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Plasma antioxidant enzymes as markers of smoke injury

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Trudy Forte, Ph.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6IT-0437 Award: $123,073
Subject Area: Cardiovascular Disease
Award Type: Inno Dev & Exp Awards (IDEAS)

Initial Award Abstract
The proposed research examines the health related effects of cigarette smoking on heart disease. It is well known that smoking is a prime factor in contributing to heart disease but exactly how smoking contributes is not completely understood. It is now generally accepted that early events in heart disease, i.e., atherosclerosis, are initiated by oxidized low density lipoproteins (LDL) in the blood. Oxidative modification of LDL triggers a series of responses that include inflammation responses that subsequently result in the migration of certain white cells (monocytes) from the blood into the artery wall. There the monocytes are converted into macrophages which essentially sequester or take up the oxidized LDL. This leads to the formation of “foam” cells in the artery wall; accumulation of foam cells leads to fatty streak formation and continued insults result in atheromas which can be life threatening. Clearly, protection of LDL from oxidation is paramount to maintaining the integrity of these particles and reducing risk for atherosclerosis. However, cigarette smoke is known to contain oxidizing molecules and other components that can also stimulate oxidative bursts in the blood; both have the potential of oxidizing plasma LDL. The plasma also contains antioxidant enzymes that normally protect LDL from oxidant injury. Interestingly, some of these enzymes are actually associated with high density lipoprotein (HDL), which is known to be protective against heart disease.

The antioxidant enzymes of interest function by hydrolyzing (breaking down) the oxidized lipids which appear on LDL in the progression of oxidant injury. It is the oxidized lipids that initiate the cascade of events that lead to inflammation and foam cell formation. Our premise is that, if these enzymes are impaired as a consequence of cigarette smoking, LDL will be modified and will become atherogenic. To test this hypothesis, we plan to investigate the acute effects of cigarette smoking in a group of young smokers. The subjects will be asked to smoke 6 cigarettes over a 1 hour period and blood samples will be taken before and after that smoking period. The blood plasma will be assayed for antioxidant enzyme activity; in addition, we will directly quantify oxidized lipids on the LDL from smoking subjects and compare it with the nonsmoking state. A reduction in plasma antioxidant enzyme activity and an increase in LDL oxidized lipids will signal the likelihood that cigarette smoke has compromised these very important protective factors in the blood. These outcomes would predispose smokers to increased risk for cardiovascular disease. The results of these studies will provide new insights into mechanisms whereby smoking can contribute to heart disease. A demonstration of the deleterious effects of smoking on plasma antioxidant enzymes will enable physicians to better advise patients on the dangers of smoking to their health, particularly the cardiovascular system. This may serve as a deterrent to smoking. The information gleaned from these studies may also be useful for developing strategies for ameliorating the adverse health effects resulting from oxidation of LDL as a consequence of impaired antioxidant enzyme activity.

Final Report
The proposed research seeks to examine the health related effects of cigarette smoke (CS) on two plasma enzymes, platelet-activating factor acetylhydrolase (PAF-AH) and paraoxonase (PON) that putatively protect against in vivo oxidative events that result in inflammation and ultimately atherosclerosis. PAF-AH is associated primarily with low density lipoproteins (LDL), and PON with high density lipoproteins (HDL). It is not known whether cigarette smoking impairs the function of one or both of these enzymes in vivo; impairment of enzyme activity could potentially contribute to increased accumulation of oxidized lipids that stimulate inflammation thus initiating atherosclerosis. The major aims of this proposal are to determine (1) whether acute cigarette smoking impairs PAF-AH and PON activity in vivo and (2) whether the enzymes are sensitive to the gas phase of CS in vitro.

Acute in vivo cigarette smoking studies (4 to 6 cigarettes smoked in 1 hr) were carried out and effects on PON and PAF-AH activity analyzed. There were no significant differences in either PON or PAF-AH activities comparing the pre-smoking with the post-smoking samples (females, n=16; males, n=18). Preliminary studies suggest that cigarette smoking subtlely alters LDL since LDL isolated from post-smoking samples were able to inhibit LCAT activity compared to pre-smoking LDL. This finding suggests that smoking produces changes in LDL that inhibit LCAT activity. Small “hits” on LDL and LCAT over prolonged periods of cigarette use could potentially contribute to increased heart disease found in smokers.

To study CS effects on PON and PAF-AH in a better defined system, we carried out in vitro studies with plasma exposed to gas phase CS. The response of these enzymes to cigarette smoke was compared with lecithin cholesterol acyltransferase (LCAT) activity which is extremely sensitive to cigarette smoke and inhibited by the equivalent of one puff of CS. Gas phase of CS inhibited PAF-AH activity but not PON activity; LCAT activity, as expected, was also inhibited. From these studies we conclude that the atherogenic properties of CS may be related to: (1) an impairment of HDL cholesterol transport through the inactivation of LCAT and (2) the inhibition of PAF-AH, which normally reduces inflammatory responses. Reduction of PAF-AH activity could account for the functional modification of LDL isolated after one hour of smoking in the in vivo studies. Development of strategies and/or therapies to prevent LDL modification and PAF-AH impairment are potential future targets for counteracting the deleterious effects of cigarette smoke.

Gas-phase cigarette smoke inhibits platelet-activating factor acetylydrolase activity
Periodical: Circulation Index Medicus:
Authors: Bielicki JK, Lee BC, Knoff LJ, Forte TM ART
Yr: 1997 Vol: Suppl. 96 Nbr: 8 Abs: Pg: I-168

Gas-phase cigarette smoke inhibits platelet-activating factor acetylydrolase activity
Periodical: Circulation Index Medicus:
Authors: Bielicki JK, Lee BC, Knoff LJ, Forte TM ART
Yr: 1997 Vol: Suppl. 96 Nbr: 8 Abs: Pg: I-168