In Search of Normality for Vitamin K1: Establishing Age-Dependent Reference Intervals in the Danish Population.

BACKGROUND: A growing body of evidence suggests that vitamin K has beneficial effects on human health, especially cardiovascular and bone health. Vitamin K1 (phylloquinone), the predominant form of vitamin K in blood, is regarded as an indicator of vitamin K status, but to our knowledge no reference intervals (RIs) have been established for vitamin K1. METHODS: In this population-based study, vitamin K1 was measured in serum from 3808 Caucasian individuals without diabetes from 26 to 78 years of age. The need for gender- and age-partitioned vitamin K1 reference intervals was evaluated using Lahti's method, and exclusion criteria were defined to obtain as healthy a study group as possible. The excluded subgroups were tested for differences in mean serum vitamin K1 levels. Serum vitamin K1 levels were quantified using an in-house newly developed, validated, and highly sensitive online SPE-LC-MS/MS method with a limit of quantitation of (LOQ) 0.05 nmol/L. RESULTS: The reference interval for serum vitamin K1 was 0.22 to 3.95 nmol/L for individuals aged 26 to 44 years and 0.35 to 3.70 nmol/L for individuals aged 45 to 78. Similar age-specific reference intervals were established for vitamin K1-triglyceride ratio being 0.20 to 3.16 and 0.31 to 3.44, respectively. No significant difference was found between genders. Serum vitamin K1 was detectable in all serum samples. Individuals with known comorbidity were found to have significantly lower serum vitamin K1 compared to those without comorbidity. Current smokers had lower serum vitamin K1 compared to nonsmokers. CONCLUSION: Age-dependent reference intervals were established for serum vitamin K1 and vitamin K1-triglyceride ratio in a well-defined, healthy Caucasian population. Lower serum vitamin K1 levels were found in individuals with known comorbidity, suggesting an association between serum vitamin K1 and disease status. Further studies are needed to determine an optimal serum vitamin K1 level.

Quantitation of vitamin K1 in serum using online SPE-LC-MS/MS and the challenges of working with vitamin K.

Vitamin K1 (phylloquinone) is one of the vitamin Ks. Several studies have previously investigated the role of vitamin K1 status in respect to disease, but without consistent results. Since vitamin K deficiency has been associated with different disease states it is important to develop a biochemical analysis with sufficient sensitivity and a low limit of quantitation (LOQ). The vitamin Ks are very fat-soluble. This non-polar nature has given rise to several challenges during the method development, because vitamin K1 sticks to materials used during the process and is lost during evaporation. We found that reducing the sample preparation as much as possible offline, instead using online SPE-LC-MS/MS improves recovery and gives satisfactory chromatograms. An Protein BEH C4 column, 300 Š(50 × 2.1 mm, 1.7 µm particle size) was used as trap column and a Phenyl-Hexyl-LC-column, 100 Š(100 × 2.1 mm, 2.6 µm particle size) was used as analytical column. The mobile phases consisted of 30 µmol/L NH4F in water and 30 µmol/L NH4F in MeOH. A triple quadrupole tandem mass spectrometer with atmospheric pressure chemical ionization (APCI) ion source, positive ion mode, was used to perform the mass spectrometric measurements. The method is simple, highly sensitive and fast. The method was validated for vitamin K1 with good analytical performance. With a LOQ of 0.05 nmol/L it is to our knowledge the vitamin K1 method with lowest LOQ reported to date in the literature. It can easily be automated and applied in a routine diagnostic laboratory. Blood collection tubes with different additives were tested and showed no difference. Stability of vitamin K1 in serum was tested at different temperatures (-20 °C, 4 °C and in light and dark at 20 °C over a period of 30 days) and showed that vitamin K1 is light sensitive in serum even after only one day.