Journal Logo

Original Article

Neutrophil Superoxide Anion Generation During Atorvastatin and Fluvastatin Therapy Used in Coronary Heart Disease Primary Prevention

Kowalski, Jan MD, PhD*; Barylski, Marcin MD*; Banach, Maciej MD, PhD; Grycewicz, Joanna MD*; Irzmański, Robert MD, PhD; Pawlicki, Lucjan MD, PhD

Author Information
Journal of Cardiovascular Pharmacology: October 2006 - Volume 48 - Issue 4 - p 143-147
doi: 10.1097/01.fjc.0000246150.52382.07
  • Free

Abstract

Within the past two decades, statins have become the treatment of choice for patients with hyperlipidemia and are the most prescribed class of lipid-lowering agents. These drugs are used in both primary and secondary prevention of coronary heart disease (CHD).1-5 Statins have numerous actions beyond lipid modulation, such as pleiotropic effects. They affect targets as diverse as cell functions, coagulation, fibrinolysis, the immune system, and oxidative processes.6-9

Considering the morphotic blood elements taking part in pathogenesis of coronary heart disease and atherosclerosis, the role of platelets has been documented very well. However, an increasing number of reports are paying attention to the role of neutrophils in the initiation of these changes.10-12 The destructive activity of these cells is connected with the fact that they release proteolytic enzymes and metabolites of oxidative metabolism, which can influence vessel permeability, coronary blood flow regulation, and lipid peroxidation, leading to coronary endothelial cell damage. On the other hand, an increase in proadhesive and proaggregative properties of neutrophils resulting from hypoxia leads to disturbances of blood flow in blocked capillary vessels. This is the cause of an increase in the vessel resistance, intensity of hypoxia, as well as disturbance of endothelial mechanisms of blood flow regulation.12

Superoxide anions are the first link in the chain of free radical reactions responsible for, among others, lipid peroxidation, inhibition of prostacyclin synthesis, and disturbance in the production of nitric oxide (NO), whose fundamental biological function is relaxation of smooth muscles and inhibition of platelet aggregation.13-15 Disturbances of these functions are observed in CHD.

The aim of our study was to measure neutrophil superoxide anion generation in patients with CHD risk before and after atorvastatin and fluvastatin therapy.

PATIENTS AND METHODS

The study comprised 35 patients (20 men, 15 women, aged 35-47 years, mean 41.3) with CHD risk (mixed hyperlipidemia, body mass index >25 kg/m2, low physical activity, family history of CHD) after 4-week hypolipemic diet. The following criteria of inclusion were observed: total cholesterol (TC) >300 mg/dL, low density lipoprotein (LDL) >170 mg/dL, and triglycerides (TG) >200 mg/dL.

The patients were randomly allotted into three groups. The atorvastatin (Sortis, Pfizer Group, Berlin, Germany) group comprised 17 patients who were administered the drug orally in a daily dose of 10 mg at bed time. The fluvastatin (Lescol, Novartis, Basel, Switzerland) group consisted of 18 patients on an oral dose of 40 mg once daily at bed time. The control group comprised 12 healthy subjects with no drug administration. Our study has been approved by the Local Ethics Committee. The recommendations of the Declaration of Helsinki were observed by the physician taking blood samples. Women who were administered the statins used contraceptive drugs. Blood samples were collected from cubital vein before and after 6-week therapy and once in the control group.

Data were analyzed for statistical significance using the Student's t-test for independent groups. Values are expressed as means ± SD. The probability value of P < 0.05 was considered significant. The comparisons between each measurement were performed with the t-test for paired data.

O2 Generation

Superoxide anion O2 generation in the whole blood without and with stimulation by opsonized zymosan (OZ) (Sigma Chemical Co; 1 mg per 1 mL of blood) was determined according to Bellavite et al16 using superoxide dismutase (SOD-1) from bovine erythrocytes (Sigma Chemical Co).

Examination was performed in three tubes labeled as “Control,” “Rest,” and “Stimulated” and 0.3 ml cytochrome C (Sigma Chemical Co) was added to each tube. Then 0.2 ml phosphate-buffered saline (PBS; Polfa, Kutno, Poland) was added to the “Control” and “Rest” tubes and 0.1 ml PBS and 1 mg OZ were added to the “Stimulated” tube. The tubes were preincubated in a water bath for 5 minutes at 37°C. Then 2 ml SOD-1 (3000 U/ml) and 0.1 ml examined blood sample were added to the “Control” tube and the tube was placed on ice for 10 minutes; 0.1 ml examined blood was added to the tubes “Rest” and “Stimulated” and they were incubated in a water bath for 10 minutes. Then 2 ml SOD-1 was added to these tubes and all three tubes were centrifuged for 10 min at 2,000 rpm at 4°C. The absorbance of cell-free supernatants was then measured at 550 nm. The results were expressed as nmol of O2/cell/min.

RESULTS

In CHD-risk patients who were subjected to fluvastatin therapy (after 4-week hypolipemic diet), O2·− generation by nonstimulated and OZ-stimulated whole blood neutrophils was 6.37 ± 2.78 and 13.69 ± 5.08 nmol/cell/min, respectively. It was significantly lower (P < 0.05) than in healthy subjects (8.01 ± 2.33 and 15.87 ± 3.01 nmol/cell/min; Figs. 1 and 2).

FIGURE 1
FIGURE 1:
The effect of fluvastatin on O2 generation by nonstimulated neutrophils.
FIGURE 2
FIGURE 2:
The effect of fluvastatin on O2 generation by OZ-stimulated neutrophils.

After 6 weeks of fluvastatin therapy, O2 generation by nonstimulated and OZ-stimulated whole blood neutrophils was 7.21 ± 3.16 and 14.65 ± 6.09 nmol/cell/min, respectively, and did not differ from that observed in healthy subjects (Figs. 1 and 2).

In patients who were subjected to atorvastatin therapy (after a 4-week hypolipemic diet), O2 generation by nonstimulated and OZ-stimulated whole blood neutrophils was 6.88 ± 2.41 and 14.01 ± 3.55 nmol/cell/min, respectively. It was lower (P > 0.05) than that observed in healthy subjects (8.01 ± 2.33 and 15.87 ± 3.01, nmol/cell/min; Figs. 3 and 4).

FIGURE 3
FIGURE 3:
The effect of atorvastatin on O2 generation by nonstimulated neutrophils.
FIGURE 4
FIGURE 4:
The effect of atorvastatin on O2 generation by OZ-stimulated neutrophils.

After 6 weeks of atorvastatin therapy O2 generation by nonstimulated and OZ-stimulated whole blood neutrophils decreased in comparison to the initial values (P > 0.05; 5.23 ± 2.74 and 12.96 ± 3.10 nmol/cell/min, respectively) and was significantly lower (P < 0.05) than in healthy subjects (Figs. 3 and 4).

In patients who were subjected to statins therapy, we observed the following decrease in levels of TC, LDL-C, and TG: in the group with fluvastatin: 23.1%, −27.2%, and −36.0%, respectively, and in the group with atorvastatin: 26.3%, −32.5%, and −40.4%, respectively.

DISCUSSION

The majority of trials concerning therapy with statins were associated with CHD secondary prevention.1,3,17 Beneficial clinical effects of the therapy with this group of drugs have also been found in the programs of primary prevention. So far, two large multicenter, prospective, randomized, double-blind controlled trials have been published: West of Scotland Coronary Prevention Study (WOSCOPS) with pravastatin and Airforce/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/Tex CAPS) with lovastatin.2,5

WOSCOPS was designed to assess the effect of 5-year pravastatin treatment on coronary events in hyperlipidemic patients with no signs of myocardial infarction in the medical history. Pravastatin therapy resulted in the decrease in total cholesterol and LDL cholesterol levels by 20% and 26%, respectively, coronary events rate, nonfatal myocardial infarction risk by 31%, and CHD death by 32%. Total mortality was decreased by 22%.2

AFCAPS/TexCAPS was a primary prevention study in asymptomatic subjects with average total cholesterol and LDL cholesterol levels and below average HDL cholesterol levels with no CHD (atherogenic dyslipidemia). The study was designed to assess the efficacy of lovastatin in primary prevention of acute coronary events, which are defined as myocardial infarction, unstable angina, or sudden cardiac death. After 5 years of lovastatin therapy, decreases in LDL cholesterol level by 25% and triglycerides level by 18%, and an increase in HDL cholesterol level by 6% were found. Moreover, a significant risk reduction in the incidence of the first acute coronary event (myocardial infarction, unstable angina) was seen with lovastatin therapy. These effects were similar in women and men, and were observed from the first year of therapy. No significant differences in total mortality were observed between lovastatin and placebo group.5

Analysis of a number of trials (WOSCOPS, CARE) has demonstrated that clinical benefits from statins therapy appear earlier and are more pronounced than in other interventions lowering LDL-cholesterol concentration. Thus, a hypothesis has been advanced on beneficial pleiotropic effect of this group of drugs on atheromatous plaques stabilization exerted independently on or in addition to the hypolipemizing effect.

In our study, we have investigated the effect of fluvastatin and atorvastatin on whole blood neutrophil superoxide anion generation in patients with CHD risk.

According to inflammatory theory of atherogenesis, endothelial injury results in neutrophil activation.18-20 Enhanced active oxygen forms generation by these cells, and when the antioxidative systems are exhausted, leads to oxidative stress. When generated in excess, superoxide anion, hydrogen peroxide, or hydroxyl radical are able to oxidize lipid components of cell membrane and thus impair their functions as a biological barrier and inactivation of enzyme active centers. Superoxide anion, the first link of oxygen free-radical reaction chain, is thought to inhibit prostacyclin synthesis and endothelial nitric oxide synthase (eNOS) but activate inducible nitric oxide synthase (iNOS) and platelet aggregation. Superoxide anion is also thought to be capable of peroxidation of lipids found in excess in hyperlipidemic patients' blood.13-15

Our studies have demonstrated that peripheral blood neutrophils in patients with CHD risk generate less superoxide anion than healthy subjects. This concerned both nonstimulated as well as OZ-stimulated neutrophils.

The majority of studies on disturbed neutrophils functions were conducted in patients with documented CHD. Kowalski et al and Wysocki et al paid attention to neutrophil activation resulting in enhanced oxygen free radical generation in myocardial infarction, unstable and stable angina.12,21,22

There are not many studies concerning this problem in dyslipidemic patients. Using ultrastructural studies, morphological changes in neutrophils of hyperlipidemic children were found. The authors suggest that lipid overload results in inhibition of their directed movement.23 Hawley estimated correlation between neutrophil activity and triglycerides components. He showed that incubation of human neutrophils with various palmitic acid concentrations inhibited significantly chemotaxis. Incubation in high concentration resulted also in impairment of phagocytosis and bactericidal activity. These functional changes were accompanied by structural ones: elongated cleft-like dilation of cytoplasmic reticulum and degenerative degranulated cytoplasmic areas.24

Triglycerides, especially, are thought to be responsible for functional neutrophil impairment because of their effect on cell membrane and receptor blocking. Another theory suggests that structural changes in neutrophil membrane caused by increased triglycerides and cholesterol levels result in inhibition of cells surface receptors expression.

The results of our studies showing decreased superoxide anion generation by neutrophils in patients with mixed hyperlipidemia are in agreement with literature.

In the next stage of the study, superoxide anion generation by nonstimulated and OZ-stimulated neutrophils after 6 weeks of fluvastatin or atorvastatin therapy was estimated. In patients treated with fluvastatin, superoxide anion generation by nonstimulated and OZ-stimulated neutrophils was increased in comparison to the initial value and was comparable to that in healthy subjects. In patients treated with atorvastatin, superoxide anion generation by nonstimulated and OZ-stimulated whole blood neutrophils was still decreasing in comparison to the initial value and was significantly lower than in healthy subjects. The same direction of changes was found in superoxide anion generation by OZ-stimulated neutrophils.

Having analyzed the above-mentioned results, it could be concluded in general that fluvastatin has no significant effect on neutrophil superoxide anion generation. Increase in superoxide anion generation, observed in fluvastatin patients, might result from improvement of neutrophil activity and function related to the decrease in plasma lipid levels. These results support previous outcomes of authors; however, they are inconsistent with the observations in other available studies regarding antioxidant effect of fluvastatin.25

A diverse direction of changes was observed in patients treated with atorvastatin, suggesting that the drug diminishes superoxide anion generation by whole blood neutrophils.

Investigators have suggested that fluvastatin does not scavenge free radicals or inhibit linoleic acid peroxidation, but rather specifically binds mainly to LDL surface phospholipids. Hussein et al observed in 10 hypercholesterolemic patients that fluvastatin therapy within 24 weeks resulted in inhibition of LDL oxidizability.26 Moreover, they showed that fluvastatin specifically binds to surface phospholipids and slightly to LDL cholesterol. This interaction altered electrical charge of this lipoprotein, resulting in a 38% decrease in electrophoretic mobility of fluvastatin-treated LDL in comparison to nontreated LDL. This drug effect was observed just after 4 weeks of therapy. Binding of fluvastatin to LDL surface can prevent the diffusion of free radicals, which are generated under oxidative stress into lipoprotein molecule.

Tanaka et al27 and Suzumura et al28-30 have investigated racemate and two enantiomers of fluvastatin that, to different extents, inhibit HMG-Co reductase (3R,5S and 3S,5R). They showed that antioxidative effects of each of them were comparable. From fluvastatin metabolites, the most potent antioxidative effect was shown to M2 and M3, and it was three times more potent than that of fluvastatin.27-29 Nakashimo et al have shown that M2 and M3 metabolites were over 30 times more efficient in LDL oxidation inhibition than the nonmetabolized drug form. Their antioxidative effect was comparable to that of probucol.31

Aviram et al investigated atorvastatin metabolite's free-radical scavenging capacity as well as their metal ion chelation capacity.32 After using o-hydroxy and p-hydroxy atorvastatin metabolites, the impairment of LDL oxidation by 44% and 34%, respectively as well as increase in TBARS by 59% and 46%, respectively were found. Moreover, they showed prolongated time required for the initiation of LDL oxidation. Antioxidative activity of metabolites is thought to be related to the additional hydroxyl group in these molecules. Hydroxyl group hydrogen atom acts as a reductor in free-radical reactions. The ortho position of hydroxyl group decides about more potent reducing properties. Atorvastatin metabolites in these studies also inhibited VLDL and HDL oxidation.

Our study showed that atorvastatin, but not fluvastatin, acts as antioxidant through decrease in superoxide anion generation. However, in our other study, we observed the increase of erythrocyte antioxidative enzymatic activity (SOD-1, GSH-Px) after treatment with fluvastatin.33 In our opinion, the different mechanism of action could be responsible for antioxidative activity of fluvastatin.

REFERENCES

1. Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383-1389.
2. Shepherd J, Cobbe SM, Ford I, et al. For the West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301-1307.
3. Sacks FM, Moye LA, Davis BR, et al. Relationship between plasma LDL concentrations during treatment with pravastatin and recurrent coronary events in Cholesterol and Recurrent Events Trial. Circulation. 1998:97:1446-1452.
4. Banach M. The role of simvastatin in ischemic heart disease prevention and its complications. Geriatr Pol. 2005;1:45-53.
5. Downs JR, Clearfield M, Weis S, et al. For the AFCAPS/TexCAPS Research Group. Primary prevention of acute coronary events with lovastatin in men and women with average cholesterol levels. JAMA. 1998;279:1615-1622.
6. Rosenson RS, Tangney CC. Antiatherotrombotic properties of statins: implications for cardiovascular event reduction. JAMA. 1998;279:1643-1650.
7. Banach M, Drozdz J, Okonski P, et al. Immunological aspects of the statins' function in patients with heart failure. Cell Mol Immunol. 2005;2:433-437.
8. Szczeklik A, Musiał J, Undas A, et al. Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia. J Am Coll Cardiol. 1999;33:1286-1293.
9. Giroux LM, Davignon J, Naruszewicz M. Simvastatin inhibits the oxidation of low-density lipoproteins by activated human monocyte derived macrophages. Biochim Biophys Acta. 1993;1165:335-338.
10. Kurtel H, Fujimoto K, Zimmerman BJ, et al. Ischaemia-reperfusion-induced mucosal dysfunction - role of neutrophils. Am J Physiol. 1991;261:G490-G496.
11. Siminiak T, Ozawa T. Neutrophil mediated myocardial injury. Int J Biochem. 1993;25:147-156.
12. Kowalski J, Kośmider M, Pawlicki L, et al. The selected functions of neutrophils in patients with unstable angina. Int Rev Allergol Clin Immunol. 1995;1:19-23.
13. O'Brien K, Chait A. The biology of the artery wall in atherogenesis. Lipid Disord. 1994;78:41-46.
14. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. Brit Med Bull. 1993;49:481-493.
15. Rysz J, Banach M, Stolarek R, et al. The lipid disorders therapy in overweight patients. Arch Med Sci. 2005;1:241-245.
16. Bellavite P, Dri P, Della Bianca V, et al. The measurement of superoxide anion production by granulocytes in whole blood. A clinical test for the evaluation of fagocyte function and serum opsonic capacity. Eur J Clin Invest. 1983;13:363-368.
17. Tonkin AM. Management of the Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) study after the Scandinavian Simvastatin Survival Study (4S). Am J Cardiol. 1995;76:107C-112C.
18. De Servi S, Ricevuti G, Fioravanti A, et al. Granulocyte function in coronary artery disease. Am J Cardiol. 1991;68:64B-69B.
19. Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of low-density lipoproteins that increases its atherogenicity. N Engl J Med. 1989;320:915-924.
20. Kazuya T, Fuji H, Hoshida S, et al. Polymorphonuclear leukocytes - induced injury in hypoxic cardiac myocytes. Free Radic Biol Med. 1994;17:501-510.
21. Siminiak T, Zozulinska D, Zeromska M, et al. Evidence for plasma-mediated neutrophil superoxide anion production during myocardial infarction. Cardiology. 1993;82:377-382.
22. Kowalski J, Kośmider M, Pawlicki L, et al. Complement activates neutrophils during PTCA procedure in patients with unstable angina pectoris. Int J Cardiol. 1997;58:229-240.
23. Itoch H. Neutrophilic polymorphonuclear leukocytes in obese children. Studies by sudan black B and electron microscope. Acta Pathol Jpn. 1981;31:167-171.
24. Hawley HP, Gordon GB. The effects of long chain free fatty acids on human neutrophil function and structure. Lab Invest. 1976;34:216-222.
25. Kowalski J, Pawlicki L, Grycewicz J, et al. Estimation of antioxidative effect of atorvastatin and fluvastatin used in primary prevention of coronary heart disease-effect on lipid peroxidation. Wiad Lek. 2005;58:386-390.
26. Hussein O, Schlezinger S, Rosenblat M, et al. Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after fluvastatin therapy is associated with the hypocholesterolemic effect of the drug and its binding to the LDL. Atherosclerosis. 1997;128:11-18.
27. Tanaka K, Yasuhara M, Suzumura K, et al. Effects of fluvastatin and its major metabolites on low density lipoprotein oxidation and cholesterol estrification in macrophages. Jpn J Pharmacol. 2001;86:289-296.
28. Suzumura K, Odawara A, Yasuhara M, et al. In vitro inhibitory effects of the optical isomers and metabolites of fluvastatin on copper ion - induced LDL oxidation. Biol Pharm Bull. 1999;22:971-974.
29. Suzumura K, Yasuhara M, Narita H, et al. Superoxide anion scavenging properties of fluvastatin and its metabolites. Chem Pharm Bull (Tokyo). 1999;47:1477-1480.
30. Suzumura K, Yasuhara M, Tanaka K, et al. An in vitro study of the hydroxyl radical scavenging property of fluvastatin an HMG-CoA reductase inhibitor. Chem Pharm Bull (Tokyo). 1999;47:1010-1012.
31. Nakashima A, Ohtawa M, Masuda N. Antioxidative effects of fluvastatin and its major metabolites. Yakugaku - Zasshi. 1999;119:93-99.
32. Aviram M, Rosenblat M, Bisgaier CL, et al. Atorvastatin and gemfibrozil metabolites, but not the parent drugs are potent antioxidants against lipoprotein oxidation. Atherosclerosis. 1998;138:271-280.
33. Kowalski J, Pawlicki L, Grycewicz J, et al. Estimation of antioxidative effect of atorvastatin and fluvastatin used in primary prevention of coronary heart disease - effect on antioxidative enzymatic activity. Wiad Lek. 2005;58:275-279.
Keywords:

atorvastatin; coronary heart disease; fluvastatin; neutrophil superoxide anion generation; prevention

© 2006 Lippincott Williams & Wilkins, Inc.