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A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists
Author(s) -
Ross P. J.,
Shearer M. J.,
Diplock A. T.,
Schey S. A.
Publication year - 1991
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1991.tb07977.x
Subject(s) - vitamin k epoxide reductase , chemistry , reductase , vitamin , stereochemistry , antagonism , epoxide , biochemistry , warfarin , metabolism , enzyme , medicine , cyp2c9 , cytochrome p450 , receptor , atrial fibrillation , catalysis
Summary 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′‐ 3 H 2 ]phylloquinone (K 1 ) or [1′,2′‐ 3 H 2 ]phylloquinone epoxide (K 1 O), the cells harvested and lipid extracts fractionated by high performance liquid chromatography. [ 3 H]K 1 was converted to [ 3 H]K 1 O (about 20% of [ 3 H] lipids) and to unidentified polar metabolites (30%). [ 3 H]K 1 O was converted to [ 3 H]K 1 (3%) and to polar metabolites (50%). Cells grown with warfarin showed a marked increase in the [ 3 H]K 1 O:K 1 ratio and in the proportion of polar metabolites. The metabolic interconversion of K 1 and K 1 O and inhibitory response to warfarin provide evidence for a fibroblast pathway analogous to the vitamin K‐epoxide cycle in the liver. From the K 1 O:K 1 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 K 1 O:K 1 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.