Vitamin K is a cofactor for enzymatic modification of glutamic acid residues (Glu) to gammacarboxyglutamic acid residues (Gla) in vitamin K-dependent Gla proteins. These Gla proteins are necessary for hemostasis, bone metabolism, vascular calcification and cell proliferation. Vitamin K is found in nature as phylloquinone (vitamin K1) in green leafy vegetables, algae and some plant oils, and menaquinones (vitamin K2 also termed MK-n, where n is the number of isoprenoid units) can be found in meat, eggs and fermented food. A synthetic form of vitamin K menadione (vitamin K3) can be found as an additive in some animal feeds.
Simultaneous measurement the concentrations of 5 forms of vitamin K
Vitamin K1-Phylloquinone, Vitamin K1-2,3-Epoxide, Vitamin K2-7-Menaquinone, Vitamin K2-
2,3-Epoxide and Vitamin K2-4-Menaquinone
Pipette 400 µL of serum sample/working calibration standard into a glass centrifuge tube
Add 800 µL of Reagent 1, additionally vortex for 5 sec.
Add 25 µL of internal standard working stock solution, then 2 ml Reagent-2 and vortex for 3 sec.
Centrifuge at 4000 rpm for 5 min.
Transfer 2.4 ml of the upper phase to a new glass tube (tube-3) and evaporate under nitrogen stream. (Upper phase can be evaporated at 7 psi during ~15 min. using water bath at the temperature of 30°C)
Reconstitute tube-3 with 100 µl of Reagent-3 and transfer to a HPLC vial prior to injection