The mixture
was subjected to a brief centrifugation. 300 AI of the upper hexane phase
was removed and evaporated to dryness at room temperature. The residue
was dissolved in 100 Ml of isopropanol and 20 MI was analyzed by high
performance liquid chromatography (HPLC) using a Rainin gradient
system equipped with a Gilson 704 HPLC system manager. Separation
was achieved on a Rainin Microsorb Short C18 reversed-phase column
in 100% methanol. Absorbance of eluting materials was measured at
254 nm with an ISCO UM-5 detector equipped with an ISCO HPLC
10-Ml flow cell. Retention times for vitamin K1 and vitamin K, epoxide
were 5.8 and 3.6 min, respectively, at a flow rate of 2 ml/min. Quantitation
was based on the integrated absorption peaks when compared with peaks
from standards of vitamin K1 and vitamin K1 epoxide. Reduced vitamin
K1 was not measured in our system. Enzymatically reduced vitamin K1
was reoxidized to the quinone before being analyzed on HPLC. Reoxidation
was achieved by evaporation of the hexane layers in open tubes
over night at room temperature in the dark. Reoxidation of chemically
reduced vitamin K1 served as control. Reduced vitamin K1 (100 MM)
was added to control incubations. These were extracted immediately
with hexane, and these extracts were allowed to evaporate over night
together with the test samples. No nonenzymatic reduction of vitamin
K1 epoxide occurred in the hexane phases. Control samples from zero
time incubations contained no vitamin K1.