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I ! ! II i n I m m ! ! ! ! im ! | m m ! had significantly elevated levels of 2,3-diphosphoglycerate (DPG), which suggests that the body is attempting to compensate for hypoxia by increasing DPG level in blood to meet tissue oxygen requirements." These sweeping remarks require a close examination of the data reported by Moskowitz etal. First of all, as noted by these investigators, the bematocrit values for the ETS-exposed and non-ETS exposed children were identical, raising doubt as to whether these children are anemic. Certainly, the weight and height of the ETS-exposed group do not support such a conclusion. With respect to the argument that the DPG increase supports the conclusion that the "body is attempting to compensate for hypoxia by increasing DPG," it must first be noted that the method used by Moskowitz etal. is one already described 65 years ago. The method of Fiske and SubbaRow which Moskowitz etal. applied for determining DPG is actually designed for the colorimetric measurement of phosphorus and is not specific at all for DPG. The principle of the method takes advantage Of the fact that sugar phosphates show quite different stabilities in acid; those which are hydrolyzed completely in 1 N sulfuric acid at 100 C during a 7-minute incubation are referred to as labile, while those which are resistant to hydrolysis under the same conditions are referred to as stable. A third class of sugar phosphates including DPG, ribose 1-phosphate, etc. show extra lability toward acid and may be estimated like inorganic phosphate (Leloir and Cardine, 1957). Concentrations of 2,3-DPG are more - ii -

Ii a I itI i I I a I I I I a ! il a ! specifically measured by the stoichiometric cleavage of 2,3-DPG to 3-phosphoglyceric acid by diphosphoglycerate kinase coupled with the sequential enzymatic conversion of the monophosphoglycerate to glycerol-3-phosphate and the Oxidation of NADH to NAD (Michal, 1974). The ETS-exposed twin group showed significant reduction in cholesterol level, a decrease in LDL concentration (especially in girls), and an excellent correlation of serum thiocyanate with the number of cigarettes smoked. Because serum tbiocyanate levels are poorly correlated with the measured cotinine levels and since thiocyanate is also known to be present in certain foods, especially leafy vegetables and some nuts (USPH8, 1986), it is possible that this group had significantly different nutritional and dietary habits as compared with the nonsmoking twin group. Such a possibility should be further evaluated as one of the confounding parameters in the future. Nutrient intake is expected to influence overall protein synthesis (Castro, 1987) and could regulate the synthesis of lipoproteins and other polypeptides involved in oxygen transport, which could give the same results as shown. Glantz and Parmley next review a number of animal studies dealing with mitochondrial ultrastructural and biochemical changes of rabbits and guinea pigs upon exposure to carbon monoxide. The carbon monoxide is administered singly for short (less than one hour), intermediate (2 weeks) and long periods - 12 -

I I I iI I i I I i I I I I i i I i I specifically measured by the stoichiometric cleavage of 2,3-DPG to 3-phosphoglyceric acid by diphosphoglycerate kinase coupled with the sequential enzymatic conversion of the monophosphoglycerate to glycerol-3-phosphate and the oxidation of NADN to NAD (Michal, 1974). The ETS-exposed twin group showed significant reduction in cholesterol level, a decrease in LDL concentration (especially in girls), and an excellent correlation of serum thiocyanate with the number of cigarettes smoked. Because serum thiocyanate levels are poorly correlated with the measured cotinine levels and since thiocyanate is also known to be present in certain foods, especially leafy vegetables and some nuts (USPHS, 1986), it is possible that this group had significantly different nutritional and dietary habits as compared with the nonsmoking twin group. Such a possibility should be further evaluated as one of the confounding parameters in the future. Nutrient intake is expected to influence OVerall protein synthesis (Castro, 1987) and could regulate the synthesis of lipoproteins and other polypeptides involved in Oxygen transport, which could give the same results as shown. Glantz and Parmley next review a number of animal studies dealing with mitochondrial ultrastructural and biochemical changes of rabbits and guinea pigs upon exposure to carbon monoxide. The carbon monoxide is administered singly for short (less than one hour), intermediate (2 weeks) and long periods - 12-

I I I i ! I ! I I I II i I i iI I I (more than 2 weeks). The structuEal changes in these animals appear real and significant. However, it is often difficult to extrapolate results of animal studies directly to humans, since different species may not react in an identical fashion to the same external challenge as hypoxia. For example, Bischoff et al. (1969) investigated myocardial ultrastructure in dogs, rabbits, and rats maintained at an altitude of 4,300 m for 5 months; the structural derangements in dogs and rabbits were similar to those found in cattle with high mountain disease, whereas the rats appeared to be only marginally affected. Even among the same animal species, mitochondria isolated from different organs may show remarkably different sensitivity toward mitochondria active chemicals. Muscatello and Carafoli (1969) demonstrated a large stimulation by the nonionic detergent Lubrol on the ability of mitochondria isolated from heart and skeletal muscles of rats to oxidize endogenous and exogenous cytochrcme c by cytochrome c oxidase, while the liver mitochondria failed to respond to the same concentration of Lubrol. PLATELET FUNCTION The next area surveyed by Glantz and Parmley concerns the asserted action of ETS on platelet function. The authors maintain that ETS exposure promotes platelet hyperaggregability and "so increases the likelihood of thrombus formation." In - 13 -

I I I I I I I I I I I I I I I I I I i contrast to this assertion, there is quite an extensive literature to show that smoking actually has no influence on the development of venous thrombosis and in certain situations appears to exert a protective effect. In their study of mortality in relation to smoking in British doctors, Doll and Peto (1976) found no association between mortality from venous thromboembolism and smoking. Additionally, a protective effect of cigarette smoking on venous thromboembolic disease has been noted after myocardial infarction and surgery. For example, Handley and Teather (1974) found that the incidence of thrombosis in the patients with a history of regular smoking within the month before admission was significantly lower than that of nonsmokers. Also, Pollock and Evans (1978) noted in patients undergoing emergency or elective laparotomy for benign or malignant disease or retropubic prostatectomy that cigarette smokers had a significantly lower incidence of deep venous thrombosis than pipe smokers or nonsmokers. The relationship between the effects of cigarette smoking on platelet aggregation and the appearance of non-hematological endothelial cells in circulating blood was next assessed by the authors. Table 2 of Chapter Eleven summarizes the reported effect of ETE exposure and smoking on platelet aggregation ratio and endothelial cell count based on the published results of Davis et al. (1985, 1986, 1989, 1990). It is worth noting that previously Davis and Davis (1979) claimed that a fall in platelet - 14 -

I l I I I I I I I I I ! I I I I I I i aggregate ratio occurred after smoking cigarettes, but this could not be confirmed by others (Rang et al., 1983). In any case, the significant though small reduction in platelet aggregation ratio and the rise in endothelial cell count could not be correlated with "the level of nicotine in the blood of the experimental subjects in any of these or other related studies." To this reviewer, the failure in this correlation would tend to support the lack of importance of nicotine as an active agent in promoting platelet aggregation and increase in endothelial cell count, though the exact opposite conclusion was reached by Glantz and Parmley, who believe the data suggest that "nicotine is an important active agent." Their rationale in arriving such a conclusion simply escapes this reviewer. The authors proceed to state that "since non-tobacco cigarettes also affected platelet aggregation somewhat, however, it is possible that carbon monoxide or some other combustion products are also influencing the platelets." Indeed, platelet aggregation is known to be extremely sensitive and variable. Factors contributing to the variability include: venipuncture technique; the effects of anticoagulants; sodium citrate concentrations; platelet concentration; time interval after venipuncture; pH changes (Triplett, 1978). In addition, the estimation of platelet aggregation ratio can also be rather subtle and requires consistent and precise manipulations at all stages of the platelet aggregation study in order to avoid - 15 -

! ! ! I I I I I I I ! I I I I I ! I I artefacts. Similarly, while circulating endothelial cells may be isolated together with platelets by the method of leucoconcentration, the demonstration of the origin of circulating endothelial cells is not definite (Hladovec and Rossman, 1973). The question of whether platelets derived from smokers versus non-smokers display differential sensitivity toward chemicals affecting platelet aggregatability is also addressed by the authors. The data of Sinzinger and Kefalides (1982) showing that ETS exposure reduced platelet sensitivity to prostacyclin (PGI2) by nearly a factor of 2 in nonsmokers, but only by 20% in smokers, was suggested to reflect that "nonsmokers' platelets seem much more sensitive to a single exposure than do smokers' platelets." However, the authors fail to mention that in the original report of Sinzinger and Kefalides, it was stated that "passive smoking reduced platelet sensitivity to the antiaggregatory PGs, being much more severe in nonsmokers than in smokers. 20 min after passive smoking, platelet sensitivity started to return to basal values and this happened more quickly in nonsmokers." Thus, the quick return to basal sensitivity toward prostacyclin by platelets of nonsmokers, an effect unmatched by platelets from smokers, suggests that the effects of ETS are only transitory and are not expected to cause permanent platelet hyperaggregability. That being the case, the statement that "the resulting increase in platelet aggregation can - 16 -

I l I I I I I I I I ! I I I I I ! I I contribute to acute thrombus formation and myocardial infarction" seems subjective, elusive and speculative. In connection with this discussion, I would point out that there have been numerous studies on the influence of habitual smoking on hemostatic function. Mustard and Murphy [1963) reported that platelet survival was significantly shorter in smokers than in nonsmokers, but they were unable to detect significant differences between the smoking and nonsmoking groups in respect of the whole-blood clotting time, one-stage prothrombin time or the partial thromboplastin time. Older smokers were found by Hawkins (1972) to have platelets which aggregated to a greater extent in response to ADP, but White et al (1983] reported that heavy smokers did not differ from control subjects in respect of platelet aggregation in response to ADP or malondialdehyde production. ATHEROSCLEROSIS The last area covered by Glantz and Parmley in this chapter is the etiology and pathogenesis of atheroscletosis. By way of background, it should be appreciated that despite almost a century of scientific study, the etiology and pathogenesis of atherosclerosis remain unknown. In humans, clinical complications and sequelae occur when the lesions have evolved to produce the fibrous plaque. The main histologic features of this stage are lipid accumulation and fibrobelastic and fibromuscular - 17 -

! ! ! ! ! ! ! ! ! I l I I I I I I I I thickening, which initially are present in a patchy distribution but later become more diffuse as individual plaques coalesce. A fatty streak stage, characterized by lipid accumulation within intimal cells, either of monocyte/macrophage or arterial smooth muscle cell origin, giving them a "foam cell" appearance in histologic sections, is considered by some to represent an intermediate step in the development of the final lesion (McGill, Jr., 1984). Because of the characteristic features of lipid accumulation and intimal thickening, it is generally believed that in humans some form of endothelial injury contributes strongly to the pathogenesis (BOSS, 1986). Much of the endothelial biology today is an attempt to probe the more subtle forms of endothelial dysfunction, since obvious evidence of damage, such as morphologic stigmata at the microscopic level, is not easily found in human observations or animal experiments. In humans, however, clinical and epidemiological studies have uncovered statistically significant risk factors which are associated with advanced disease (Keys, 1970; Doll and Peto, 1976). ~espite these associations however, relatively little light has been shed on the dark shadows of pathogenesis. Moreover, even if the major risk factors are taken into account, collectively they are unable to predict the majority of new cases of the disease (Eliot, 1987). - 18 -

I I I I i I I I i I I I I I I I I I I Over the last decade, two formal hypotheses of atherogenesls have been formulated. The first hypothesis discussed by Glantz and Parmley, starting on page ii of Chapter ii, is what has been termed the response to injury hypothesis of atherosclerosis (ROSS, 1986). This hypothesis suggests that various systemic and local changes occur in the arterial network in association with the different risk factors commonly shown to be related to increased incidence of atherosclerosis, and that these changes result in various forms of injury to the endothelial cell lining the arterial tree. This injury to the endothelium may take several forms because the endcthelium is not only a blood container but a source of vasoactive substance, a permeability barrier, and a nonthrombogenic surface. The results of these various forms of injury may culminate in a series of changes in the endothelium that at one end of the spectrum may lead to minor alterations in functional capacities of the endothelial cells with no apparent morphological alteration, whereas at the other end of the spectrum the changes may result in endothelial cell-cell detachment and cell-connective detachment, leading to oportunities at particular anatomical sites (such as branches and bifurcations) for endothelial cell detachment, desquamation into the blood stream, and exposure of denuded areas of the artery wall. The hypothesis suggests that these denuded areas lead to interactions between elements from the blood (including plasma constituents, platelets, and monocytes) and the artery wall at these sites. - 19 -