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Abstract

A novel application of the liquid chromatography method combined with the triple quadrupole tandem mass spectrometry method was developed for the quantification of vitamin K1 and two forms of vitamin K2 (menaquinone-4, menaquinone-7) in human serum. Total chromatography time for each run was 9 min. Time required for the sample pretreatment procedures was approximately 4 h. The coefficients of variation (CVs) of intra-assay were 10.4%, 3.2 % and 2.3% for vitamin K1 in three levels of quality control samples; were 14.3%, 3.2% and 6.7% for menaquinone-4; and were 11.1%, 6.0% and 7.0% for menaquinone-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for vitamin K1; were 15.2%, 9.2% and 8.7% for menaquinone-4; and were 13.2%,11.1% and 7.2% for menaquinone-7. No interference was found between K1, menaquinone-4 and menaquinone-7, nor any deuterated internal standards. This method was then used to determine reference values for Caucasian populations of central European origin. Samples were measured from 191 healthy volunteers (51.2 ± 16.2 years (mean ± SD)) and the values concerning K1 were 0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. The values for menaquinone-4 and menaquinone-7 did not exhibit any differences between women and men, and were 0.050–1.598 and 0.074–0.759 ng/mL, respectively.

Subject and study design

Volunteers were determined to be healthy according to their medical history, physical examination and standard laboratory procedures. Samples were measured from 191 volunteers (51.2 ± 16.2 years (mean ± SD)), with 149 participants being female and 42 male. Ethical approval for this study was obtained from Ethics committee of University Hospital Motol (Ref.No.: EK-1422/13). Written informed consent was obtained from the participants at recruitment prior to their inclusion in the study.

Results

Our LC–MS/MS method for analysis of K1, MK-4 and MK-7 in human serum has been successfully validated following the criteria of the . Calibration curves were established by plotting the peak area ratios K1, MK-4 and MK-7 against IS at 10-point concentrations. Pooled serum samples exposed to UV light were spiked with 0.03, 0.05, 0.1, 0.3, 0.6, 1.0, 1.5, 2.0, 4.0 and 10.0 ng/mL to obtain calibration curves. The assays were linear across the whole range of concentrations with mean correlation coefficients R2 of 0.980 for K1, 0.994 for MK-4 and 0.978 for MK-7. The first point of calibration curves (0.03 ng/mL) corresponds to the lower limit of quantification (LLOQ). Limits of detection (LoD) were determined by a further dilution of the first calibration point (LLOQ). Concentrations of 0.0019 ng/mL for K1 and MK-7 and 0.00375 ng/mL for MK-4 were obtained.

Results

We measured the vitamin K levels in a healthy Central European population. The group of patients consisted of 191 volunteers, 22% of them male, ages 51 ± 16 years (mean ± SD). Serum levels of K1, MK-4 and MK-7 from the healthy Central European population were: 0.03–2.19 (0.20) ng/mL, 0.05–2.79 (0.19) ng/mL, and 0.05–1.39 (0.09) ng/mL, respectively (min—max (median)).

Methods

In this randomized, double‐blind, placebo‐controlled clinical trial, 84 PCOS patients were randomly assigned into the treatment (90 µg Menaquinone‐7 daily for 8 weeks) and placebo groups. Insulin resistance, lipid profile, endocrine biomarkers, and body composition of the participants were measured before and after the intervention. This study was performed in Ghadir Mother & Child Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran.

Results

Menaquinone‐7 supplementation, when compared to placebo, significantly decreased serum fasting insulin (p = .002), homeostasis model of assessment insulin resistance (p = .002), and homeostasis model of assessment β‐cell function (p = .02) in addition to a significant increase in quantitative insulin sensitivity check index (p = .001). Also, MK‐7 administration led to significant declines in serum triglyceride (p = .003) and dihydrotestosterone (DHT; p = .03) levels, free androgen index (p < .001), waist circumference (p = .03), and body fat mass (p < .001) as well as significant increases in skeletal muscle (p < .001) and sex hormone binding globulin (SHBG, p < .001).

Study design

Polycystic ovary syndrome participants were randomized into the control and treatment arms by block randomization with fixed block size of four by means of random allocation software (Saghaei, ). The treatment group consumed one vitamin K2 capsule, containing 90 µg of Menaquinone‐7 (MK‐7), daily for 8 weeks; the dosage was administered in a previous study (Razavi et al., ). Patients in the control group received a placebo capsule (avesil) daily for 8 weeks. The MK‐7 and placebo capsules were identical in color (white), smell, size (size 0), and weight which were prepared for this study by Arian Salamat Sina Company to blind the participants, researchers, and the Infertility Clinic staff to group allocation (Fermented soybean called natto was the food source of MK‐7 used in this study). A booklet containing PCOS dietary recommendation was given to both groups. The participants were requested not to change their regular physical activity level and diet and in the course of study.