Long-term follow-up of biomarkers of vascular calcification after switch from traditional hemodialysis to online hemodiafiltration.

Rapid progression of vascular calcification (VC) in hemodialysis (HD) patients is caused by several factors including inflammation and an imbalance between active inducers and inhibitors of VC. Growing evidence shows that online hemodiafiltration (ol-HDF), a combination of diffusive and convective solute transport, has positive effects on the uremic environment that affects patients on dialysis. However, we recently reported that serum 25-hydroxyvitamin D (25(OH)D) decreased after a switch from HD to ol-HDF. As a consequence of this finding, the present study was undertaken to investigate if inducers and inhibitors of VC (i.e. the inactive matrix Gla protein fractions dp-ucMGP and t-ucMGP, fetuin-A, Gla-rich protein (GRP), osteopontin (OPN), bone-specific alkaline phosphatase (BALP), and osteoprotegerin (OPG)) also are affected by ol-HDF. This non-comparative prospective study comprised 35 prevalent patients who were investigated 6, 12, and 24 months after their switch from HD to ol-HDF. Most patients had increased levels of the calcification inhibitors OPN and OPG; and of the inactive calcification inhibitor dp-ucMGP during the study period irrespective of the dialysis modality. BALP and t-ucMGP were mostly within the reference interval, but fetuin-A was mostly below the reference interval during the study period. OPN was significantly associated with BALP and parathyroid hormone, r = 0.62 and r = 0.65 (p < .001), respectively. In conclusion, in contrast to decreased 25(OH)D levels, no differences were found for any of the measured biomarkers of VC following the switch from HD to ol-HDF. Further studies are needed to elucidate how these biomarkers can contribute to calcification risk assessment.

Menaquinone Content of Cheese.

Vitamin K2 (menaquinone) concentrations were measured in a wide range of cheeses and the effects of fat content, ripening and origin of the cheeses were investigated. Moreover, the menaquinone content of cheese was compared with that of other foods known to contain vitamin K2. It was found that cheese and curd are the most important sources of long-chain menaquinones in the Western diet and, in general, hard cheeses are richer in menaquinones than soft cheeses. However, the actual menaquinone content varies substantially and is dependent on the type of cheese, the time of ripening, the fat content and the geographic area where the cheeses are produced. Given the fact that poor vitamin K status has been mentioned as a risk factor for cardiovascular disease and mortality, while there is no clear evidence for adverse cardiovascular effects of dairy fats, cheese should be considered as a recommendable component in a heart-healthy diet.

Inactive Matrix Gla Protein, Arterial Stiffness, and Endothelial Function in African American Hemodialysis Patients.

BACKGROUND: Matrix Gla protein (MGP) is a vascular calcification inhibitor dependent upon vitamin K for activation. Evidence suggests that elevated plasma inactive MGP levels (desphospho-uncarboxylated MGP, dp-ucMGP; indicating poorer vascular vitamin K status) are associated with greater cardiovascular disease (CVD) risk. Despite African Americans experiencing highest rates of kidney failure and CVD events, relationships between dp-ucMGP and CVD risk markers have not been examined in this population. We investigated vascular vitamin K status (via plasma dp-ucMGP) between African American hemodialysis (HD) patients and healthy controls, and the associations of dp-ucMGP with arterial stiffness and endothelial function in HD patients only. METHODS: In 37 African American HD patients and 37 age- and race-matched controls, plasma dp-ucMGP was measured by enzyme immunoassay as a marker of vascular vitamin K status. Carotid-femoral pulse wave velocity (PWV; arterial stiffness measurement) and brachial artery flow-mediated dilation (FMD; endothelial function measurement) were assessed by applanation tonometry and ultrasound, respectively, in HD patients only. RESULTS: Mean dp-ucMGP levels were 5.6 times higher in HD patients vs. controls (2,139 ± 1,102 vs. 382 ± 181 pmol/l, P < 0.01). Multiple linear regression, adjusting for age, sex, dialysis vintage, diabetes mellitus, CVD history, body mass index, and blood pressure, revealed that dp-ucMGP was independently related to PWV (standardized ß = 0.49) and FMD (standardized ß = -0.53) (both P < 0.01). CONCLUSIONS: Our data suggest that the higher plasma dp-ucMGP concentrations found in African American HD patients may be associated with greater arterial stiffness and endothelial dysfunction.

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo.

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.

The abnormal status of uncarboxylated matrix Gla protein species represents an additional mortality risk in heart failure patients with vascular disease.

BACKGROUND: Matrix Gla protein (MGP) is a natural inhibitor of tissue calcification. In a previous study, we observed the positive association between abnormal concentrations of uncarboxylated MGP species and increased mortality risk in stable vascular patients. We explore whether co-incidence of abnormal status of uncarboxylated MPG and heart failure (HF) affects the mortality risk. METHODS: We examined 799 patients (mean age 65.1 years) with stable vascular disease and followed them in a prospective study. Both, desphospho-uncarboxylated and total uncarboxylated MGP (dp-ucMGP or t-ucMGP) were quantified by pre-commercial ELISA assays. RESULTS: Elevated (>100 ng/L) circulating brain natriuretic peptide (BNP) and abnormal status of plasma uncarboxylated MGP species (i.e.: dp-ucMGP ≥ 977 pmol/L or t-ucMGP ≤ 2825 nmol/L) were all identified as robust predictors of all-cause 5-year mortality. However, their co-incidence represented a substantial additional risk. We observed the highest mortality risk in patients with elevated BNP plus high dp-ucMGP compared to those with normal BNP plus low dp-ucMGP; fully adjusted HRR's were 4.86 (3.15-7.49). Likewise, the risk was increased when compared with patients with elevated BNP plus low dp-ucMGP; HRR 2.57 (1.60-4.10). Similar result we observed when co-incidence of elevated BNP and low t-ucMGP was analyzed [corresponding HRR's were 4.16 (2.62-6.61) and 1.96 (1.24-3.12)]. CONCLUSIONS: The concomitant abnormality of uncarboxylated MGP and mild elevation of BNP leads in chronic patients with vascular disease to about two-fold increase of the relative mortality risk. We hypothesize that abnormal homeostasis of MGP is involved in the pathophysiology of HF.

Yogurt drink fortified with menaquinone-7 improves vitamin K status in a healthy population.

Population-based studies have shown an inverse association between dietary menaquinones (MK-n, vitamin K2) intake, coronary calcification and CHD risk, suggesting a potential role of vitamin K in vascular health. To date, the effects of increased menaquinone intake on (markers of) vascular health have been investigated using predominantly food supplements. Dairy products contain many essential nutrients and can serve as a good matrix for food fortification in order to support health. We were therefore interested to study the effects of a menaquinone-fortified yogurt drink (menaquinone as menaquinone-7 (MK-7); 28 µg MK-7/yogurt drink) on vitamin K status and markers of vascular health. The yogurt drink was also fortified with n-3 PUFA, vitamin D, vitamin C, Ca and Mg to support vascular and/or general health. Healthy men (n 32) and postmenopausal women (n 28) with a mean age of 56 (sd 5) years received either basic or fortified yogurt drink twice per d for 12 weeks. MK-7 was efficiently absorbed from the fortified yogurt drink. Levels of circulating MK-7 were significantly increased from 0·28 to 1·94 ng/ml. In accordance, intake of the fortified yogurt drink improved vitamin K status, as measured by significant decreases in uncarboxylated osteocalcin and desphospho-uncarboxylated matrix Gla-protein. No effects were, however, seen on markers of inflammation, endothelial dysfunction and lipid metabolism. In summary, consumption of a yogurt drink fortified with low doses of among others MK-7 for 3 months significantly improved vitamin K status in a healthy population.

Inactive Matrix Gla-Protein Is Associated With Arterial Stiffness in an Adult Population-Based Study.

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 7.87±2.10 m/s for PWV and 0.43±0.20 nmol/L for dp-ucMGP. PWV was positively associated with dp-ucMGP both before and after adjustment for sex, age, body mass index, height, systolic and diastolic blood pressure (BP), heart rate, renal function, low- and high-density lipoprotein, glucose, smoking status, diabetes mellitus, BP and cholesterol lowering drugs, and history of cardiovascular disease (P≤0.01). In conclusion, high levels of dp-ucMGP are independently and positively associated with arterial stiffness after adjustment for common cardiovascular risk factors, renal function, and age. Experimental studies are needed to determine whether vitamin K supplementation slows arterial stiffening by increasing MGP carboxylation.

Menaquinone-7 supplementation improves arterial stiffness in healthy postmenopausal women. A double-blind randomised clinical trial.

Observational data suggest a link between menaquinone (MK, vitamin K2) intake and cardiovascular (CV) health. However, MK intervention trials with vascular endpoints are lacking. We investigated long-term effects of MK-7 (180 µg MenaQ7/day) supplementation on arterial stiffness in a double-blind, placebo-controlled trial. Healthy postmenopausal women (n=244) received either placebo (n=124) or MK-7 (n=120) for three years. Indices of local carotid stiffness (intima-media thickness IMT, Diameter end-diastole and Distension) were measured by echotracking. Regional aortic stiffness (carotid-femoral and carotid-radial Pulse Wave Velocity, cfPWV and crPWV, respectively) was measured using mechanotransducers. Circulating desphospho-uncarboxylated matrix Gla-protein (dp-ucMGP) as well as acute phase markers Interleukin-6 (IL-6), high-sensitive C-reactive protein (hsCRP), tumour necrosis factor-α (TNF-α) and markers for endothelial dysfunction Vascular Cell Adhesion Molecule (VCAM), E-selectin, and Advanced Glycation Endproducts (AGEs) were measured. At baseline dp-ucMGP was associated with IMT, Diameter, cfPWV and with the mean z-scores of acute phase markers (APMscore) and of markers for endothelial dysfunction (EDFscore). After three year MK-7 supplementation cfPWV and the Stiffness Index ßsignificantly decreased in the total group, whereas distension, compliance, distensibility, Young's Modulus, and the local carotid PWV (cPWV) improved in women having a baseline Stiffness Index ß above the median of 10.8. MK-7 decreased dp-ucMGP by 50 % compared to placebo, but did not influence the markers for acute phase and endothelial dysfunction. In conclusion, long-term use of MK-7 supplements improves arterial stiffness in healthy postmenopausal women, especially in women having a high arterial stiffness.

The association between uncarboxylated matrix Gla protein and lipoprotein-associated phospholipase A2.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is independently associated with cardiovascular risk, probably via inflammatory activity in sclerotic plaque. We speculated whether Lp-PLA2 has a role in the aetiology of vascular calcifications, estimated from circulating uncarboxylated matrix Gla protein (MGP) species and whether we could find a potential interaction of Lp-PLA2 and MGP in terms of mortality. MATERIALS AND METHODS: We examined 798 patients (mean age 65.1 years) with stable vascular disease and followed them in a prospective study. Both, desphospho-uncarboxylated and total MGP (dp-ucMGP or t-ucMGP) were quantified by pre-commercial ELISA assays, developed by VitaK (Maastricht, The Netherland) RESULTS: Lp-PLA2 activity was independently positively associated with desphospho-uncarboxylated MGP (dp-ucMGP) [ß coeff = 0.098, p=0.006]. 1SD of Lp-PLA2 activity was associated with 37% increased risk (p=0.001) of elevated dp-ucMGP (≥977 pmol/L, top quartile). In the Cox proportional hazard model adjusted for conventional risk factors, the patients in the highest quartile of dp-ucMGP or lowest quintile of total-uncarboxylated ucMGP (<2660 nmol/L) had higher risk of all-cause mortality [HRR 2.79 (95% CI 1.97-3.94) and HRR 1.69 (95% CI 1.18-2.42), respectively]. We observed no effect of high Lp-PLA2 activity (≥195 nmol/min/mL) on total mortality. CONCLUSIONS: We assume that Lp-PLA2 is involved in vascular calcification and that dp-ucMGP is a more appropriate biomarker of residual risk than Lp-PLA2 itself.

Vitamin K status and mortality after kidney transplantation: a cohort study.

BACKGROUND: Vitamin K modulates calcification by activating calcification inhibitors such as matrix Gla protein (MGP). In kidney transplant recipients, vitamin K insufficiency is common, but implications for long-term outcomes are unclear. STUDY DESIGN: Single-center observational study with a longitudinal design. SETTING & PARTICIPANTS: 518 stable kidney transplant recipients; 56% men; mean age, 51±12 (SD) years; and a median of 6 (IQR, 3-12) years after kidney transplantation. FACTOR: Plasma desphosphorylated-uncarboxylated MGP (dp-ucMGP) levels, reflecting vitamin K status. OUTCOMES: All-cause mortality and transplant failure. RESULTS: At inclusion, median dp-ucMGP level was 1,038 (IQR, 733-1,536) pmol/L, with 473 (91%) patients having vitamin K insufficiency (defined as dp-ucMGP>500pmol/L). During a median follow-up of 9.8 (IQR, 8.5-10.2) years, 152 (29%) patients died and 54 (10%) developed transplant failure. Patients in the highest quartile of dp-ucMGP were at considerably higher mortality risk compared with patients in the lowest quartile (HR, 3.10; 95% CI, 1.87-5.12; P for trend<0.001; P for quartile 1 [Q1] vs Q4<0.001). After adjustment for potential confounders, including kidney function and exclusion of patients treated with a vitamin K antagonist, this association remained significant. Patients in the highest quartile also were at higher risk of developing transplant failure (HR, 2.61; 95% CI, 1.22-5.57; P for trend=0.004; P for Q1 vs Q4=0.01), but this association was lost after adjustment for baseline kidney function (HR, 1.20; 95% CI, 0.52-2.75; P for trend=0.6; P for Q1 vs Q4=0.7). LIMITATIONS: Although MGP exists as various species, only dp-ucMGP was measured. No data were available for vascular calcification as an intermediate end point. CONCLUSIONS: Vitamin K insufficiency, that is, a high circulating level of dp-ucMGP, is highly prevalent in stable kidney transplant recipients and is associated independently with increased risk of mortality. Future studies should address whether vitamin K supplementation may lead to improved outcomes after kidney transplantation.

Inactive matrix Gla protein is causally related to adverse health outcomes: a Mendelian randomization study in a Flemish population.

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 doubling of the nadir (1.43 and 0.97 µg/L, respectively). With higher dp-ucMGP, cardiovascular mortality log-linearly increased (hazard ratio for dp-ucMGP doubling, 1.14 [1.01-1.28]; P=0.027), but coronary events log-linearly decreased (0.93 [0.88-0.99]; P=0.021). dp-ucMGP levels were associated (P≤0.001) with MGP variants rs2098435, rs4236, and rs2430692. For non-cancer mortality and coronary events (P≤0.022), but not for total and cardiovascular mortality (P≥0.13), the Mendelian randomization analysis suggested causality. Higher dp-ucMGP predicts total, non-cancer and cardiovascular mortality, but lower coronary risk. For non-cancer mortality and coronary events, these associations are likely causal.

Association of vitamin K status with adiponectin and body composition in healthy subjects: uncarboxylated osteocalcin is not associated with fat mass and body weight.

Osteocalcin (OC) is a vitamin K-dependent protein found in bone and in circulation. High serum γ-carboxylated OC reflects a high, and high uncarboxylated OC (ucOC) reflects a low vitamin K status. A revolutionary hypothesis is that ucOC acts as a hormone improving glucose handling and reducing fat mass. The objective was to test the logical extrapolation of the ucOC hormone hypothesis to humans that elevated ucOC is associated with higher body weight, BMI and fat mass. In a cross-sectional analysis, the associations of vitamin K status with circulating adiponectin and body composition were investigated in 244 postmenopausal women (study I). The effects of vitamin K treatment on adiponectin, body weight and BMI were investigated in archived samples from forty-two young men and women who received varying doses of menaquinone-7 during 12 weeks (study II) and from a cohort of 164 postmenopausal women who participated in a 3-year placebo-controlled trial on 45 mg menaquinone-4 (MK-4) (study III). No association was found between vitamin K status and circulating adiponectin before or after vitamin K supplementation. A higher carboxylation of OC was significantly correlated with lower body weight, BMI and fat mass of the trunk. Women taking MK-4 maintained their baseline body weight and BMI, whereas women taking placebo showed significant increases in both indices. These findings demonstrate that a high vitamin K status of bone has no effect on circulating adiponectin in healthy people and long-term vitamin K supplementation does not increase weight in healthy postmenopausal women.

Vitamin K-containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7.

Vitamin K is a cofactor in the production of blood coagulation factors (in the liver), osteocalcin (in bone), and matrix Gla protein (cartilage and vessel wall). Accumulating evidence suggests that for optimal bone and vascular health, relatively high intakes of vitamin K are required. The synthetic short-chain vitamin K(1) is commonly used in food supplements, but recently the natural long-chain menaquinone-7 (MK-7) has also become available as an over-the-counter (OTC) supplement. The purpose of this paper was to compare in healthy volunteers the absorption and efficacy of K(1) and MK-7. Serum vitamin K species were used as a marker for absorption and osteocalcin carboxylation as a marker for activity. Both K(1) and MK-7 were absorbed well, with peak serum concentrations at 4 hours after intake. A major difference between the 2 vitamin K species is the very long half-life time of MK-7, resulting in much more stable serum levels, and accumulation of MK-7 to higher levels (7- to 8-fold) during prolonged intake. MK-7 induced more complete carboxylation of osteocalcin, and hematologists should be aware that preparations supplying 50 mug/d or more of MK-7 may interfere with oral anticoagulant treatment in a clinically relevant way.

Novel conformation-specific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification.

OBJECTIVE: Matrix gamma-carboxyglutamic acid (Gla) protein (MGP), a vitamin K-dependent protein, is a potent in vivo inhibitor of arterial calcification. We hypothesized that low endogenous production of MGP and impaired carboxylation of MGP may contribute to the development or the progression of vascular disease. METHODS AND RESULTS: Novel conformation-specific antibodies against MGP were used for immunohistochemistry of healthy and sclerotic arteries. In healthy arteries, MGP was mainly displayed around the elastin fibers in the tunica media. The staining colocalized with that for carboxylated MGP, whereas undercarboxylated MGP (ucMGP) was not detected. In atherosclerotic arteries, ucMGP was found in the intima, where it was associated with vesicular structures. In Mönckeberg's sclerosis of the media, ucMGP was localized around all areas of calcification. The results indicate that ucMGP is strongly associated with vascular calcification of different etiologies. In a separate study, serum MGP concentrations in a cohort of 172 subjects who had undergone percutaneous coronary intervention were significantly reduced compared with an apparently healthy population. CONCLUSIONS: These data show that impaired carboxylation of MGP is associated with intimal and medial vascular calcification and suggest the essentiality of the vitamin K modification to the function of MGP as an inhibitor of ectopic calcification.

Characteristics and performance of an immunosorbent assay for human matrix Gla-protein.

BACKGROUND: Matrix gammacarboxyglutamate (Gla)-protein (MGP) is a strong inhibitor of soft tissue calcification and is mainly produced by chondrocytes and vascular smooth muscle cells (VSMCs). MGP-deficient mice have extensive calcifications of cartilage and arteries leading to osteopenia, fractures and blood vessel ruptures. Promotor polymorphisms resulting in decreased expression levels were found to be associated with an increased risk for cardiovascular disease in humans. METHODS: Recently, an ELISA-based assay has become available with which MGP may be detected in the circulation. The principle of the test kit is that of a competitive immunoassay using a monoclonal antibody against MGP bound to the microtiter plate. RESULTS: Here, we report on a critical evaluation of this assay and its potential diagnostic utility in diseases associated with the degeneration of the arterial vessel wall and cartilage. The biochemical performance of the kit is satisfactory, and significant differences were found between a number of patient cohorts and the reference population. Serum MGP concentrations were significantly decreased in patients with angina pectoris and in various cartilage diseases. CONCLUSIONS: The assay allows comparison of groups and may become a suitable marker for risk assessment or diagnosis in cardiovascular disease and osteoarthritis.

Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study.

Vitamin K-dependent proteins, including matrix Gla-protein, have been shown to inhibit vascular calcification. Activation of these proteins via carboxylation depends on the availability of vitamin K. We examined whether dietary intake of phylloquinone (vitamin K-1) and menaquinone (vitamin K-2) were related to aortic calcification and coronary heart disease (CHD) in the population-based Rotterdam Study. The analysis included 4807 subjects with dietary data and no history of myocardial infarction at baseline (1990-1993) who were followed until January 1, 2000. The risk of incident CHD, all-cause mortality, and aortic atherosclerosis was studied in tertiles of energy-adjusted vitamin K intake after adjustment for age, gender, BMI, smoking, diabetes, education, and dietary factors. The relative risk (RR) of CHD mortality was reduced in the mid and upper tertiles of dietary menaquinone compared to the lower tertile [RR = 0.73 (95% CI: 0.45, 1.17) and 0.43 (0.24, 0.77), respectively]. Intake of menaquinone was also inversely related to all-cause mortality [RR = 0.91 (0.75, 1.09) and 0.74 (0.59, 0.92), respectively] and severe aortic calcification [odds ratio of 0.71 (0.50, 1.00) and 0.48 (0.32, 0.71), respectively]. Phylloquinone intake was not related to any of the outcomes. These findings suggest that an adequate intake of menaquinone could be important for CHD prevention.

Factors affecting bone loss in female endurance athletes: a two-year follow-up study.

BACKGROUND: Low bone mass leading to stress fractures is a well-known and yet unsolved problem among female athletes. PURPOSE: To quantify the rate of bone loss in healthy female athletes and investigate the effects of estrogen and vitamin K supplementation on bone loss. STUDY DESIGN: Prospective cohort study. METHODS: We classified 115 female endurance athletes into amenorrheic, eumenorrheic, or estrogen-supplemented groups and randomized them to receive either placebo or vitamin K(1). The bone mineral densities of the subjects' femoral neck and lumbar spine were measured at baseline and after 2 years. RESULTS: Bone mineral density in the lumbar spine remained constant, but bone density in the femoral neck had decreased significantly after 2 years in all three subgroups. The decrease was higher in amenorrheic (-6.5% +/- 4.0%) than in eumenorrheic (-3.2% +/- 4.1%) and estrogen-supplemented athletes (-3.9% +/- 3.1%). Supplementation with vitamin K did not affect the rate of bone loss. CONCLUSIONS: The rate of bone loss in all three subgroups of female athletes was unexpectedly high; neither estrogen nor vitamin K supplementation prevented bone loss. CLINICAL RELEVANCE: High-intensity training maintained over several years must be regarded in women as a risk factor for osteoporosis, and protocols for optimal treatment should be developed.