Vitamin K intake and status are low in hemodialysis patients.

Vitamin K is essential for the activity of γ-carboxyglutamate (Gla)-proteins including matrix Gla28 protein and osteocalcin; an inhibitor of vascular calcification and a bone matrix protein, respectively. Insufficient vitamin K intake leads to the production of non-carboxylated, inactive proteins and this could contribute to the high risk of vascular calcification in hemodialysis patients. To help resolve this, we measured vitamin K(1) and K(2) intake (4-day food record), and the vitamin K status in 40 hemodialysis patients. The intake was low in these patients (median 140 µg/day), especially on days of dialysis and the weekend as compared to intakes reported in a reference population of healthy adults (mean K(1) and K(2) intake 200 µg/day and 31 µg/day, respectively). Non-carboxylated bone and coagulation proteins were found to be elevated in 33 hemodialysis patients, indicating subclinical hepatic vitamin K deficiency. Additionally, very high non-carboxylated matrix Gla28 protein levels, endemic to all patients, suggest vascular vitamin K deficiency. Thus, compared to healthy individuals, hemodialysis patients have a poor overall vitamin K status due to low intake. A randomized controlled trial is needed to test whether vitamin K supplementation reduces the risk of arterial calcification and mortality in hemodialysis patients.

Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin K status, but had no effect on thrombin generation in healthy subjects.

Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteocalcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal conditions, 10-40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose-response effects of extra intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily dose of 10, 20, 45, 90, 180 or 360 µg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA (Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection against age-related diseases needs further investigation in specifically designed intervention trials.

Effect of vitamin K2 supplementation on functional vitamin K deficiency in hemodialysis patients: a randomized trial.

BACKGROUND: Vascular calcification is a predictor of cardiovascular morbidity and mortality. Hemodialysis patients experience severe vascular calcifications. Matrix Gla protein (MGP) is a central calcification inhibitor of the arterial wall; its activity depends on vitamin K-dependent γ-glutamate carboxylation. Uncarboxylated MGP, formed as a result of vitamin K deficiency, is associated with cardiovascular disease. Recent studies suggest poor vitamin K status in hemodialysis patients. We therefore aimed to investigate whether daily vitamin K supplementation improves the bioactivity of vitamin K-dependent proteins in hemodialysis patients, assessed by circulating dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and uncarboxylated prothrombin (PIVKA-II [protein induced by vitamin K absence II]). STUDY DESIGN: Interventional randomized non-placebo-controlled trial with 3 parallel groups. SETTING & PARTICIPANTS: 53 long-term hemodialysis patients in stable conditions, 18 years or older. 50 healthy age-matched individuals served as controls. INTERVENTIONS: Menaquinone-7 (vitamin K(2)) treatment at 45, 135, or 360 µg/d for 6 weeks. OUTCOMES: Plasma levels of dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and PIVKA-II. MEASUREMENTS: Plasma levels were assessed using enzyme-linked immunosorbent assays. RESULTS: At baseline, hemodialysis patients had 4.5-fold higher dephosphorylated-uncarboxylated MGP and 8.4-fold higher uncarboxylated osteocalcin levels compared with controls. PIVKA-II levels were elevated in 49 hemodialysis patients. Vitamin K(2) supplementation induced a dose- and time-dependent decrease in circulating dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and PIVKA-II levels. Response rates in the reduction in dephosphorylated-uncarboxylated MGP levels were 77% and 93% in the groups receiving 135 µg and 360 µg of menaquinone-7, respectively. LIMITATIONS: Small sample size. CONCLUSIONS: This study confirms that most hemodialysis patients have a functional vitamin K deficiency. More importantly, it is the first study showing that inactive MGP levels can be decreased markedly by daily vitamin K(2) supplementation. Our study provides the rationale for intervention trials aimed at decreasing vascular calcification in hemodialysis patients by vitamin K supplementation.

Circulating nonphosphorylated carboxylated matrix gla protein predicts survival in ESRD.

The mechanisms for vascular calcification and its associated cardiovascular mortality in patients with ESRD are not completely understood. Dialysis patients exhibit profound vitamin K deficiency, which may impair carboxylation of the calcification inhibitor matrix gla protein (MGP). Here, we tested whether distinct circulating inactive vitamin K-dependent proteins associate with all-cause or cardiovascular mortality. We observed higher levels of both desphospho-uncarboxylated MGP (dp-ucMGP) and desphospho-carboxylated MGP (dp-cMGP) among 188 hemodialysis patients compared with 98 age-matched subjects with normal renal function. Levels of dp-ucMGP correlated with those of protein induced by vitamin K absence II (PIVKA-II; r = 0.62, P < 0.0001). We found increased PIVKA-II levels in 121 (64%) dialysis patients, indicating pronounced vitamin K deficiency. Kaplan-Meier analysis showed that patients with low levels of dp-cMGP had an increased risk for all-cause and cardiovascular mortality. Multivariable Cox regression confirmed that low levels of dp-cMGP increase mortality risk (all-cause: HR, 2.2; 95% CI, 1.1 to 4.3; cardiovascular: HR, 2.7; 95% CI, 1.2 to 6.2). Furthermore, patients with higher vascular calcification scores showed lower levels of dp-cMGP. In 17 hemodialysis patients, daily supplementation with vitamin K2 for 6 weeks reduced dp-ucMGP levels by 27% (P = 0.003) but did not affect dp-cMGP levels. In conclusion, the majority of dialysis patients exhibit pronounced vitamin K deficiency. Lower levels of circulating dp-cMGP may serve as a predictor of mortality in dialysis patients. Whether vitamin K supplementation improves outcomes requires further study.

Characterisation and potential diagnostic value of circulating matrix Gla protein (MGP) species.

Matrix γ-carboxyglutamate (Gla) protein (MGP) is an important local inhibitor of vascular calcification, which can undergo two post-translational modifications: vitamin K-dependent γ-glutamate carboxylation and serine phosphorylation. While carboxylation is thought to have effects upon binding of calcium-ions, phosphorylation is supposed to affect the cellular release of MGP. Since both modifications can be exerted incompletely, various MGP species can be detected in the circulation. MGP levels were measured with two commercially available competitive and two novel sandwich assays in healthy controls, in patients with rheumatic disease, aortic valve disease, and end-stage renal disease, as well as in volunteers after vitamin K supplementation (VKS) and treatment with vitamin K antagonists (VKA). Major differences were found between the MGP assays, including significantly different behaviour with regard to vascular disease and the response to VKA and VKS. The dual-antibody assay measuring non-phosphorylated, non-carboxylated MGP (dp-ucMGP) was particularly sensitive for these changes and would be suited to assess the vascular vitamin K status. We conclude that the different assays for particular circulating MGP species allows the assessment of various aspects of the MGP system.

Relation of circulating Matrix Gla-Protein and anticoagulation status in patients with aortic valve calcification.

Matrix-Gla Protein (MGP) is a vitamin K-dependent protein acting as a local inhibitor of vascular calcification. Vitamin K-antagonists (oral anticoagulant; OAC) inhibit the activation of MGP by blocking vitamin K-metabolism. The aim of this study was to investigate the effect of long-term OAC treatment on circulating MGP levels in humans and on MGP expression in mice. Additionally, we tested the association between circulating inactive MGP (ucMGP) levels and the presence and severity of AVC in patients with aortic valve disease (AVD). We analysed circulating ucMGP levels in 191 consecutive patients with echocardiographically proven calcific AVD and 35 control subjects. The extent of AVC in the patients was assessed by multislice spiral computed tomography. Circulating ucMGP levels were significantly lower in patients with AVD (348.6 +/- 123.1 nM) compared to the control group (571.6 +/- 153.9 nM, p < 0.001). Testing the effect of coumarin in mice revealed that also the mRNA expression of MGP in the aorta was downregulated. Multifactorial analysis revealed a significant effect of glomerular filtration rate and long-term OAC therapy on circulating ucMGP levels in the patient group. Subsequently, patients on long-term OAC had significantly increased AVC scores. In conclusion, patients with calcific AVD had significantly lower levels of circulating ucMGP as compared to a reference population, free of coronary and valvular calcifications. In addition, our data suggest that OAC treatment may decrease local expression of MGP, resulting in decreased circulating MGP levels and subsequently increased aortic valve calcifications as an adverse side effect.

Association of kidney function and uncarboxylated matrix Gla protein: data from the Heart and Soul Study.

BACKGROUND: Vascular calcification is highly prevalent in persons with chronic kidney disease (CKD) and predicts cardiovascular disease (CVD) events. Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification, and lower levels of its precursor-uncarboxylated MGP (ucMGP)--are associated with vascular calcification and atherosclerosis. Whether mild to moderate decrements in kidney function are associated with lower serum ucMGP is unknown. METHODS: In a cross-sectional study among 842 outpatients with stable CVD, estimated glomerular filtration rate (eGFR), serum cystatin-C and urine albumin-to-creatinine ratio (ACR) were measured and serum ucMGP levels were determined by ELISA. Multivariate linear regression evaluated the association of each kidney function measure with serum ucMGP levels. RESULTS: The mean eGFR was 76 +/- 23 mL/min/1.73 m(2), and 186 subjects (22%) had moderate CKD (eGFR <60 mL/min/1.73 m(2)). The mean +/- SD ucMGP level was 3289 +/- 1177 nM. In unadjusted analysis, each 10 mL/ min/1.73 m(2) lower eGFR was associated with 101 nM lower ucMGP level. This association was only minimally attenuated in final multivariate models wherein each 10 mL/ min/1.73 m(2) lower eGFR was associated with 79 nM lower ucMGP (95% confidence interval [CI]; 44 to 115; P < 0.001) after adjustment for age, sex, race, body mass index, blood pressure, smoking, hypertension, diabetes; and serum albumin, calcium, phosphorus, and fetuin-A levels. Similarly, in models adjusted for identical covariates, each 0.1 mg/L higher cystatin-C was associated with 39 nM lower ucMGP (95% CI 23 to 55; P < 0.001). In contrast, no significant association was observed between ACR and ucMGP in either unadjusted or adjusted analyses (adjusted P = 0.17). All associations were similar among subjects with or without diabetes (P-values for interaction > 0.50). CONCLUSIONS: Among outpatients with stable CVD, a reduced glomerular filtration rate is associated with a decreased serum ucMGP level. In contrast, ACR is not associated with ucMGP levels. Whether ucMGP is a useful marker of vascular calcification and CVD event risk in persons with CKD deserves future study.

Uncarboxylated matrix Gla protein (ucMGP) is associated with coronary artery calcification in haemodialysis patients.

Matrix gamma-carboxyglutamate (Gla) protein (MGP) is a potent local inhibitor of cardiovascular calcification and accumulates at areas of calcification in its uncarboxylated form (ucMGP). We previously found significantly lower circulating ucMGP levels in patients with a high vascular calcification burden. Here we report on the potential of circulating ucMGP to serve as a biomarker for vascular calcification in haemodialysis (HD) patients. Circulating ucMGP levels were measured with an ELISA-based assay in 40 HD patients who underwent multi-slice computed tomography (MSCT) scanning to quantify the extent of coronary artery calcification (CAC). The mean ucMGP level in HD patients (193 +/- 65 nM) was significantly lower as compared to apparently healthy subjects of the same age (441 +/- 97 nM; p < 0.001) and patients with rheumatoid arthritis (RA) without CAC (560 +/- 140 nM; p < 0.001). Additionally, ucMGP levels correlated inversely with CAC scores (r = -0.41; p = 0.009), and this correlation persisted after adjustment for age, dialysis vintage and high-sensitivity C-reactive protein (hs-CRP). Since circulating ucMGP levels are significantly and inversely correlated with the extent of CAC in HD patients, ucMGP may become a tool for identifying HD patients with a high probability of cardiovascular calcification.

Matrix Gla-protein: the calcification inhibitor in need of vitamin K.

Among the proteins involved in vascular calcium metabolism, the vitamin K-dependent matrix Gla-protein (MGP) plays a dominant role. Although on a molecular level its mechanism of action is not completely understood, it is generally accepted that MGP is a potent inhibitor of arterial calcification. Its pivotal importance for vascular health is demonstrated by the fact that there seems to be no effective alternative mechanism for calcification inhibition in the vasculature. An optimal vitamin K intake is therefore important to maintain the risk and rate of calcification as low as possible. With the aid of conformation-specific antibodies MGP species in both tissue and the circulation have been detected in the healthy population, and significant differences were found in patients with cardiovascular disease (CVD). Using ELISA-based assays, uncarboxylated MGP (ucMGP) was demonstrated to be a promising biomarker for cardiovascular calcification detection. These assays may have potential value for identifying patients as well as apparently healthy subjects at high risk for CVD and/or cardiovascular calcification and for monitoring the treatment of CVD and vascular calcification.

The circulating inactive form of matrix Gla Protein (ucMGP) as a biomarker for cardiovascular calcification.

OBJECTIVE: Matrix gamma-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. METHODS AND RESULTS: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. CONCLUSION: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification.

Vitamin K: the coagulation vitamin that became omnipotent.

Vitamin K, discovered in the 1930s, functions as cofactor for the posttranslational carboxylation of glutamate residues. Gammacarboxy glutamic acid (Gla)-residues were first identified in prothrombin and coagulation factors in the 1970s; subsequently, extra-hepatic Gla proteins were described, including osteocalcin and matrix Gla protein (MGP). Impairment of the function of osteocalcin and MGP due to incomplete carboxylation results in an increased risk for developing osteoporosis and vascular calcification, respectively, and is an unexpected side effect of treatment with oral anticoagulants. It is conceivable that other side effects, possible involving growth-arrest-specific gene 6 (Gas6) protein will be identified in forthcoming years. In healthy individuals, substantial fractions of osteocalcin and MGP circulate as incompletely carboxylated species, indicating that the majority of these individuals is subclinically vitamin K-deficient. Potential new application areas for vitamin K are therefore its use in dietary supplements and functional foods for healthy individuals to prevent bone and vascular disease, as well as for patients on oral anticoagulant treatment to offer them protection against coumarin-induced side effects and to reduce diet-induced fluctuations in their INR values.