Increase in Circulating Products off Lipid Peroxidation ... - Diabetes Care

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Increase in Circulating Products off Lipid Peroxidation in Smokers With IDDM GlACOMO ZOPPINI, MD

ELENA PASQUALINI, PHD

GIOVANNI TARGHER, MD

CARLO MARTINELLI, MD

TlZIANO MONAUNI, MD

MARIA L. ZENARI, PHD

GIOVANNI FACCINI, MD

MlCHELE MUGGEO, MD

(MDA), a product of lipid peroxidation, in a group of young IDDM patients with no evidence of macro vascular disease.

RESEARCH DESIGN AND OBJECTIVE— To measure plasma malondialdehyde (MDA) concentration, a product of lipid peroxidation, both in IDDM patients and in healthy control subjects and to examine whether smoking has a negative impact on the plasma MDA levels in diabetic patients. RESEARCH DESIGN AND METHODS — Plasma total MDA concentration (as a thiobarbituric acid adduct by high-performance liquid chromatography) was measured in 56 young IDDM patients and in a group of 32 age-, sex-, BMI-, and smoking habit-matched healthy subjects. RESULTS— Plasma MDA concentration in IDDM patients was significantly higher than that in healthy control subjects (mean ± SE: 0.95 ± 0.03 vs. 0.54 ± 0.03 umol/1; P < 0.0001). After stratification by smoking status, it was seen that diabetic smokers had values of age, BMl, serum lipids, blood pressure, metabolic control, and diabetes duration and its chronic complications superimposable on those of their nonsmoking counterparts. Nevertheless, plasma MDA concentration was significantly higher in IDDM patients who smoked than in IDDM patients who didn't smoke (1.03 ± 0.4 vs. 0.87 ± 0.03 umol/1; P = 0.002), without any sex difference with regard to MDA levels. CONCLUSIONS — These data show an increase in circulating products of lipid peroxidation in young diabetic smokers, thus further supporting the clinical importance of discouraging the initiation of smoking as well as promoting its cessation in people with IDDM.

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orbidity and mortality in diabetes is typically associated with the development of atherosclerotic cardiovascular diseases and diabetes-related late complications (1,2). In these pathological events, lipid peroxidation of cellular structures, a consequence of increased free radical activity, is thought to play an important role (3-5). A number of studies have suggested that diabetes is a state of elevated free radical activity (3-10). Mechanisms that might lead to the formation of free radicals in diabetes are currently unclear. Recently, because cigarette smoke is known to contain a large number of oxidants, it has been hypothesized that at least a part of the adverse effects of cigarette smoking on the cardiovascular sys-

tem may result from oxidative damage to critical biological substances by free radicals. In this context, increased circulating products of lipid peroxidation have been demonstrated in nondiabetic smokers as compared with nonsmokers in some studies (11-13), though not all (14). To our knowledge, available data regarding the impact of cigarette smoking on lipid peroxidation in young patients with IDDM are lacking. On the other hand, the clarification of that may help to explain underlying mechanisms and may be of clinical importance for undertaking preventive and therapeutic strategies. Thus, the main purpose of the present study was to evaluate the impact of cigarette smoking on plasma levels of malondialdehyde

I;rom the Division of Endocrinology and Metabolic Diseases (G.Z., G.T., T.M., M.M.) and the Institute of Clinical Chemistry (G.E, E.R, CM., M.L.Z.), University of Verona, Verona, Italy. Address correspondence and reprint requests to Giacomo Zoppini, MD, Divisione di Endocrinologia e Malattie del Mctabolismo, Ospedale Civile Maggiore, Piazzale Stefani, 1,1-37126 Verona, Italy. Received for publication 14 December 1995 and accepted in revised form 13 June 1996. AliR, albumin excretion rate; MDA, malondialdehyde; TBA, thiobarbituric acid.

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METHODS— The study group consisted of 56 IDDM patients attending the Division of Endocrinology and Metabolic Diseases, University of Verona. A control group was established from 32 healthy volunteers (recruited from hospital staff and relatives) who were age-, sex-, weight-, and smoking habit-matched to the group of diabetic patients. All participants underwent a medical history and physical examination. In particular, none of them had a history of recent acute disease or had clinical evidence suggestive of any cardiovascular event, kidney disease, or liver disease. All IDDM patients were treated with insulin and diet and had stable metabolic control; none of them, including healthy control subjects, were taking any other drugs. BMl was calculated by dividing weight in kilograms by height in meters squared (15). Blood pressure was measured with a standard mercury manometer by a trained member of staff using phase V (disappearance of Korotkoffs sound) as a criterion for diastolic blood pressure. Information on smoking habits and daily alcohol intake was obtained in all subjects from a questionnaire. Smoking habits were categorized into those who had never smoked and current smokers (more than five cigarettes per day). Daily alcohol consumption was quantitated and expressed as grams consumed per day. Venous blood was drawn in the morning (8:00-8:30 A.M.) after an overnight fast and at least 8 h of abstention from smoking. Plasma glucose, creatinine, uric acid, triglyceride, and total cholesterol concentrations were determined by an automatic colorimetric method (DAX 96, Bayern Diagnostics, Milan, Italy). HDL cholesterol was assayed according to Warnick et al (16). HbAlc was assayed by high-performance liquid chromatography (HPLC) (17); normal range values in our laboratory are 3.0-5.5%. Fructosamine was assayed by a colorimetric method. Blood for lipid peroxidation analysis was collected into HDTAcontaining Vacutainer tubes and immedi1233

MDA in IDDM smokers

Table 1—Main clinical characteristics of IDDM patients and healthy control subjects

n Age (years) Sex (M/F) BMI (kg/m2) Smokers (%) Total MDA (umol/l) HbAlt. (%) Diabetes duration (years)

IDDM patients

Control subjects

P values

56

32 31 ±2 18/14 24 ±0.7 46.9 0.54 ±0.03 — —

NS NS NS NS 200 ug/min). Nine IDDM patients were microalbuminuric, while most of them (84%) had normal AER values. Retinopathy was confirmed by fundoscopy by a single ophthalmologist after pupillary dilatation. About two-thirds of patients had no diabetic retinopathy, while 19 patients (33.9%) had background retinopathy. Data analyses were performed with StatView SE+ Graphics statistical software. The following statistical tests were done: Student's t test for unpaired data, Pearsons product-moment correlation, and \ 2 t e s t (for categorical variables). When the distribution of continuous variables was skewed, logarithmic transformations were carried out. However, because the differences in 1234

the results were extremely small, we presented only the statistical analyses using untransformed variables. Nonparametric statistical tests (i.e., Spearman's rank correlation and Mann Whitney U test) were also performed, but because the results obtained with parametric and nonparametric statistical procedures were similar, only the former are presented. Data are presented as mean ± SE. P values 27 kg/m2), three subjects had plasma total cholesterol and/or triglyceride levels exceeding 240 mg/dl (6.2 mmol/1) and 250 mg/dl (2.8 mmol/1), and one subject had systolic and diastolic blood pressure values exceeding 140/90 mmHg. After stratifying by smoking status (Table 2), although diabetic smokers had slightly lower HDL cholesterol concentration than those who never smoked, the two groups of patients were comparable for all potential confounders. In particular, no significant differences were found in age, sex, BMI, lipids, blood pressure, metabolic control, diabetes duration, and chronic complications. Nevertheless, plasma MDA concentration was significantly higher in IDDM patients who smoked than in IDDM patients who were nonsmokers, without any sex difference with regard to plasma MDA concentration (Fig. 1). These results remained substantially unchanged when allowance was made for HDL cholesterol concentration (not shown). Similarly, healthy people who smoked had significantly higher plasma levels of total MDA than healthy nonsmokers (Fig. 1).

Table 2—Clinical and biochemical characteristics of IDDM patients grouped according to smoking habit IDDM patients Nonsmokers n Sex (M/F) Age (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/1) Fasting glucose (mmol/1) Fructosamine (umol/1) HbAlc (%) Creatinine (umol/1) Diabetes duration (years) Retinopathy Urinary AER 20 200 ug/min

30 12/18 31 ±2 24 ±0.5 121 ±2 78 ±1.5 4.85 ±0.2 0.87 ±0.05 1.63 ±0.1 13.0 ±1.0 400.0 ±12.0 7.0 ±0.21 88.4 ±1.8 13 ±1.4 11 24 6 0

Smokers 26 17/9 29 ±2 23 ±0.5 123 ±2 78 ±1.5 4.50 ±0.2 0.95 ±0.1 1.37 ±0.07* 12.8 ±1.0 412.0 ±15.0 7.5 ±0.23 88.4 ±1.8 10 ±1.3 8 23 3 0

Data are means ± SE or n. *P = 0.05. Note that all other differences were not statistically significant.

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Zoppini and Associates

2.0-/

All

n=I7 n=15

Controls (n=32)

All

n=30 n=26

IDD-patients (n=56)

Figure 1—Plasma MDA concentration in 56 IDDM patients and 32 healthy control subjects subdivided according to smoking habit (smokers[o] and nonsmokers [nj). The IDDM patients were also subdivided according to sex (upper right panel). Data are presented as means ± SE. §P < 0.0001 vs. healthy control subjects; #P = 0.002 and *P < 0.05 vs. IDDM nonsmokers.

Because the diabetic group included individuals with microvascular complications and because these conditions might alter the levels of plasma MDA, statistical analyses excluding the participants with these conditions were repeated. The results were substantially unchanged, being that plasma total MDA concentration was higher in IDDM smokers than in their nonsmoking counterparts (1.08 ± 0.02 vs. 0.89 ± 0.01 umol/1; P = 0.005). When diabetic patients were considered all together, plasma MDA levels did not show any significant univariate correlation with age, BMI, serum lipids (including HDL cholesterol), blood pressure, metabolic control, or other study variables (data not shown). CONCLUSIONS— Several epidemiological studies have reported that diabetic patients have an elevated plasma lipid peroxidation, thus supporting the evidence that diabetes is a state of increased free radical activity (3-10). Mechanisms that contribute to the formation of free radicals in diabetes may include increased nonenzymatic and autoxidative glycosylation, metabolic stress resulting from changes in energy metabolism, alterations in sorbitol pathway activity, changes in the level of inflammatory mediators, and the status of antioxidant defense (3-5). The most widely used test for oxidative stress is the measurement of MDA, a product of lipid

peroxidation, by the TBA-reacting substances assay (20). In the present study, we measured plasma total MDA levels by using a TBA reaction and subsequent HPLC analysis, thus improving the specificity of our TBA test (18-20). Recently, it has been hypothesized that many of the adverse effects of cigarette smoking on the cardiovascular system may result from oxidative damage to critical biological substances by free radicals. In this context, a number of studies have reported that nondiabetic smokers have significantly higher plasma levels of lipid peroxides than nonsmokers (11-13). Although the mechanisms by which smoking may increase plasma lipid peroxides remain still speculative, in vitro studies have supported the evidence of an increased formation of lipid peroxides after exposure of plasma to the gas phase of cigarette smoke (21). Other evidence suggesting that smokers are subjected to oxidative stress includes the findings that they have lower levels of the antioxidant ascorbic acid than nonsmokers and that the risk to smokers of getting coronary artery disease correlates inversely with their intake of the antioxidants vitamin E and beta carotene (22,23). The present study was designed to investigate the impact of cigarette smoking on lipid peroxidation in young IDDM patients without any clinical evidence of macrovascular complications, which

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would complicate the interpretation of data. Moreover, because our patients were predominantly normotensive and normolipidemic (thus excluding the potential confounding effects of these pathological events), we believe that this could enhance the validity of our findings. In line with previous studies (3-5,9), we found that plasma levels of total MDA were significantly higher in IDDM patients than in a matched group of healthy control subjects. After stratifying by smoking status, smoking increased plasma MDA comparably in control and diabetic individuals. In particular, plasma MDA levels were significantly higher in IDDM patients who smoked than in nonsmokers. Importantly, the two subgroups of diabetic patients were comparable for potential confounding variables, such as age, sex, BMI, serum lipids, blood pressure, metabolic control, diabetes duration, and complication status. In agreement with previous reports (24,25), current smokers had lower levels of HDL cholesterol concentration, but the adjustment for this potential confounding variable did not substantially alter the present results. Although the levels of MDA tended to be higher in heavier smokers than lighter smokers, we did not find any significant relation between plasma MDA levels and number of cigarettes smoked per day. It is possible that differences in antioxidant defense capacity affect susceptibility to the oxidant effects of cigarette smoke (13). No significant sex difference was found with regard to plasma MDA levels according to smoking status. This finding is partly consistent with earlier observations demonstrating that there are no sex differences with regard to plasma MDA levels among diabetic patients (26). The conclusions from this and other studies (3,4,8-10) are in contrast with those of Leonard et al. (27) who have recently reported that young adult patients with IDDM did not significantly differ in their plasma MDA levels as compared with an age-matched group of healthy control subjects. They also reported that cigarette smoking did not affect the plasma levels of MDA of diabetic subjects, suggesting that any increase in free radical activity due to cigarette smoke was adequately scavenged in young adults with IDDM (27). In that study, however, diabetic smokers and nonsmokers were comparable for some but not for other important potential confounders, such as plasma lipids, which might have complicated the interpretation of data.

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MDA in IDDM smokers

Overall, therefore, the evidence from this and other studies suggests that patients with IDDM differ in their plasma MDA concentrations as compared with healthy control subjects and that smoking itself plays a role in the elevation of plasma MDA levels both in diabetic patients and in healthy individuals. Because diabetic nonsmokers, however, had a significantly higher (+60%) plasma MDA concentration than healthy control subjects (both smokers and nonsmokers), these results also support the possibility that the increase of plasma MDA concentration found in patients with IDDM is only partly explained by cigarette smoking and that other specific and diabetes-related mechanisms may be involved, as suggested previously (3-5). The relatively small number of subjects examined did not allow us to do full analyses by complication status. Previous epidemiological studies have reported increased plasma lipid peroxides in diabetic patients with micro- or macrovascular complications (3,8-10), thus suggesting a role for oxidative stress in the development of complications in diabetes. On the other hand, although the evidence for a relationship between smoking status and diabetic complications is still conflicting, a number of studies have reported a strong relationship between smoking and microvascular disease (28-30), thus supporting the possibility that quitting smoking would be effective in reducing the incidence of complications.

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