The Influence of Pravastatin and Atorvastatin on Markers of Oxidative Stress in Hypercholesterolemic Humans Bonnie Ky, MD,* Anne Burke, MD,* Sotirios Tsimikas, MD, Mahlet G. Tadesse, SCD,* Philippe O. Szapary, MD, etc Philadelphia, Pennsylvania Vol. 51, No. 17, 2008, JACC

Background  HMG-Co A reductase inhibitors reduce LDL-C & Cardiovascular risk

 Effects on circulating biomarkers of reactive oxidative stress are not well-defined  Smaller studies : Statin therapy might reduce the urinary excretion or plasma levels of the isoprostane 8 - iso - IPF2-III

 Impact of statins on oxidative stress Many of the aforementioned studies : not well controlled and require confirmation with rigorous methodology The question of statin dose-response on oxidative stress has not been addressed. Equivalent doses of statins have never been compared with each other in a human study with regard to effects on oxidative stress

Objectives  Determine the effects of pravastatin and atorvastatin on markers of oxidative stress in plasma.

Methods  Subjects  120 healthy subjects ( 21 and 80 years )  LDL-C ; 130 ~ 220 mg/dl ( no lipid-lowering therapy )  Exclusion criteria  Exposure to cholesterol-modulating drugs ( 6wks )  Known CHD, diabetes or fasting glucose 126 mg/dl  Significant renal or hepatic disease  Triglycerides 500 mg/dl  Familial hypercholesterolemia  Pregnant or lactating women

Study design  Randomization in a 1:1:1:1  Pravastatin 40 mg/day ( Prava40 )  Atorvastatin 10 mg/day ( Atorva10 )  Atorvastatin 80 mg/day ( Atorva80 )  Placebo  After 8wks & 16 wks  Fasting blood work  12-h urine collection  Adverse event reporting & study medication  Laboratory analyses

Laboratory measurements  Lipid parameters  Plasma total cholesterol ( TC )  High-density lipoprotein cholesterol ( HDL-C )  TG levels  LDL-C  VLDL levels  Apolipoprotein B ( apoB )  Concentrations of oxidized LDL ( OxLDL )  Phospholipase A2 ( Lp-PLA2 )  Content of OxPL/apoB  OxPL/apoB & IC/apoB  8, 12-iso-iPF2-VI  the most abundant isoprostane detected in human urine

Results  Baseline characteristics and study completion  Lipid parameters  Urinary 8, 12-iso-iPF2-VI  Oxidized LDL  Lp-PLA2  OxPL/apoB assay  Autoantibodies to MDA-LDL and apoB immune complexes  Correlation between markers of oxidative stress at baseline  Correlation between changes in markers of oxidative stress after statin therapy

Baseline characteristics & study completion

Lipid parameters  No significant differences in baseline lipid values among the groups.  All statin-treated groups experienced significant reductions in TC, LDL-C, apoB, and non–HDL-C

Urinary 8, 12-iso-iPF2-VI  No significant difference in the log transformed excretion of the F2 isoprostane across the 4 treatment groups

Oxidized LDL (Mercodia assay)  No significant differences in baseline OxLDL  After 16 weeks: all statin-treated groups experienced a reduction in OxLDL

Lp-PLA2  Both pravastatin and atorvastatin led to significant decreases in Lp-PLA2 mass  Lp-PLA2 mass correlated strongly with LDL-C and apoB levels atorva10 & prava40 remained significant

OxPL/apoB assay  No statistically significant differences in OxPL/apoB at baseline  After 16 weeks : significant increases were noted across the 3 groups with prava40, atorva80 increase in OxPL/apoB  Lp(a) increased significantly in parallel with OxPL/apoB:

Autoantibodies to MDA-LDL and apoB immune complexes  No statistically significant differences in MDA-LDL autoantibodies or IC formation at baseline.  After 16 weeks : statin therapy there were no significant changes in IgG or IgM autoantibodies to MDA-LDL or IgG or IgM IC/apoB  All 3 statin-treated arms led to a trend in reduction in total apoB-IC content

Correlation between markers of oxidative stress at baseline  Mercodia OxLDL correlated strongly with apoB & LDL at baseline

Correlation between changes in markers of oxidative stress after statin therapy  Changes in Mercodia OxLDL strongly correlated with changes in apoB, LDL-C, Lp-PLA2

Discussion  Statins reduced LDL-C and non–HDL-C as expected but had variable effects on levels of several distinct biomarkers of oxidative stress  Isoprostanes : sensitive and reliable measures of lipid peroxidation in vivo

 Possible explanations for the lack of change in isoprostane excretion in our study  The most likely is that statin therapy did not suppress lipid peroxidation in this population  Only when endogenous antioxidant defenses are depleted  In spite of the study randomization, the distribution of smokers > greater proportion in the placebo arm  The Mercodia OxLDL assay

 Bruneck study : OxPL/apoB were significantly associated with cardiovascular events  Statistically significant increase in OxPL/apoB has been observed in response to diet or treatment with atorvastatin or pravastatin > paradoxical  During early atherosclerosis regression, the increase in OxPL/apoB levels in plasma might reflect a beneficial effect of diet and perhaps statins on the vessel wall

 Lp-PL A2 : proatherogenic and proinflammatory  Levels of Lp-PLA2 : strong association with cardiovascular risk  Lp- PLA2 activity potentiated the increased risk of death and cardiac events : associated with elevated OxPL/apoB or Lp(a)

Study limitations  Prospective study and the first to measure all these oxidative biomarkers in the same subset  Modest size  These findings will need to be validated in larger studies

Conclusions  Statin therapy results in variable effects on oxidative stress markers in hypercholesterolemic subjects.  Future outcome studies should collectively assess various oxidative markers to define clinical utility