GAS-PHASE CIGARETTE-SMOKE INHIBITS PLASMA LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVITY BY MODIFICATION OF THE ENZYMES FREE THIOLS

Cigarette smoking is associated with an increased risk of premature at herosclerosis. The underlying mechanisms responsible for this associat ion are unknown. Recent work from this laboratory has shown that ex vi vo exposure of plasma to gas-phase cigarette smoke (CS) produces a rap id inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and mo dified HDL. When dialyzed human plasma (12 ml) was exposed to the gas- phase of an equivalent of 1/8 of a cigarette (one 'puff) at 15 min int ervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to con trols; supplementation of plasma with glutathione produced a dose-depe ndent protection of LCAT activity where at the highest concentration ( 1 mM) 78% protection was observed. A similar protection was obtained w ith N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apol ipoproteins were crosslinked after 3 h exposure of plasma to CS; cross linking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl argi nine, and aminoguanidine failed to protect LCAT and HDL indicating a s pecificity with regard to the ability of free thiols to buffer the del eterious components of CS which inhibited LCAT and crosslinked HDL-apo lipoproteins. Since LCAT contains two free cysteine residues (Cys-31 a nd -184) near the active site of the enzyme, we tested whether pretrea tment of plasma with the reversible sulfhydryl modifying compound, 5,5 '-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-ind uced inhibition. Plasma pretreated with DTNB prior to CS exposure reta ined an additional 50% of LCAT activity compared to CS treatment in th e absence of DTNB. These results indicate that covalent modification o f free cysteine residues of LCAT was, in part, responsible for the inh ibition of plasma LCAT activity by CS.