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disproportionately high LDL, then high total cholesterol is associated with increased heart disease risk. If, on the other hand, total cholesterol is high because HDL is high, then it does not have this association with increased heart disease risk. The process by which LDL contributes to atherosclerosis is not entirely clear. It is believed that LDLs are oxidatively modified during the development of atherosclerosis, resulting in alteration of their gross physical structure and chemical properties (32). This may be caused in vivo by free radical attack of the polyunsaturated free fatty acids which proceeds via a chain reaction (33). The extent of oxidation appears to be• influenced by the ratio of lipid components and antioxidant levels in the LDL of the individual and is thought to occur in three phases: an initial lag phase when endogenous LDL antioxidants such as vitamin E are consumed; a propagation phase with rapid oxidation of unsaturated fatty acids to lipid hydroperoxides; and a decomposition phase, when hydroperoxides are converted to reactive aldehydes (e.g., malondialdehyde and 4- hydroxynonenal) (34). Interaction of these aldehydes with positively charged epsilon-amino groups of lysine residues in the apolipoprotein B-100 (apo B-100) moiety renders the LDL more negatively charged, resulting in decreased affinity for LDL receptors and increased affinity for scavenger receptors (35), which in turn allows delivery of an excess of cholesteryl esters to target cells via a receptor-independent mechanism. This process, coupled with the fact that oxidized LDLs are cytotoxic ~ ~ 11 ~ 05 ~

,I .I as well as chemotactic for monocytes, probably explains to some degree how L'DL contributes to atherosclerosis (36-38). Although it has been suggested that cigarette smoking contributes to oxidation of LDL by releasing free radicals (39- 42), a contrary result has recently been demonstrated experimentally. Specifically, an aqueous cigarette smoke extract was reported to have antioxidant properties that actually inhibited the oxidative modification of LDL resulting from incubation with either copper or 2,21-azo-bis(2-amidinopropane) hydrochloride (43). These data are in the opposite direction from that predicted on the basis of the claim that ETS exposure contributes to lipid formation and atherosclerosis resulting from oxidation of LDL. Before any definitive conclusions can be drawn about whether ETS exposure is related to LDL, however, further analysis is required of other LDL subgroups as well as the influence of external factors on LDL levels. Many studies report greater , proportional elevations of inean apolipoprotein B (apo B) than LDL cholesterol ("LDL-C") in patients with clinical coronary heart disease (44, 45). This suggests that specific apolipoproteins, such as apo B, may be more strongly associated with atherosclerosis than LDL-C. Additionally, numerous studies have reported that HDL and LDL levels can be affected by diet, alcohol consumption, and physical activity (46-49). In one recent study, treatment of hypercholesteremic rats with ascorbate was associated with reductions in both HDL-C and LDL-C (50). 12

i1 I Accompanying the decrease in the cholesterol, triglycerides, and protein content of all plasma lipoproteins in ascorbate-treated rats was a marked modification of the apoprotein pattern of all lipoprotein classes, with an increase in apo E in LDL and a decrease of C, AI and B in VLDL-IDL and of apo C in LDL. By contrast, it was found that ascorbate induces an increase in C apoproteins and a decrease of E.and B apoprotein in HDL fractions. The need for further study is highlighted by the animal studies that Dr. Glantz invokes in support of his claim that ETS :. _ .. exposure contributes to atherosclerosis by promoting cholesterol induced lipid accumulation. Unrealistic and stressful exposure conditions in these animal studies introduce numerous confounding problems. Thus, these studies provide no insight into the biological connection, if any, between ETS exposure and lipid accumulation. Furthermore, these studies used either fresh sidestream smoke or aged mainstream smoke rather than ETS. As noted below, different forms of cigarette smoke are not comparable, particularly with regard to potential associations with effects on the cardiovascular system. Finally, these studies used unrealistically high doses of smoke. This is particularly significant given that CHD is a chronic disease with long latency periods and the studies investigated only short term exposure. The significance of cardiovascular measurements and/or changes as part of a short-term response to unrealistic 13

environmental exposures in relation to the eventual manifestation of CHD is open to serious question. In sum, there is little question that additional research needs to be performed before any definitive conclusions can be drawn about the relationship, if any, between ETS exposure and cholesterol in promoting atherosclerosis. Accordingly, Dr. Glantzf claim that ETS contributes to atherosclerosis by promoting cholesterol induced lipid accumulation is, at best, premature. 3. Platelet aggregation and thrombus formation 11 I Finally, Dr..Glantz contends that ETS exposure contributes to CHD by increasing platelet aggregation and thrombus formation. However, although it has been suggested that spontaneous and induced increases in platelet aggregability may - contribute to CHD, attempts to relate the effects of smoking to changes in platelet function have produced only conflicting results. Some investigators have reported a positive association between smoking and platelet adhesiveness and aggregability (51, 52) while others have failed to demonstrate any differences between smokers and non-smokers (53, 54). Moreover, those studies reporting positive associations suffer from experimental defects which render their results highly suspect. First, the studies did not control for diet. Plasma or serum lipids have been associated with changes in platelet aggregability. Vitamin E has an inhibiting effect an the platelet release reaction (55) and may also play an indirect 14

i ! I role in platelet function by influencing prostacyclin (56) and thromboxane (57) production. Foo et al. (54) recently studied habitual smoking in relation to whole blood platelet aggregation and production of prostacyclin and thromboxane AZ in young adult males under controlled dietary conditions. According to their data, the mean platelet aggregation was significantly lower in smokers than non-smokers. These results suggest that smoking does not directly enhance aggregation and may be associated with a reduction in platelet aggregability when diet is taken into account. Second, the few studies reporting positive associations between ETS exposure and platel:et aggregability were generally performed in vitro. Substantial differences have been observed in platelet aggregability, however, depending on whether reactions were performed in vitro or in vivo. Larsson et al. (58) studied platelet aggregability in healthy volunteers during mental stress and low- and high-dose..adrenaline infusion using ex vivo (filtragometry) and conventional in vitro (aggregometry) methods. Results of their experiments show that the conventional in vitro techniques are not representative of platelet aggregability in vivo. This difference between the results of in vitro and in vivo studies is likely due, in part, to the selection of less sensitive platelets in in vitro'studies, owing to greater loss or artifactual activation of platelets during blood sample preparation. Additionally, the difference in experimental 15

1 I I I results may also be related to the failure of in vitro studies to account for all of the complex processes that regulate platelet aggregability in vivo. For example, as noted earlier, it has been claimed that ETS exposure may impair the oxidation of LDL in the body (43). Recent studies suggest that oxidation of LDL may be one of the primary mechanisms contributing to platelet aggregation (59). Secause the in vitro studies fail to account for any influences on platelet aggregability by indirect mechanisms such as that involving LDL, they do not accurately reflect platelet function in vivo. When properly analyzed by using in vivo studies that control for dietary effects, an association between ETS exposure and platelet aggregability has not been reported. Finally, Dr. Glantz completely omits any discussion of the potential vasodilatory effects of ETS that may completely offset any vascular constriction resulting from increased platelet aggregation. NO, a cigarette smoke constituent (60), has recently been established as a key EDRF having a pivotal role in endothelial cell function_and in signal transduction. It is thus of interest that the inhaled gas phase of cigarette smoke has been reported to relax the pulmonary circulation in pigs in almost an identical fashion as NO (61-63). To a lesser extent, vasodilatory responses have also been associated with the particulate phase of cigarette smoke. In a recent study, the vasodilation was assessed during/following continuously administered cigarette smoke in concentrations 16

''I I I I 1 II relevant to normal smoking (64). The relative importance of nicotine versus other particulate phase constituents of cigarette smoke in counteracting the gas-phase induced pulmonary vasodilation was also examined. The study reported that unfiltered cigarette smoke induced variable responses in the pulmonary circulation whereas inhalation of filtered smoke was consistently associated with pulmonary vasodilation. The major part of the vasodilatory response was attributed to NO. This apparent effect of NO was partially opposed in the unfiltered smoke by the particulate phase (but not by nicotine) presumably through a mechanism involving the induction of sympathetic reflexes. In a somewhat related study, Murohara et al. (65) studied stable contraction of pig coronary artery rings, incubated in organ chambers with prostaglandin FZa, in the absence or presence of cigarette smoke extracts (CSE). They reported that CSE induced an initial contraction followed by a relaxation of the coronary artery rings. They proposed that the initial contraction may be, at least in part, mediated through the degradation of basally released EDRFs by superoxide anions derived from CSE. Taken as a whole, these studies suggest that if ETS exposure has any relationship with vascular tone, it is highly complex and poorly understood. Dr. Glantz' uncritical and oversimplified claim regarding a vasoconstrictive effect of ETS exposure, therefore, reflects a clear bias and lack of scientific candor that is incompatible with accepted scientific procedure. 17 I

III. CONCLUSION I I It has been suggested on the basis of epidemiological studies that prolonged exposure to ETS may increase the risk of CHD. Nonetheless, studies focusing on this issue fall far short of providing conclusive evidence for a causal association. Potential confounders may account for spurious positive results and the multifactorial nature of CHD often makes it difficult to determine which confounders are likely to be the most important. For this reason, examining and identifying the biological mechanisms that could account for the association, if any, between ETS exposure and CHD is of critical importance. Only in this way can scientists gain an accurate understanding of the potential significance of ETS exposure as a risk factor for this disease. As the discussion in this comment demonstrates, researchers are beginning to explore the biological processes which might be relevant to an association between ETS exposure and the cardiovascular system. Such studies may eventually enable us to appropriately evaluate whether there is a biological relationship between ETS exposure and adverse cardiovascular effects. At present, however, the results of these studies'are equivocal at best, with the findings varying both in direction and magnitude of association. Accordingly, whether ETS exposure has an adverse impact an the cardiovascular system and contributes to CHD is a question that can not be scientifically resolved on the basis of the data currently available. 18 I

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