, PIVKA-II and sKlotho did not associate with CAC scores and arterial stiffness. fT4 and TSH levels were both inversely associated with CAC scores, but not with arterial stiffness. The positive associations between fT3 and dp-ucMGP and sKlotho suggest that synthesis of MGP and Klotho is influenced by thyroid hormones, and supports a link between non-thyroidal illness and alterations in calcification inhibitor levels. However, the absence of an association between serum calcification inhibitor levels and coronary calcification/arterial stiffness and the fact that MGP and Klotho undergo post-translational modifications underscore the complexity of this association. Further studies, measuring total levels of MGP and membrane bound Klotho, should examine this proposed pathway in further detail.

The Biochemical journal, 1987

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction o... more Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 fr...

British Journal of Nutrition, 2002

The effect of maternal phylloquinone supplementation on vitamin K in breast milk was studied to e... more The effect of maternal phylloquinone supplementation on vitamin K in breast milk was studied to establish: (1) if phylloquinone is the source of menaquinone-4 in breast milk; (2) the doseeffect relationship between intake and obtainable levels. Four groups of lactating mothers with a full-term healthy infant participated and took oral phylloquinone supplements of 0·0 (n 8), 0·8 (n 8), 2·0 (n 8), and 4·0 (n 7) mg/d for 12 d, starting at day 4 post-partum. Milk samples were collected on days 4, 8, 16, and 19. Blood samples were collected on days 4 and 16. Vitamin K and vitamin E concentrations, the latter for reason of comparison, were assayed. Phylloquinone and menaquinone-4 were present in all milk samples: 5·84 (SD 2·31) and 2·98 (SD 1·51) nmol/l (n 31) respectively, in colostrum (day 4 sample). A strong correlation between the vitamers was found (r 0·78, P,0·001). Breast-milk phylloquinone levels were raised in a dose-dependent manner: 4-, 12-, and 30-fold on day 16 for the 0·8, 2·0, and 4·0 mg group respectively. In addition, menaquinone-4 levels were higher: 2·5-(P,0·05) and 7-fold (P,0·001) in the 2·0 and 4·0 mg groups respectively. Plasma of supplemented subjects contained 3-, 5-, and 10-fold higher phylloquinone levels on day 16. Detectable menaquinone-4 was found in ten of thirtyone day 4 plasma samples. All day 16 plasma samples of the 4 mg supplemented group contained the vitamin. There was no correlation between the K-vitamers in plasma. Vitamin E and phylloquinone appear to differ in their distribution in breast milk, milk:plasma concentration ratios were #1 and 3 -5 for vitamin E and phylloquinone respectively. The milk:plasma concentration ratio of menaquinone-4 was .10. In conclusion, dietary phylloquinone is a source of menaquinone-4 in breast milk. Phylloquinone supplementation to lactating mothers may be of benefit to the newborn infant, since both phylloquinone and menaquinone-4 are raised by supplementation.

Journal of Investigative Dermatology, 1986

Journal of Clinical Epidemiology, 2002

The risk of hemorrhage when using coumarin anticoagulants sharply increases when the Internationa... more The risk of hemorrhage when using coumarin anticoagulants sharply increases when the International Normalized Ratio (INR) is 3=6.0. We performed a case-control study among oulpatients of an anticoagulation clinic to identify sociodemographic-, lifestyle-, and dietary factors related to overanticoagulation. Three hundred cases with an INR 5=6.0 were compared with 302 randomly selected matched controls with an INR within the target zone. Age, sex, and level of education were not associated with overanticoagulation. Body mass index was negatively related to overanticoagulation, a beneath-average level of physical activity was positively related to overanticoagulation and never-smokers were more likely to have an INR 3=6.0 compared with smokers. Habitual alcohol consumption, even heavy drinking, was not related to overanticoagulation. However, a recent decreasc of alcohol intake increased the risk of an INR 3=6.0. In addition, weight loss and a vacation were risk factors for overanticoagulation. Dietary factors were not associated with overanticoaguation. If risk factors can not be avoided, increased monitoring of INR values could prevent overanticoagulation and potential bleeding complications.

Food & Nutrition Research, 2012

Vitamin K is essential for the synthesis of proteins belonging to the Gla-protein family. To the ... more Vitamin K is essential for the synthesis of proteins belonging to the Gla-protein family. To the members of this family belong four blood coagulation factors, which all are exclusively formed in the liver. The importance of vitamin K for hemostasis is demonstrated from the fact that vitamin K-deficiency is an acute, life-threatening condition due to excessive bleeding. Other members of the Gla-protein family are osteocalcin, matrix Gla-protein (MGP), and Gas6 that play key functions in maintaining bone strength, arterial calcification inhibition, and cell growth regulation, respectively. In total 17 Gla-proteins have been discovered at this time. Recently, it was observed that the dietary vitamin K requirement for the synthesis of the coagulation factors is much lower than for that of the extra-hepatic Gla-proteins. This forms the basis of the triage theory stating that during poor dietary supply, vitamins are preferentially utilized for functions that are important for immediate survival. This explains why in the healthy population all clotting factors are synthesized in their active form, whereas the synthesis of other Gla-proteins is sub-optimal in nonsupplemented subjects. Prolonged sub-clinical vitamin K deficiency is a risk factor for osteoporosis, atherosclerosis, and cancer. Present recommendations for dietary intake are based on the daily dose required to prevent bleeding. Accumulating scientific data suggests that new, higher recommendations for vitamin K intake should be formulated.

Food & Function, 2014

Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for c... more 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.

FEBS Letters, 1987

In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of p... more In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of proteinbound glutamic acid residues, is recycled by the action of one or more dithiol-dependent reductases. In vitro synthetic dithiols may serve as a cofactor for these enzymes, but the physiological reductant has not yet been found. In this paper we report that in vitro the commercially available thioredoxin/thioredoxin reductase from E. coli can replace the synthetic dithiols during the various reactions of the vitamin K cycle. Based on the assumption that in vivo thioredoxin also plays a role in the regeneration of vitamin K hydroquinone from the epoxide, an extension of the generally accepted vitamin K cycle is proposed.

Biochemical Pharmacology, 1995

AbstractRa1.s were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rig... more AbstractRa1.s were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rigorously preventing coprophagy. After one week, circulating prothrombin concentrations were between 5 and 10% of initial values, and various amounts of phylloquinone, menaquinone-4, and menaquinone-9 were given in a single dose either subcutaneously, orally, or colorectally. The relative 'vitamin K activities' of these compounds were assessed by comparing their ability to support prothrombin synthesis after subcutaneous injection. Intestinal and colonic absorption were deduced from the difference between subcutaneous and either oral or colorectal administration of the vitamers. It is concluded that the colonic absorption of all three forms of vitamin K is extremely poor. suggesting that physiological menaquinones in the colon do not contribute substantially to vitamin K status in rats. Furthermore, the stimulation of prothrombin synthesis by menaquinone-9 lasted much longer than that by the two other K-vitamers, resulting in a substantially higher 'vitamin K activity' of menaquinone-9.

Biochemical and Biophysical Research Communications, 1987

The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containin... more The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containing proteins of various origin. In this paper we demonstrate that: Gla-residues are required for the inhibitory activity; the inhibition is effected by a protein which in vivo is bound to calcified tissue (osteocalcin) as well as by proteins occurring in blood plasma (factor X) and urine (the urinary Gla-protein); The inhibitor concentration required for 50% precipitation-inhibition varied slightly from one salt to the other, but no marked differences were observed between the effects of the various Gla-containing proteins used; Precipitation-inhibition occurred in all phosphates (Be, Ca, Mn and Zn) and in all calcium salts (phosphate, oxalate and carbonate) tested.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

. To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinon... more . 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 mmol l y1 . 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. q 1998 Elsevier Science B.V.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may diffe... more Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone-quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K and MK-4 would be expected to influence the 1 stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones. q 1998 Elsevier Science B.V.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

x Two forms of vitamin K phylloquinone K and menaquinone-4 MK-4 were added to vitamin K-deficient... more x Two forms of vitamin K phylloquinone K and menaquinone-4 MK-4 were added to vitamin K-deficient rat food in 1 varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary Ž . requirements MDR to attain maximal prothrombin synthesis were determined to be 0.6 and 6-10 mgrg of food for K 1 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 mgrg of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K and MK-4 was found in all 1 tissues investigated, but both the absolute tissue concentration and the ratio between K and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no g-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 K exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its 1 mechanism are known. q 1998 Elsevier Science B.V.

BioMed Research International, 2014

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1985

Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of war... more Two types of solid-phase carboxylase, SPC-II and SPC-X, have been prepared from the livers of warfarintreated cows. Their enzymatic activities were compared with substrate-free carboxylase in microsomes from normal cows and substrate-bound carboxylase in microsomes from warfarin-treated cows. A number of exogenous substrates for carboxylase have been purified and tested. We found that large substrates, such as descarboxyprothrombin, are carboxylated only by substrate-free carboxylase and not by the substrate-bound enzyme. No differences in apparent K m values between solid-phase carboxylases II and X were observed.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2001

In the present study, we examined the expression of regulators of bone formation and osteoclastog... more In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification. (Arterioscler Thromb Vasc Biol.

Nephrology Dialysis Transplantation, 2013

Haemodialysis patients suffer from accelerated vascular calcification. The vitamin K-dependent ma... more Haemodialysis patients suffer from accelerated vascular calcification. The vitamin K-dependent matrix Gla protein (MGP) is one of the most powerful inhibitors of vascular calcification. Haemodialysis patients have high levels of the inactive form of MGP (desphosphorylated-uncarboxylated-MGP, dp-uc-MGP) and may benefit from pharmacological doses of vitamin K2 (menaquinone) to improve the calcification inhibitory activity of MGP. To determine the optimal dose of menaquinone-7 (MK-7) for MGP activation, 200 chronic haemodialysis patients were recruited to randomly receive 360, 720 or 1080 µg of MK-7 thrice weekly for 8 weeks. Dp-uc-MGP was measured at baseline and after 8 weeks. Dietary intake of vitamin K1 (phylloquinone) and menaquinone was estimated based on a detailed questionnaire. At baseline, dp-uc-MGP was not associated with phylloquinone intake (P = 0.92), but correlated inversely with menaquinone intake (P = 0.023). MK-7 supplementation dose dependently reduced dp-uc-MGP. The levels decreased by 17, 33 and 46% in the respective groups. Drop-outs were mainly due to gastrointestinal side-effects related to the unpleasant smell of the tablets. Chronic haemodialysis patients have high levels of inactive MGP, possibly related to a low dietary vitamin K intake. Pharmacological doses of MK-7 dose-dependently reduce dp-uc-MGP. Menaquinone supplementation may be a novel approach to prevent vascular calcifications in chronic haemodialysis patients.

Meat Science, 2014

Meat is a natural source of vitamin K, a vitamin associated with reduced bone loss and prevention... more Meat is a natural source of vitamin K, a vitamin associated with reduced bone loss and prevention of osteoporosis. Whether vitamin K content varies between breeds and muscles in cattle is not known. In the present study, contents of vitamin K1 (phylloquinone) and K2 (menaquinone, MK) were analysed in three different muscles from steers of two different breeds, Norwegian Red and Jersey, respectively. Results showed that MK4 was the most dominant of the vitamin K2 analogues, while only traces were found of MK6 and MK7. Both breeds had higher levels of MK4 in M. biceps femoris (BF) and M. longissimus dorsi (LD) compared to M. psoas major (PM). The results also showed significantly higher MK4 levels in muscles from Jersey compared to Norwegian Red. Furthermore, MK4 was not associated with intramuscular fat, suggesting a physiological role for MK4 in skeletal muscle cells. There were no association between vitamin K content and tenderness.

Hypertension, Jan 18, 2015

Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor ... more Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor of mortality. Matrix Gla-protein (MGP) is a vascular calcification inhibitor that needs vitamin K to be activated. Inactive MGP, known as desphospho-uncarboxylated MGP (dp-ucMGP), can be measured in plasma and has been associated with various cardiovascular markers, cardiovascular outcomes, and mortality. In this study, we hypothesized that high levels of dp-ucMGP are associated with increased PWV. We recruited participants via a multicenter family-based cross-sectional study in Switzerland. Dp-ucMGP was quantified in plasma by sandwich ELISA. Aortic PWV was determined by applanation tonometry using carotid and femoral pulse waveforms. Multiple regression analysis was performed to estimate associations between PWV and dp-ucMGP adjusting for age, renal function, and other cardiovascular risk factors. We included 1001 participants in our analyses (475 men and 526 women). Mean values were 7...

Hypertension, 2015

Matrix Gla-protein is a vitamin K-dependent protein that strongly inhibits arterial calcification... more Matrix Gla-protein is a vitamin K-dependent protein that strongly inhibits arterial calcification. Vitamin K deficiency leads to production of inactive nonphosphorylated and uncarboxylated matrix Gla protein (dp-ucMGP). The risk associated with dp-ucMGP in the population is unknown. In a Flemish population study, we measured circulating dp-ucMGP at baseline (1996-2011), genotyped MGP, recorded adverse health outcomes until December 31, 2012, and assessed the multivariable-adjusted associations of adverse health outcomes with dp-ucMGP. We applied a Mendelian randomization analysis using MGP genotypes as instrumental variables. Among 2318 participants, baseline dp-ucMGP averaged 3.61 μg/L. Over 14.1 years (median), 197 deaths occurred, 58 from cancer and 70 from cardiovascular disease; 85 participants experienced a coronary event. The risk of death and non-cancer mortality curvilinearly increased (P≤0.008) by 15.0% (95% confidence interval, 6.9-25.3) and by 21.5% (11.1-32.9) for a dou...

PLOS ONE, 2015

Vascular calcification is a common, serious and elusive complication of end-stage renal disease (... more Vascular calcification is a common, serious and elusive complication of end-stage renal disease (ESRD). As a pro-calcifying risk factor, non-thyroidal illness may promote vascular calcification through a systemic lowering of vascular calcification inhibitors such as matrix-gla protein (MGP) and Klotho. In 97 ESRD patients eligible for living donor kidney transplantation, blood levels of thyroid hormones (fT3, fT4 and TSH), total uncarboxylated MGP (t-ucMGP), desphospho-uncarboxylated MGP (dp-ucMGP), descarboxyprothrombin (PIVKA-II), and soluble Klotho (sKlotho) were measured. The degree of coronary calcification and arterial stiffness were assessed by means of cardiac CT-scans and applanation tonometry, respectively. fT3 levels were inversely associated with coronary artery calcification (CAC) scores and measures of arterial stiffness, and positively with dp-ucMGP and sKlotho concentrations. Subfractions of MGP, PIVKA-II and sKlotho did not associate with CAC scores and arterial stiffness. fT4 and TSH levels were both inversely associated with CAC scores, but not with arterial stiffness. The positive associations between fT3 and dp-ucMGP and sKlotho suggest that synthesis of MGP and Klotho is influenced by thyroid hormones, and supports a link between non-thyroidal illness and alterations in calcification inhibitor levels. However, the absence of an association between serum calcification inhibitor levels and coronary calcification/arterial stiffness and the fact that MGP and Klotho undergo post-translational modifications underscore the complexity of this association. Further studies, measuring total levels of MGP and membrane bound Klotho, should examine this proposed pathway in further detail.

The Biochemical journal, 1987

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction o... more Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 fr...

British Journal of Nutrition, 2002

The effect of maternal phylloquinone supplementation on vitamin K in breast milk was studied to e... more The effect of maternal phylloquinone supplementation on vitamin K in breast milk was studied to establish: (1) if phylloquinone is the source of menaquinone-4 in breast milk; (2) the doseeffect relationship between intake and obtainable levels. Four groups of lactating mothers with a full-term healthy infant participated and took oral phylloquinone supplements of 0·0 (n 8), 0·8 (n 8), 2·0 (n 8), and 4·0 (n 7) mg/d for 12 d, starting at day 4 post-partum. Milk samples were collected on days 4, 8, 16, and 19. Blood samples were collected on days 4 and 16. Vitamin K and vitamin E concentrations, the latter for reason of comparison, were assayed. Phylloquinone and menaquinone-4 were present in all milk samples: 5·84 (SD 2·31) and 2·98 (SD 1·51) nmol/l (n 31) respectively, in colostrum (day 4 sample). A strong correlation between the vitamers was found (r 0·78, P,0·001). Breast-milk phylloquinone levels were raised in a dose-dependent manner: 4-, 12-, and 30-fold on day 16 for the 0·8, 2·0, and 4·0 mg group respectively. In addition, menaquinone-4 levels were higher: 2·5-(P,0·05) and 7-fold (P,0·001) in the 2·0 and 4·0 mg groups respectively. Plasma of supplemented subjects contained 3-, 5-, and 10-fold higher phylloquinone levels on day 16. Detectable menaquinone-4 was found in ten of thirtyone day 4 plasma samples. All day 16 plasma samples of the 4 mg supplemented group contained the vitamin. There was no correlation between the K-vitamers in plasma. Vitamin E and phylloquinone appear to differ in their distribution in breast milk, milk:plasma concentration ratios were #1 and 3 -5 for vitamin E and phylloquinone respectively. The milk:plasma concentration ratio of menaquinone-4 was .10. In conclusion, dietary phylloquinone is a source of menaquinone-4 in breast milk. Phylloquinone supplementation to lactating mothers may be of benefit to the newborn infant, since both phylloquinone and menaquinone-4 are raised by supplementation.

Journal of Investigative Dermatology, 1986

Journal of Clinical Epidemiology, 2002

The risk of hemorrhage when using coumarin anticoagulants sharply increases when the Internationa... more The risk of hemorrhage when using coumarin anticoagulants sharply increases when the International Normalized Ratio (INR) is 3=6.0. We performed a case-control study among oulpatients of an anticoagulation clinic to identify sociodemographic-, lifestyle-, and dietary factors related to overanticoagulation. Three hundred cases with an INR 5=6.0 were compared with 302 randomly selected matched controls with an INR within the target zone. Age, sex, and level of education were not associated with overanticoagulation. Body mass index was negatively related to overanticoagulation, a beneath-average level of physical activity was positively related to overanticoagulation and never-smokers were more likely to have an INR 3=6.0 compared with smokers. Habitual alcohol consumption, even heavy drinking, was not related to overanticoagulation. However, a recent decreasc of alcohol intake increased the risk of an INR 3=6.0. In addition, weight loss and a vacation were risk factors for overanticoagulation. Dietary factors were not associated with overanticoaguation. If risk factors can not be avoided, increased monitoring of INR values could prevent overanticoagulation and potential bleeding complications.

Food & Nutrition Research, 2012

Vitamin K is essential for the synthesis of proteins belonging to the Gla-protein family. To the ... more Vitamin K is essential for the synthesis of proteins belonging to the Gla-protein family. To the members of this family belong four blood coagulation factors, which all are exclusively formed in the liver. The importance of vitamin K for hemostasis is demonstrated from the fact that vitamin K-deficiency is an acute, life-threatening condition due to excessive bleeding. Other members of the Gla-protein family are osteocalcin, matrix Gla-protein (MGP), and Gas6 that play key functions in maintaining bone strength, arterial calcification inhibition, and cell growth regulation, respectively. In total 17 Gla-proteins have been discovered at this time. Recently, it was observed that the dietary vitamin K requirement for the synthesis of the coagulation factors is much lower than for that of the extra-hepatic Gla-proteins. This forms the basis of the triage theory stating that during poor dietary supply, vitamins are preferentially utilized for functions that are important for immediate survival. This explains why in the healthy population all clotting factors are synthesized in their active form, whereas the synthesis of other Gla-proteins is sub-optimal in nonsupplemented subjects. Prolonged sub-clinical vitamin K deficiency is a risk factor for osteoporosis, atherosclerosis, and cancer. Present recommendations for dietary intake are based on the daily dose required to prevent bleeding. Accumulating scientific data suggests that new, higher recommendations for vitamin K intake should be formulated.

Food & Function, 2014

Vitamin K's recommended dietary allowance (RDA) is based on the hepatic requirement for c... more 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.

FEBS Letters, 1987

In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of p... more In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of proteinbound glutamic acid residues, is recycled by the action of one or more dithiol-dependent reductases. In vitro synthetic dithiols may serve as a cofactor for these enzymes, but the physiological reductant has not yet been found. In this paper we report that in vitro the commercially available thioredoxin/thioredoxin reductase from E. coli can replace the synthetic dithiols during the various reactions of the vitamin K cycle. Based on the assumption that in vivo thioredoxin also plays a role in the regeneration of vitamin K hydroquinone from the epoxide, an extension of the generally accepted vitamin K cycle is proposed.

Biochemical Pharmacology, 1995

AbstractRa1.s were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rig... more AbstractRa1.s were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rigorously preventing coprophagy. After one week, circulating prothrombin concentrations were between 5 and 10% of initial values, and various amounts of phylloquinone, menaquinone-4, and menaquinone-9 were given in a single dose either subcutaneously, orally, or colorectally. The relative 'vitamin K activities' of these compounds were assessed by comparing their ability to support prothrombin synthesis after subcutaneous injection. Intestinal and colonic absorption were deduced from the difference between subcutaneous and either oral or colorectal administration of the vitamers. It is concluded that the colonic absorption of all three forms of vitamin K is extremely poor. suggesting that physiological menaquinones in the colon do not contribute substantially to vitamin K status in rats. Furthermore, the stimulation of prothrombin synthesis by menaquinone-9 lasted much longer than that by the two other K-vitamers, resulting in a substantially higher 'vitamin K activity' of menaquinone-9.

Biochemical and Biophysical Research Communications, 1987

The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containin... more The precipitation of insoluble salts containing divalent metal ions is inhibited by Gla-containing proteins of various origin. In this paper we demonstrate that: Gla-residues are required for the inhibitory activity; the inhibition is effected by a protein which in vivo is bound to calcified tissue (osteocalcin) as well as by proteins occurring in blood plasma (factor X) and urine (the urinary Gla-protein); The inhibitor concentration required for 50% precipitation-inhibition varied slightly from one salt to the other, but no marked differences were observed between the effects of the various Gla-containing proteins used; Precipitation-inhibition occurred in all phosphates (Be, Ca, Mn and Zn) and in all calcium salts (phosphate, oxalate and carbonate) tested.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

. To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinon... more . 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 mmol l y1 . 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. q 1998 Elsevier Science B.V.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may diffe... more Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone-quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K and MK-4 would be expected to influence the 1 stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones. q 1998 Elsevier Science B.V.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1998

x Two forms of vitamin K phylloquinone K and menaquinone-4 MK-4 were added to vitamin K-deficient... more x Two forms of vitamin K phylloquinone K and menaquinone-4 MK-4 were added to vitamin K-deficient rat food in 1 varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary Ž . requirements MDR to attain maximal prothrombin synthesis were determined to be 0.6 and 6-10 mgrg of food for K 1 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 mgrg of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K and MK-4 was found in all 1 tissues investigated, but both the absolute tissue concentration and the ratio between K and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no g-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 K exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its 1 mechanism are known. q 1998 Elsevier Science B.V.