A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists.
The metabolism and antagonism of vitamin K has been studied in cultured fibroblasts. Monolayers of 3T3 mouse fibroblasts (grown in the absence or presence of warfarin or other putative antagonists) were incubated for 24 h with [1',2'-3H2]phylloquinone (K1) or [1',2'-3H2]phylloquinone epoxide (K1O), the cells harvested and lipid extracts fractionated by high performance liquid chromatography. [3H]K1 was converted to [3H]K1O (about 20% of [3H] lipids) and to unidentified polar metabolites (30%). [3H]K1O was converted to [3H]K1 (3%) and to polar metabolites (50%). Cells grown with warfarin showed a marked increase in the [3H]K1O:K1 ratio and in the proportion of polar metabolites. The metabolic interconversion of K1 and K1O and inhibitory response to warfarin provide evidence for a fibroblast pathway analogous to the vitamin K-epoxide cycle in the liver. From the K1O:K1 ratios it was possible to grade the antagonism of vitamin K epoxide reductase activity by known and suspected inhibitors. Inhibitory ratios were seen for racemic warfarin down to 10(-8) M. S-warfarin was a more potent antagonist than the R-enantiomer. Consistently low K1O:K1 ratios were observed for N-methyl-thiotetrazole and antibiotics with (moxalactam) or without (cefotaxime) this side chain suggesting that none of these compounds are direct inhibitors of vitamin K epoxide reductase. Fibroblasts grown in cell culture provide a useful model to study the extrahepatic role of vitamin K and the mode of action of vitamin K antagonists.
Ross
P J
PJ
Division of Haematology, United Medical School, Guy's Hospital, London.
Shearer
M J
MJ
Diplock
A T
AT
Schey
S A
SA
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D008954:Models, Biological; D010837:Vitamin K 1 / Q000031:analogs & derivatives / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology
C016186:vitamin K1 oxide; D014859:Warfarin; D010837:Vitamin K 1; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases