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(Circulation. 1999;99:224-229.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

In Vivo Formation of 8-Iso-Prostaglandin F₂ and Platelet Activation in Diabetes Mellitus

Effects of Improved Metabolic Control and Vitamin E Supplementation

Giovanni Davì, MD; Giovanni Ciabattoni, MD; Agostino Consoli, MD; Andrea Mezzetti, MD; Antonio Falco, MD; Stella Santarone, MD; Elsa Pennese, MD; Ester Vitacolonna, MD; Tonino Bucciarelli, MD; Fabrizio Costantini, MD; Fabio Capani, MD; Carlo Patrono, MD

From the Departments of Medicine and Aging (G.D., A.C., A.M., S.S., E.P., E.V., F. Costantini, F. Capani, C.P.) and Biomedical Sciences (T.B.), University of Chieti "G. D'Annunzio" School of Medicine; the Division of Internal Medicine, Civil Hospital of Popoli (A.F.); and the Department of Pharmacology (G.C.), Catholic University School of Medicine; Rome, Italy.

Correspondence to Dr Patrono, Department of Medicine and Aging, University of Chieti "G. D'Annunzio," via dei Vestini 31, 66013 Chieti, Italy. E-mail cpatrono{at}unich.it

	Abstract

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Background—Diabetes mellitus (DM) is associated with enhanced lipid peroxidation and persistent platelet activation. We tested the hypothesis that the in vivo formation of the F₂-isoprostane 8-iso-prostaglandin (PG)F₂, a bioactive product of arachidonic acid peroxidation, is enhanced in DM and contributes to platelet activation.

Methods and Results—Urine samples were obtained from 85 diabetic patients and 85 age- and sex-matched healthy subjects for measurement of immunoreactive 8-iso-PGF₂ and 11-dehydro-thromboxane B₂ (TXM), an in vivo index of platelet activation. Sixty-two had non–insulin-dependent (NID)DM, and 23 had insulin-dependent (ID) DM. Vitamin E supplementation, metabolic control, and cyclooxygenase inhibitors were used to investigate the mechanisms of formation of 8-iso-PGF₂ in this setting. Urinary 8-iso-PGF₂ excretion was significantly higher (P=0.0001) in NIDDM patients (419±208 pg/mg creatinine; range 160 to 1014) than in age-matched control subjects (208±92; 41 to 433). Urinary 8-iso-PGF₂ was linearly correlated with blood glucose and urinary TXM. 8-iso-PGF₂ excretion was also significantly (P=0.0001) higher in IDDM patients (400±146; 183 to 702) than in control subjects (197±69; 95 to 353). Vitamin E supplementation (600 mg/d for 14 days) was associated with a statistically significant reduction in both urinary 8-iso-PGF₂ (by 37%) and TXM (by 43%) in 10 NIDDM patients. Improved metabolic control was associated with a significant (P=0.0001) reduction in 8-iso-PGF₂ and TXM excretion by 32% and 41%, respectively, in 21 NIDDM patients. 8-iso-PGF₂ was unchanged after 2-week dosing with aspirin and indobufen despite profound suppression of TXM excretion.

Conclusions—We conclude that DM is associated with increased formation of F₂-isoprostanes, as a correlate of impaired glycemic control and enhanced lipid peroxidation. This may provide an important biochemical link between impaired glycemic control and persistent platelet activation. These results provide a rationale for dose-finding studies of antioxidant treatment in diabetes.

Key Words: diabetes mellitus • platelets • lipids • thromboxane • antioxidants

	Introduction

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There is a well-established association of diabetes mellitus with the development of atherosclerosis and its thromboembolic complications and with the occurrence of disorders of the microvasculature.¹ However, the mechanism(s) responsible for accelerated atherogenesis remain elusive. Altered lipoprotein levels; changes in lipoprotein composition, possibly affecting low-density lipoprotein (LDL) binding to its receptors; and reduced LDL clearance resulting from impaired receptor recognition of glycated LDL have been described in diabetic patients (reviewed in Reference 2² ). Moreover, both high glucose levels and protein glycation enhance LDL oxidation by metal ions, and these reactions also yield advanced glycosylation end (AGE) products.^{2 3} In fact, LDLs isolated from non–insulin-dependent diabetics (NIDDM) contain higher levels of AGE products and conjugated dienes and are more easily oxidized by copper than native LDL.⁴ In addition, plasma from patients whose insulin-dependent diabetes mellitus (IDDM) is poorly controlled has less antioxidant capacity.⁵

Recently, a series of bioactive prostaglandin (PG) F₂-like compounds (isoprostanes) has been discovered,⁶ which are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation, catalyzed by oxygen free radicals on cell membranes and LDL particles (reviewed in Reference 7⁷ ). Among these products, of particular importance is 8-iso-PGF₂, which induces vasoconstriction⁸ and modulates the function of human platelets.^{9 10 11}

F₂-isoprostanes can be reliably measured in both plasma and urine^{12 13} and have been shown to be increased in association with advanced age,¹³ hypercholesterolemia,¹⁴ and cigarette smoking.^{15 16}

We have previously reported biochemical evidence of persistent platelet activation, as reflected by enhanced 11-dehydro-TXB₂ (TXM) excretion, in NIDDM patients with macrovascular complications and shown that this abnormality was partially reversible in association with improved glycemic control.¹⁷ We speculated that increased oxidant stress in diabetes could induce enhanced generation of 8-iso-PGF₂ and other biologically active iso-eicosanoids and that these compounds could in turn contribute to platelet activation in this setting. Therefore, in the present study, we investigated whether 8-iso-PGF₂ formation is altered in NIDDM patients when compared with age-matched nondiabetic subjects and whether it correlates with the rate of TXA₂ biosynthesis. We also measured F₂-isoprostane formation in IDDM patients as compared with their age-matched control subjects to assess the relative contribution to oxidant stress of the diabetic metabolic abnormality per se, vis-à-vis the macrovascular complications often accompanying NIDDM. Moreover, we examined the effects of 2 interventions potentially capable of reducing oxidant stress, that is, improvement in metabolic control and vitamin E supplementation, by assessing time-related changes in urinary 8-iso-PGF₂ and TXM excretion in NIDDM.

The results of the present study suggest that enhanced peroxidation of arachidonic acid to form biologically active isoprostanes may represent an important biochemical link between impaired glycemic control and persistent platelet activation in this setting.

	Methods

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Subjects
Eighty-five patients with diabetes mellitus were studied on several occasions between May 1995 and December 1997. Sixty-two had NIDDM and 23 had IDDM, as defined in accordance with the criteria of the American Diabetes Association.¹⁸ Sixty-two and 23 healthy subjects, age- and sex-matched with the NIDDM and IDDM patients, respectively, were also studied. The baseline characteristics of patients and control subjects are detailed in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Diabetic Patients and Healthy Subjects

Of the 62 NIDDM patients, 20 patients had a history or physical examination positive for evidence of macrovascular complications. Twelve patients had stable angina pectoris or had had a myocardial infarction, and 8 had peripheral vascular disease. Patients with coronary heart disease were in a stable phase. Patients with peripheral vascular disease were in Fontaine stage II (intermittent claudication, ankle-arm pressure index of <0.85, and no resting pain), with a constant level of pain while walking. At the time of the study, NIDDM patients were being treated by diet alone (4 patients), insulin alone (6 patients), or by diet plus oral hypoglycemic agents (metformin and/or sulfonylureas) (43 patients); in 9 patients, insulin was added to the oral hypoglycemic agents. Twenty-four patients had arterial hypertension, defined as current systolic/diastolic blood pressure >140/90 mm Hg. Twenty-six patients were hypercholesterolemic (blood cholesterol level >240 mg/dL).

None of the 23 IDDM patients had a history or physical examination positive for evidence of macrovascular complications. Nine patients had diabetic retinopathy as determined by direct ophthalmoscopy through a dilated pupil. Only 1 IDDM patient was hypertensive as defined above. At the time of the study, all IDDM patients were being treated with insulin therapy.

Patients with renal insufficiency or proteinuria (by serum creatinine levels and urinalysis), altered hepatic function (by liver enzymes), or alcohol abuse (by clinical history and laboratory measurements) were excluded. Both diabetic patients and healthy subjects were selected for being nonsmokers at the time of study to eliminate a potential confounder.

Informed consent was obtained from each participating subject, and the protocol was approved by the ethical committee of the University of Chieti Medical School.

Design of the Studies
In the first study, a cross-sectional comparison of urinary 8-iso-PGF₂ and TXM, a major enzymatic metabolite of TXA₂, was performed between patients and control subjects. All the subjects were studied as outpatients after a 12-hour fast. Each patient performed an overnight urine collection, immediately before blood sampling. Urine samples were added with the antioxidant 4-hydroxy-tempo (1 mmol/L) (Sigma Chemical Co) and stored at -20°C until extraction.

Because small amounts of 8-iso-PGF₂ can be formed by human platelets and monocytes through a cyclooxygenase-dependent mechanism,⁷ a second study was performed to evaluate whether inhibition of cyclooxygenase activity had any influence on 8-iso-PGF₂ excretion in diabetes. For this purpose, 6 of the 62 NIDDM patients (3 women and 3 men; 56 to 75 years of age) were given 50 mg acetylsalicylic acid once daily and 200 mg indobufen (a reversible cyclooxygenase inhibitor) twice daily, each drug for 7 days in 2 successive weeks, according to a randomized sequence. These patients collected overnight urine samples before dosing and on the last day of each treatment for measurement of 8-iso-PGF₂ and TXM excretion.

To assess the potential influence of improved metabolic control on F₂-isoprostane formation and platelet activation, a third study was performed in 21 NIDDM patients (13 women and 8 men; 39 to 75 years of age). At the time of the cross-sectional study, these 21 NIDDM patients were in poor metabolic control (fasting blood glucose >200 mg/dL, glycohemoglobin [HbA_1c] >9%) despite taking oral antidiabetic agents for months. These patients were examined twice weekly over a 4-week period for blood glucose monitoring, and oral antidiabetic therapy was adjusted accordingly and/or insulin therapy was instituted to achieve improved metabolic control. Throughout the study, patients followed an isocaloric diet that provided 50% of calories as carbohydrates, 30% as fat, and 20% as protein. The level of dietary cholesterol was 0.3 g per day. Physical activity was encouraged, and patients were instructed to walk at least 30 minutes after each meal. No patient experienced a hypoglycemic reaction during the study period. Blood and overnight urine samples were obtained before and at the end of this intensive monitoring and treatment program for determination of fasting glucose and HbA_1c levels and 8-iso-PGF₂ and TXM excretion, respectively.

To investigate the short-term effects of antioxidant intervention on urinary 8-iso-PGF₂ and TXM excretion, vitamin E was given to 10 (6 women and 4 men; 42 to 69 years of age) of the 62 NIDDM patients. They were given 600 mg/d, l--tocopherol acetate (Evion) daily for 2 weeks after a baseline evaluation. This dose of vitamin E is 60-fold higher than the recommended daily dietary allowance. Before and after vitamin E supplementation, they collected an overnight urine sample for measurement of 8-iso-PGF₂ and TXM and had a fasting blood sample drawn for lipid levels, plasma and LDL vitamin E, and oxidation of isolated LDL in vitro.

Urinary Eicosanoid Assays
Urinary 8-iso-PGF₂ and TXM were measured by previously described radioimmunoassay methods.^{13 19} Measurements of urinary 8-iso-PGF₂ and TXM by these radioimmunoassays have been validated with the use of different antisera and by comparison with gas chromatography/mass spectrometry, as detailed elsewhere.^{13 19}

Lipid Measurements
All blood samples for lipid studies were drawn into EDTA (1 mg/mL and separated within 1 hour after sampling. Total, LDL and high-density lipoprotein cholesterol, triglycerides, and vitamin E were determined as previously described.¹⁴ Aliquots of the plasma in EDTA, immediately after separation, were stored at -80°C until LDL isolation. LDL was isolated by single vertical spin density gradient ultracentrifugation, and LDL protein, cholesterol, and vitamin E were determined as previously described.¹⁴ To induce oxidation, LDL (0.2 mg cholesterol/mL) was incubated with 5 mmol/L CuSO₄ in PBS, pH 7.4, at 37°C. The formation of conjugated dienes was determined spectrophotometrically.¹⁴

Clinical Laboratory Measurements
Fasting plasma glucose was measured by the glucose oxidase method. HbA_1C level was measured by automated high-performance liquid chromatography.

Statistical Analysis
The data were analyzed by nonparametric methods to avoid assumptions about the distribution of the measured variables. ANOVA was performed with the Kruskal-Wallis method. Subsequent pairwise comparisons were made with the Mann-Whitney U test. The differences between baseline and posttreatment values were analyzed with the Wilcoxon signed rank test. Moreover, the association of eicosanoid measurements with other biochemical parameters was assessed by the Spearman rank correlation test. All values are reported as mean±1 SD.

	Results

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Urinary 8-iso-PGF₂ excretion was significantly (P=0.0001) higher in NIDDM patients (419±208 pg/mg creatinine; mean±SD, n=62) than in age-matched healthy subjects (208±92 pg/mg creatinine) (Figure 1). Twenty-six of the 62 NIDDM had blood cholesterol >240 mg/dL (292±37) and abnormal LDL cholesterol levels (189±38 mg/dL). The urinary excretion of 8-iso-PGF₂ was not significantly higher in these hypercholesterolemic patients than in the 36 normocholesterolemic NIDDM patients (438±178 vs 401±227 pg/mg creatinine). Similarly, no statistically significant differences in urinary 8-iso-PGF₂ were found between hypertensive and normotensive NIDDM patients (429±219 vs 412±203 pg/mg creatinine) nor between those with and those without macrovascular complications (459±233 vs 400±195 pg/mg creatinine).

View larger version (18K): [in this window] [in a new window]   Figure 1. Urinary excretion rates of 8-iso-PGF2 in 62 NIDDM patients and 62 age-matched healthy subjects. Dots represent individual measurements; horizontal bars represent mean value for each group.

Consistent with previous findings, NIDDM patients had significantly enhanced TXM excretion versus that in control subjects (1103±1068 vs 415±244 pg/mg creatinine; P=0.0001). A statistically significant correlation was found between TXM and 8-iso-PGF₂ excretion in NIDDM patients (r=0.39, P=0.0023).

The urinary excretion of 8-iso-PGF₂ was also increased in the group of IDDM patients (400±146 pg/mg creatinine; n=23) as compared with their age-matched control subjects (197±69 pg/mg creatinine; P=0.0001) (Figure 2).

View larger version (15K): [in this window] [in a new window]   Figure 2. Urinary excretion rates of 8-iso-PGF2 in 23 IDDM patients and 23 age-matched healthy subjects. Dots represent individual measurements; horizontal bars represent mean value for each group.

Effects of Cyclooxygenase Inhibition
Two structurally unrelated cyclooxygenase inhibitors, aspirin and indobufen, were used to investigate the mechanism(s) of F₂-isoprostane formation in NIDDM. As shown in Figure 3, urinary 8-iso-PGF₂ excretion was largely unaffected during 2 successive weeks of cyclooxygenase inhibition achieved with either agent, despite normalization of TXM excretion. This finding is consistent with a noncyclooxygenase mechanism of F₂-isoprostane formation as characterized in other clinical settings.^{13 14 20}

View larger version (17K): [in this window] [in a new window]   Figure 3. Urinary excretion of 8-iso-PGF2 (top) and 11-dehydro-TXB2 (bottom) before and after cyclooxygenase inhibition with aspirin and indobufen. Eicosanoid levels were measured in 6 NIDDM patients on the day before dosing and on the last day of each week of consecutive treatment with either aspirin (50 mg daily) or indobufen (200 mg twice daily). Solid bars depict mean±1 SD values for the whole group of patients. Cyclooxygenase inhibition produced no detectable changes in urinary 8-iso-PGF2 excretion.

This study also allowed assessing the reproducibility of 8-iso-PGF₂ excretion in NIDDM on the basis of 3 different urine collections performed over a 2-week period in 6 patients. The intrasubject coefficient of variation of these measurements averaged 19±11%.

Influence of Metabolic Control
Twenty-one NIDDM patients who had not achieved adequate metabolic control (fasting blood glucose >200 mg/dL and HbA_1c >9%) despite oral antidiabetic therapy were subjected to intensive monitoring and treatment over 4 weeks, and blood and urine samples were obtained before and after improved metabolic control. At the end of this period, HbA_1c fell from 9.9±1.3% to 7.5±1.0% (P<0.0001), and fasting blood glucose was significantly reduced from 306±118 to 159±47 mg/dL (P<0.0001). Improvement in metabolic control was associated with a statistically significant reduction in both 8-iso-PGF₂ (from 533±276 to 365±226 pg/mg creatinine; P=0.0001) (Figure 4) and TXM (from 1643±1269 to 975±726 pg/mg creatinine; P=0.0005) excretion.

View larger version (24K): [in this window] [in a new window]   Figure 4. Changes in F2-isoprostane formation associated with improved metabolic control in NIDDM. Individual measurements of 8-iso-PGF2 excretion are represented for 17 patients who achieved improved metabolic control (ie, blood glucose <200 mg/dL and HbA1c <9%) after intensive monitoring and treatment.

Effects of Vitamin E Supplementation
We also examined the effects of vitamin E supplementation (600 mg daily for 2 weeks) on the urinary excretion of 8-iso-PGF₂ and TXM to test the hypothesis of a cause-and-effect relation between enhanced lipid peroxidation and platelet activation in NIDDM. Vitamin E supplementation was associated with statistically significant changes in plasma vitamin E levels, vitamin E content of LDL, and lag time for LDL oxidation (Table 2). As depicted in Figure 5, vitamin E supplementation was associated with a statistically significant reduction in urinary 8-iso-PGF₂ and TXM, by 37% and 43%, respectively. A statistically significant correlation was found between the 2 sets of measurements (r=0.582; P=0.0112). Individual values of 8-iso-PGF₂ fell within the normal range in all NIDDM patients treated with vitamin E.

View this table: [in this window] [in a new window]   Table 2. Concentrations of Vitamin E in Plasma, Vitamin E Content of LDL, and Lag Time for LDL Oxidation in NIDDM Patients Before and After Vitamin E Supplementation

View larger version (16K): [in this window] [in a new window]   Figure 5. Effects of vitamin E supplementation on urinary excretion of 8-iso-PGF2 (bottom) and 11-dehydro-TXB2 (top) in NIDDM patients. Eicosanoid levels were measured in 10 NIDDM patients twice before starting supplementation (baseline) and at the end of 2-week daily dosing with 600 mg vitamin E. Solid bars depict mean±1 SD values for the whole group of patients. *P=0.005 for comparison of vitamin E vs baseline for both eicosanoids.

	Discussion

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We have previously demonstrated enhanced TXA₂ biosynthesis in NIDDM patients and provided evidence for its platelet origin and its reduction in response to tight metabolic control.¹⁷ Furthermore, a recently completed study²¹ suggests that the metabolic disorder rather than the attendant vascular disease is responsible for persistent platelet activation in NIDDM. In the present report, we performed a series of studies to test the hypothesis that persistent platelet activation may, at least in part, be related to enhanced formation of biologically active products of arachidonic acid peroxidation.

Previous evidence for enhanced lipid peroxidation in diabetes mellitus is largely indirect and based on crude measurements of lipid oxidation products in plasma^{22 23 24} or the susceptibility of the patient's LDL to oxidation in vitro.^{25 26 27} Our study has used the urinary excretion of the F₂-isoprostane 8-iso-PGF₂ as a marker of in vivo lipid peroxidation.⁷

We found that the formation and urinary excretion of 8-iso-PGF₂ was abnormally elevated in the vast majority of a relatively large group of NIDDM patients carefully characterized for other variables potentially influencing lipid peroxidation. Thus we excluded the contribution of advanced age by adequate age-matching of individual patients and control subjects and of cigarette smoking by only recruiting nonsmokers into the study. Similarly, differences in 8-iso-PGF₂ formation between diabetic patients and healthy subjects could not be accounted for by the presence of macrovascular complications, arterial hypertension, or hypercholesterolemia.

We found a highly significant correlation between blood glucose and urinary 8-iso-PGF₂, suggesting that lipid peroxidation may be, at least in part, related to the determinants of glycemic control. This observation is consistent with the in vitro findings of Natarajan et al,²⁸ who demonstrated enhanced formation and release of 8-iso-PGF₂ by porcine vascular smooth muscle cells cultured under hyperglycemic conditions. Gopaul et al²⁹ previously reported increased plasma levels of esterified 8-iso-PGF₂ in NIDDM that did not correlate with fasting glucose or with HbA_1c. Whether the greater variability in plasma versus urinary measurements of this F₂-isoprostane or the small sample size of the latter study contribute to this apparent discrepancy remains unanswered by the present study.

That impaired glycemic control rather than the attendant macrovascular complications is responsible for enhanced formation of F₂-isoprostanes in NIDDM is also supported by the similar findings in IDDM patients (Figure 2). Catella-Lawson et al³⁰ have recently reported a trend toward increased urinary 8-iso-PGF₂ excretion in a group of 18 diabetics, with statistically significant elevations in patients with diabetic ketoacidosis.

We further examined the relation between metabolic control and F₂-isoprostane formation by studying 21 NIDDM patients with inadequate glycemic control, before and after intensive antidiabetic treatment, and closer monitoring. Reduced blood glucose levels were associated with a fall in urinary 8-iso-PGF₂ excretion rates, the average extent of which showed a remarkably good fitting, with the linear relation between blood glucose and urinary 8-iso-PGF₂, as established in the whole group of NIDDM patients under baseline conditions. Improvement of metabolic control in these patients was accompanied by a statistically significant reduction in TXM excretion by 41% as compared with 45% in the previous study carried out with a similar protocol.¹⁷ On the basis of these findings, it is tempting to speculate that changes in the rate of arachidonate peroxidation to form biologically active iso-eicosanoids, such as 8-iso-PGF₂, may represent an important biochemical link between altered glycemic control, oxidant stress, and platelet activation in NIDDM.

That changes in F₂-isoprostane formation are not merely a consequence of persistent platelet activation is clearly indicated by the study with 2 structurally unrelated cyclooxygenase inhibitors. Thus 70% reduction in TXM excretion-corresponding to complete normalization of TXA₂ biosynthesis-was not accompanied by any detectable change in urinary 8-iso-PGF₂ excretion. This finding is consistent with the noncyclooxygenase mechanism of formation of 8-iso-PGF₂ in other clinical settings characterized by enhanced lipid peroxidation.^{7 14}

Having established that formation of 8-iso-PGF₂ is largely independent of cyclooxygenase activity, we set out to assess the reversibility of its increase in response to vitamin E supplementation. We choose a pharmacological dose of vitamin E, 600 mg daily, based on the results of a similar study performed in hypercholesterolemic patients¹⁴ demonstrating dose-dependent reduction in F₂-isoprostane formation in response to short-term vitamin E supplementation. Similar to the results obtained in hypercholesterolemic patients, we found virtually complete normalization of 8-iso-PGF₂ excretion in NIDDM in the present study. Moreover, changes in F₂-isoprostane formation were accompanied by similar reductions in TXM excretion, consistent with a cause-and-effect relation between enhanced lipid peroxidation and persistent platelet activation in this setting.

Concentrations of 8-iso-PGF₂ in the range of 1 nmol/L to 1 µmol/L induce a dose-dependent increase in platelet shape change, calcium release from intracellular stores, and inositol phosphates.^{9 10 11} Moreover, 8-iso-PGF₂ increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of nitric oxide.³¹ Furthermore, 8-iso-PGF₂ causes dose-dependent, irreversible platelet aggregation in the presence of concentrations of collagen, ADP, arachidonic acid, and PGH₂/TXA₂ analogues that when acting alone fail to aggregate platelets.¹¹ Although platelet-active concentrations of 8-iso-PGF₂ may not be achieved in circulating blood, it should be pointed out that this compound is but one of a series of biologically active iso-eicosanoids formed through a similar noncyclooxygenase mechanism of lipid peroxidation.³²

We believe that our results have potential clinical implications for the prevention of atherothrombosis in diabetic patients. Given the relatively limited impact of conventional antiplatelet prophylaxis in this setting,³³ the present findings suggest the opportunity of exploring combined preventive strategies based on the combination of low-dose aspirin and vitamin E supplementation. The urinary excretion of 8-iso-PGF₂ provides a noninvasive, reproducible biochemical end point for dose-finding studies of vitamin E supplementation.⁷ This may help resolve the present uncertainty about the optimal dose of vitamin E and provide a more rational basis for dose selection for large-scale intervention trials.

	Acknowledgments

 
This study was supported by grants from Progetto Strategico "Infarto," Consiglio Nazionale delle Ricerche (CNR) (96.052685.T74 and 97.04882.ST74), and Progetto Finalizzato Prevenzione e Controllo dei Fattori di Malattia, CNR (SP8: 95.00882.PF41 and 95.00807.PF41). We wish to thank Domenico De Cesare, Salvatore Roccaforte, Antonio Colangelo, Andrea Salvati, Antonina Legnino, Lia Cucco, and Lorella Cesarone for assistance with the clinical studies.

Received June 18, 1998; revision received September 18, 1998; accepted October 1, 1998.

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