The vitamin K (VK) levels vary greatly among different populations and in different regions. Currently, there is a lack of reference intervals for VK levels in healthy individuals, The aim of this study is to establish and validate the reference intervals of serum vitamin K1 (VK1) and vitamin K2 (VK2, specifically including menaquinone-4 (MK4) and menaquinone-7 (MK7)) levels in some healthy populations in Beijing. Serum VK1, MK4, and MK7 were firstly measured by high-performance liquid chromatography and mass spectrometry in 434 subjects. The reference intervals for three indicators were established by calculating the data of 2.5 and 97.5 percentiles. Finally, preliminary clinical validation was conducted on 60 apparent healthy individuals undergoing physical examination. In the young, middle-aged, and elderly groups, the reference intervals of VK1 were 0.180 ng/mL ∼ 1.494 ng/mL, 0.247 ng/mL ∼ 1.446 ng/mL, and 0.167 ng/mL ∼ 1.445 ng/mL, respectively. The reference intervals of MK4 were 0.009 ng/mL ∼ 0.115 ng/mL, 0.002 ng/mL ∼ 0.103 ng/mL, and 0.003 ng/mL ∼ 0.106 ng/mL, respectively. The reference intervals of MK7 were 0.169 ng/mL ∼ 0.881 ng/mL, 0.238 ng/mL ∼ 0.936 ng/mL, and 0.213 ng/mL ∼ 1.012 ng/mL, respectively. The reference intervals had been validated by the samples of healthy individuals for physical examination. In conclusion, the reference intervals of VK established in this study with different age groups have certain clinical applicability, providing data support for further multicentre studies.

Menaquinone-7 (MK-7), a multipotent vitamin K2, possesses a wide range of biological activities, a precise curative effect and excellent safety. A simple and rapid LC-APCI-MS/MS method for the determination of MK-7 in human plasma with single liquid–liquid extraction (LLE) extraction and 4·5-min analysis time has been developed and validated. Four per cent bovine serum albumin (BSA) was used as surrogate matrix for standard curves and endogenous baseline subtraction. This method was reproducible and reliable and was used to analyse of MK-7 in human plasma. The endogenous circadian rhythm and bioavailability of MK-7 were investigated in two randomised single-dose, open, one-way clinical trials (Study I and Study II). A total of five healthy male subjects were enrolled in Study I and 12 healthy male subjects in Study II. Single-dose (1 mg) of MK-7 was given to each subject under fasting condition, and all eligible subjects were given a restricting VK2 diet for 4 d prior to drug administration and during the trial. The experiment results of Study I demonstrated that endogenous MK-7 has no circadian rhythm in individuals. Both studies showed MK-7 are absorbed with peak plasma concentrations at about 6 h after intake and has a very long half-life time.

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.

Menaquinone-7 (MK-7), a member of the vitamin K2 family, performs several functions, all related to its recognised effect on post-translational carboxylation of certain protein-bound glutamate residues. Due to its lipophilic structure MK-7 is soluble in olive oil, so the aim of the present study was to test whether extra-virgin (EV) olive oil enriched with MK-7 significantly increases MK-7 plasma levels and has an effect on osteocalcin and its carboxylation status. Healthy young volunteers (n 12) were administered 20 ml EV olive oil per d for 2 weeks, followed by 2 weeks of the same amount of olive oil enriched with 45 μg and then 90 μg MK-7, with an appropriate washout time in between. Blood was collected and plasma separated in each phase of the study. We found that integration of the diet with EV olive oil alone did not produce any significant variation of MK-7 plasma levels compared with baseline. Supplementation with MK-7-enriched olive oil resulted in a significant and dose-dependent increase in plasma levels. The high dose also significantly increased carboxylated osteocalcin (cOC) and decreased undercarboxylated osteocalcin (ucOC) plasma levels, resulting in a significant increase in the cOC:ucOC ratio. A significant correlation was also found between percentage variation of plasma cOCA:ucOC ratio and increase in plasma MK-7 levels. We conclude that regular consumption of MK-7-enriched olive oil may constitute a valid approach in order to preserve some key biochemical mechanisms controlling bone mineralisation.

Vitamin K contributes to bone health, probably through its role as cofactor in the carboxylation of osteocalcin. Intervention studies in adults have demonstrated that markedly higher osteocalcin carboxylation is obtained by intakes of vitamin K well above the current recommended dietary intake. However, the relationship between increased vitamin K2 intake and enhanced osteocalcin carboxylation has never been shown in healthy children. The objective was to study the effect of 45 μg menaquinone-7 (MK-7; one of the vitamin K2 species) on the circulating levels of undercarboxylated osteocalcin (ucOC) and carboxylated osteocalcin (cOC) in healthy prepubertal children. We hypothesised that MK-7 supplementation will reduce the ucOC:cOC ratio (UCR), indicating an improved vitamin K status. The present study is a double-blind randomised placebo-controlled trial examining the effect of 8 weeks MK-7 supplementation on the carboxylation of osteocalcin in healthy children (n 55). Serum levels of ucOC, cOC and MK-7 were measured at baseline and after 8 weeks, together with bone markers and coagulation parameters. The UCR was used as an indicator of vitamin K status. In the MK-7-supplemented group (n 28), the circulating concentration of inactive ucOC reduced and the UCR improved whereas the concentration of MK-7 increased. Within the placebo group, ucOC, cOC, UCR and MK-7 did not significantly change over time. In both groups, bone markers and coagulation parameters remained constant over time. These findings demonstrate that in healthy, prepubertal children, modest supplementation with MK-7 increases circulating concentrations of MK-7 and increases osteocalcin carboxylation.