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Division of Clinical Chemistry (K.W., I.F., M.M.H., H.W.), Department of Medicine, Albert Ludwigs-University, D-79106 Freiburg, Germany; Novartis Pharma GmbH (C.A.), D-90327 Nürnberg, Germany; and Department of Clinical Chemistry (W.M.), University of Graz, A-8036 Austria
Address all correspondence and requests for reprints to: Karl Winkler, M.D., Department of Medicine, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail: kwinkler{at}.ukl.uni-freiburg.de.
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Abstract |
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A multicenter, double-blind, randomized comparison of fluvastatin XL (80 mg) (n = 42) and placebo (n = 47), each given once-daily for 8 wk, in 89 patients with T2DM, was conducted. At baseline and on treatment, lipoproteins, including lipoprotein (a) [Lp(a)] and LDL subfractions, and the activity of PAF-AH were measured.
Increasing PAF-AH activity was significantly associated with a positive history of CAD (+0.7% per IU/liter PAH-AH; P = 0.010), the odds ratio estimate adjusted for age, gender, and body mass index of the highest quartile being 10.6 (P = 0.036). At baseline and at study end, PAF-AH activity was associated with the apolipoprotein B (apoB) content in dLDL (LDL-5 and LDL-6) (r = 0.447; P < 0.001 and r = 0.651; P < 0.001, respectively) and with non-HDL cholesterol at baseline (r = 0.485; P < 0.001). However, after additional adjustment for apoB in dLDL and non-HDL cholesterol at baseline, the odds ratio increment for CAD across PAF-AH quartiles was 2.09 (95% confidence interval, 1.02–4.29; P = 0.043). Fluvastatin treatment decreased the activity of PAF-AH by 22.8% compared with an increase of 0.4% in the placebo group (P < 0.001). This effect was independent of changes of Lp(a) concentrations.
In patients with T2DM, PAF-AH activity is associated with a positive history of CAD. Fluvastatin not only decreases atherogenic dLDL but also PAF-AH activity, emphasizing the significance of fluvastatin treatment in T2DM.
The antiatherogenic potential of fluvastatin in T2DM may thus be greater than expected from its effects on LDL-C and triglycerides alone.
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Introduction |
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Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammatory diseases (6) as well as in atherogenesis (7). In plasma, PAF is hydrolized and inactivated by PAF-acetylhydrolase (PAF-AH, EC 3.1.1.47), a Ca2+-independent phospholipase A2 (8). Plasma PAF-AH is complexed to lipoproteins in vivo (9, 10); thus it is also denoted as lipoprotein-associated phospholipase A2 (11).
Several reports link PAF-AH to atherogenesis and increased CAD risk (12, 13). However, the role of this enzyme in inflammatory and atherosclerotic diseases still remains to be established; PAF-AH may represent a potent antiinflammatory and antiatherogenic enzyme because it degrades PAF and proinflammatory oxidized phospholipids (PL) (14). To the contrary, PAF-AH may also generate bioactive oxidized free fatty acids (11) and lysophosphatidylcholine (15). In fact, phospholipase A2/PAF-AH activity liberates arachidonic acid, a precursor of eicosanoids including prostaglandins and leukotrienes (16), thus exerting proinflammatory and proatherogenic actions. A recent clinical study is in support of the latter view: In a nested case-control evaluation of 1160 patients of the West of Scotland Coronary Prevention (WOSCOP) Study, elevated PAF-AH levels appeared to be a strong independent risk factor for CAD (17). The observation that PAF-AH inhibition in rabbits slows atherosclerosis progression provided further support for the association of PAF-AH with atherogenesis (18).
In patients with T2DM, there is a preponderance of atherogenic dense low-density lipoprotein (dLDL) (19, 20). Because PAF-AH activity resides on dLDL (9, 15), this enzyme activity may play a crucial role for CAD risk, particularly in T2DM. This is supported by a study that shows that PAF-AH activity is increased in patients with T2DM compared with patients with dyslipidemia or healthy control subjects (21).
Recently it was shown that fluvastatin XL reduced the concentration of dLDL in patients with T2DM (20). Taking the same study population, we now assess the effect of fluvastatin on PAF-AH activity. However, because it was shown that PAF-AH activity may reside on lipoprotein (a) [Lp)(a)] as well (22), we also evaluate whether a putative effect of fluvastatin on PAF-AH activity may be related to changes of Lp(a) concentrations. To our knowledge, this is the first study that investigates the effect of a statin on PAF-AH activity in a placebo-controlled setup.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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This was a double-blind, randomized, placebo-controlled, 12-wk study comprising a 4-wk dietary run-in phase followed by a treatment period of 8 wk on fluvastatin XL (80 mg) (n = 42) or placebo (n = 47) as reported previously (20). T2DM [glycosylated hemoglobin, 6–10% (mean, 7.2 ± 1.0%)] had to be diagnosed at least 12 wk before the dietary run-in. The study protocol was approved by the Ethics Committee of the University of Freiburg, Germany, and the institutional review boards at each study center. All patients gave their informed, written consent.
Laboratory assessments
Before randomization (wk -4) and after 8 wk of treatment, venous blood was sampled from fasted patients and subsequently assayed for biochemistry. LDL subfractions were measured at baseline (wk 0) and after 8 wk of treatment. All laboratory procedures were performed in the Department of Clinical Chemistry, University of Freiburg, Germany.
Measurement of PAF-AH activity
Measurement of PAF-AH activity was performed as described by Kosaka et al. (23) by the Azwell Auto PAF-AH kit (Azwell Inc., Osaka): PAF-AH hydrolizes the sn-2 position of the substrate [1-myristoyl-2-(4-nitrophenyl succinyl) phosphatidylcholine], producing 4-nitrophenyl succinate, which is immediately degraded to 4-nitrophenol and subsequently measured spectrophotometrically.
Lipoprotein separation
Lipoproteins were isolated by sequential preparative ultracentrifugation using the following densities: d < 1.006 kg/liter for very LDL, 1.006 < d < 1.019 kg/liter for IDL, 1.019 < d < 1.063 kg/liter for LDL, and 1.063 < d < 1.21 kg/liter for high-density lipoprotein (HDL). LDL subfractions were separated according to Baumstark (24); total LDL (1.019 < d < 1.063 kg/liter) was fractionated into six density classes by equilibrium density gradient centrifugation. Density ranges of LDL subfractions were: LDL-1, <1.031 kg/liter; LDL-2, 1.031–1.034 kg/liter; LDL-3, 1.034–1.037 kg/liter; LDL-4, 1.037–1.040 kg/liter; LDL-5, 1.040–1.044 kg/liter; LDL-6, >1.044 kg/liter. Atherogenic LDL-5 and LDL-6 are summarized as dLDL (20). The assay of apoB in LDL-5 and LDL-6 is linear to a concentration less than 50 mg/liter, and the intraassay coefficient of variance (CV) at these concentration levels is around 2.5% (20). The interassay CV for apoB for the six LDL subfractions is 5% or less (25).
Lipoprotein chemistry
Cholesterol (CH) and triglycerides (TG) were determined enzymatically with the CHOD-PAP and the GPO-PAP method (Roche Diagnostics, Mannheim, Germany), respectively. Free cholesterol (FC) and PL were determined enzymatically with the COD-PAP method and by phospholipase D, cholineoxidase and peroxidase, respectively, with commercially available reagents (Wako Chemicals, Osaka, Japan). The concentration of esterified cholesterol was calculated from the difference of CH and FC. Concentrations of Lp(a) and apolipoproteins were determined by turbidimetry on a Olympus AU 640 analyzer (Olympus Diagnostica GmbH, Hamburg, Germany) using polyclonal antisera (Rolf Greiner Biochemica, Flacht, Germany) specific for the respective antigens. Non-HDL cholesterol was calculated as the difference of total cholesterol minus HDL cholesterol.
Stratification by baseline dLDL
Baseline data are shown according to predefined baseline dLDL level: patients were classified as having high levels of dLDL at baseline (apoB in LDL-5 and LDL-6 > 250 mg/liter) or as having low levels of dLDL at baseline (apoB in LDL-5 and LDL-6 
250 mg/liter) as described previously (20).
Mean LDL diameter
The mean diameter of LDL was calculated using the molar concentrations of FC, esterified cholesterol, PL, TG, and apoB-100 in the LDL fraction (1.019 kg/liter < d < 1.063 kg/liter) as validated by x-ray small-angle scattering (24). The intraassay CV of the mean LDL diameter is around 5% (26).
Mean LDL density
The mean density of total LDL was calculated as the weighted (by apoB-100 content) mean of the densities of each of the LDL subfractions according to the following equation: mean LDL density = [(apoB in LDL-1 x 1.025) + (apoB in LDL-2 x 1.0325) + (apoB in LDL-3 x 1.0355) + (apoB in LDL-4 x 1.0385) + (apoB in LDL-5 x 1.042) + (apoB in LDL-6 x 1.0535)]/(apoB in total LDL) (kg/liter). The intraassay CV of the mean LDL diameter is around 5% (26).
Statistical analysis
The primary aim of this study was to investigate whether fluvastatin XL (80 mg) was efficacious in reducing PAF-AH activity. Changes in PAF-AH activities between baseline (wk 0) and wk 8 were compared between treatment groups using the nonparametric Mann-Whitney U test. A positive history of CAD was defined by clinical or electrocardiographical signs of CAD, or previous myocardial infarction, angioplasty, or bypass grafting. The odds ratio estimate for CAD per unit increase of PAF-AH (IU/liter) was assessed by binary logistic regression with positive history of CAD as dependent variable and PAF-AH activity (IU/liter) as continuous covariate. In addition, the adjusted odds ratio estimate for CAD of PAF-AH quartiles as established in the patients without CAD was assessed by binary logistic regression with positive history of CAD as dependent variable; quartiles of PAF-AH activity as categorial covariate; and age, gender, and BMI as covariates. Furthermore, the odds ratio increment adjusted for age, gender, BMI, apoB in dLDL, and non-HDL cholesterol for positive history of CAD across PAF-AH quartiles was also determined by logistic regression analysis. Associations of variables were analyzed by Pearson’s correlation coefficients. Changes of Lp(a) concentrations between baseline (wk 0) and wk 8 were compared between treatment groups using the nonparametric Mann-Whitney U test. A general linear model (GLM) was used to assess whether changes in Lp(a) in total serum, in the HDL fraction, or in the dLDL fraction (LDL-5 and LDL-6) influenced the effect of fluvastatin (vs. placebo) on PAF-AH activity. PAF-AH activity was used as dependent variable, treatment group (fluvastatin or placebo) as fixed factor, and percent changes of Lp(a) concentrations (either total, in the HDL, or in the dLDL fraction) were used as covariates. Changes were considered statistically significant at P < 0.05. All calculations were performed using SPSS for Windows (version 11.0).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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At baseline, PAF-AH activity was associated with LDL size (r = -0.270; P = 0.015) (Fig. 2A) and mean LDL density (r = 0.314; P = 0.004) (Fig. 2B). In addition, the apoB content in dLDL (LDL-5 and LDL-6) at baseline (r = 0.447; P < 0.001) (Fig. 2C) and at the end of the study (r = 0.651; P = < 0.001) (Fig. 2D) was correlated with PAF-AH activity. Furthermore, PAF-AH activity was associated with non-HDL cholesterol at baseline as well (r = 0.485; P < 0.001; data not shown).
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). Even after additional adjustment for apoB in dLDL and non-HDL cholesterol, the odds ratio increment for CAD across PAF-AH quartiles was 2.09 (95% CI, 1.02–4.29; P = 0.043).
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). Lp(a) concentrations at baseline were not significantly different between treatment groups (Table 1
). From baseline to wk 8, treatment with fluvastatin resulted in a change of Lp(a) concentrations from 234 ± 320 mg/liter (Table 1) to 249 ± 360 mg/liter (+6%) (data not shown) and from 346 ± 351 mg/liter (Table 1) to 351 ± 327 mg/liter (+1%) with placebo (data not shown). These changes were not significant.
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).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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PAF-AH activity was significantly associated with a positive history of CAD; the adjusted odds ratio estimate of the highest quartile of PAF-AH activity was 10.6 compared with the reference quartile (Fig. 1). This is in accordance with previous findings (17); however, the previously reported risk of CAD associated with PAF-AH in the WOSCOP study was only about 2-fold for the highest, compared with the lowest, quintile of PAF-AH (17). This might be due to the fact that PAF-AH is associated with dLDLs, which are thought to confer increased CAD-risk. This is especially true for patients with T2DM. In fact, in the WOSCOP study, the prevalence of patients with T2DM was very low (1%) (27). Thus, the tremendous association of PAF-AH activity with a positive history of CAD seen in this study might be due to the different patient population studied.
The association of PAF-AH activity with CAD may be explained by the fact that PAF-AH shows also transacetylase activity, producing PAF and presumably other PAF-like species from lyso-PAF (28). These proinflammatory compounds may be released at sites of inflammation from proinflammatory cells. Therefore, the transacetylase reaction associated with LDL may transform lyso-PAF into the biologically active PAF and its analogs. In addition, PAF-AH/phospholipase A2 activity may liberate arachidonic acid, the precursor for eicosanoid synthesis (16). In macrophages and other major immunoinflammatory cells, arachidonic acid released by phospholipase A2 leads to immediate prostaglandin synthesis (29).
However, PAF-AH/phospholipase A2 resides on dLDL (9), and LDL particles, in turn, are an important source for the essential compound arachidonic acid (30). Because dLDLs show increased binding to the proteoglycans of the vessel wall (31), one might speculate whether dLDL may contribute to atherogenesis by functioning as a shuttle providing both the essential substrate and the crucial enzyme activity necessary for the production of proinflammatory prostaglandins and leukotrienes. In fact, dLDLs were shown to increase thromboxane formation in endothelial cells (32). It was shown previously that the use of aspirin reduces the risk of a first myocardial infarction, suggesting that antiinflammatory agents may prevent cardiovascular disease (3). One might, therefore, speculate whether, by reducing the formation of proinflammatory compounds in the atherosclerotic plaque, the beneficial effects of aspirin may, at least in part, be linked to atherogenic dLDL.
Eight weeks of fluvastatin treatment decreased the activity of PAF-AH by 23% compared with virtually no change in the placebo group (P < 0.001) (Fig. 3). Similar results have previously been obtained with lovastatin in patients with T2DM (33) and with atorvastatin in patients with type 2 hyperlipidemia (34), although both studies were not placebo-controlled. The reduction of PAF-AH activity by atorvastatin was not only due to a reduction in plasma LDL levels but also to a decrease in the enzyme activity associated with dLDL subspecies (34).
It was suggested that the increased removal of LDL by atorvastatin from the circulation in patients with type 2 hyperlipidemia may represent the main mechanism by which statins reduce plasma PAF-AH activity (34). However, as shown previously (9, 15) and in this study (Fig. 2, A and B), there is a strong association of PAF-AH activity with dLDL. In patients with T2DM and atherogenic lipoprotein phenotype, it was suggested that dLDL may be reduced by statin therapy via decreased synthesis of dLDL (20). Therefore, the reduction of PAF-AH activity in patients with T2DM may be associated as well with decreased synthesis of dLDL from triglyceride-rich precursors.
PAF AH activity was associated with LDL density, small LDL size, and the apoB content in dLDL (LDL-5 and LDL-6), and non-HDL cholesterol. Thus, PAF-AH activity may provide a good surrogate for atherogenic dLDL and the atherogenic lipoprotein phenotype. However, in this study CAD risk associated with PAF-AH activity was still significant after additional adjustment for apoB in dLDL and non-HDL cholesterol. PAF-AH has also been reported to be associated with Lp(a) (22). Therefore, one might speculate whether the association of PAF-AH activity seen with dLDL may actually be rather an association with Lp(a). The effect of statins on Lp(a) levels is controversial (35); however, in this study, fluvastatin did not lower Lp(a) concentrations. In contrast, dLDL and PAF-AH activity levels were lowered by 28% (20) and 23% (Fig. 3), respectively. In addition, the reduction of PAF-AH activity was independent from changes of Lp(a) concentrations in either total plasma, in HDL, or in dLDL (Table 2).
In conclusion, this is the first placebo-controlled trial assessing the effect of a statin on the activity of lipoprotein-associated phospholipase A2 / PAF-AH. Fluvastatin XL substantially lowered the concentration of dLDL (20) and, at the same time, reduced PAF-AH activity which is significantly associated with a positive history of coronary artery disease. The antiatherogenic potential of fluvastatin XL in patients with type 2 diabetes may thus be greater than that expected from its effects on LDL-C and TG levels alone.
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Acknowledgments |
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Footnotes |
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Abbreviations: apoB, Apolipoprotein B; CAD, coronary artery disease; CH, cholesterol; CI, confidence interval; CV, coefficient of variance; dLDL, dense LDL; FC, free cholesterol; GLM, general linear model; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); PAF-AH, platelet-activating factor acetyl hydrolase; PL, phospholipids; T2DM, type 2 diabetes mellitus; TG, triglycerides; WOSCOP Study, West of Scotland Coronary Prevention Study.
Received August 29, 2003.
Accepted November 21, 2003.
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