Karl Winkler

Plasma PCSK9 is increased by Fenofibrate and Atorvastatin in a non-additive fashion in diabetic patients

OBJECTIVEnProprotein convertase subtilisin kexin/type 9 (PCSK9) is an inhibitor of the low density (LDL) lipoprotein receptor. Plasma PCSK9 is increased by fenofibrate and statins. Here, we determined how standard dose of statin and combined therapy with fenofibrate modulate PCSK9.nnnMETHODSnRandomized, open-label cross-over study investigating the effect of fenofibrate (160 mg), atorvastatin (10 mg), and combination of both in patients with type 2 diabetes mellitus and atherogenic dyslipidemia. After the single administration of atorvastatin and fenofibrate for 6 weeks, patients received both for another 6 weeks. PCSK9, lipids and lipoproteins levels were determined at day 1, weeks 6, 9 and 12.nnnRESULTSnUpon 6 weeks of treatment, atorvastatin decreased LDL-cholesterol by 30% (p<0.001) and fenofibrate decreased triglyceride level by 31% (p<0.01) and increased HDL-cholesterol by 13% (p<0.05). Combination did not show further benefit. Atorvastatin increased PCSK9 by 24% at day 1 and by 14% at week 6 (p < or = 0.01). Fenofibrate increased PCSK9 by 26% at week 6 (p < or = 0.01), but had no effect at day 1. Three weeks of combination therapy increased PCSK9 by 42%, 6 weeks by 19% (p < or = 0.01). PCSK9 changes were not different between treatments over 6-week periods.nnnCONCLUSIONnFenofibrate and atorvastatin increased circulating PCSK9 in diabetic patients, with no additive effect after 6 weeks of combined therapy.

Effect of atorvastatin on inflammation and outcome in patients with type 2 diabetes mellitus on hemodialysis

Statins have multiple effects, including anti-inflammatory actions, lowering C-reactive protein levels, and reducing coronary events. We performed a post hoc analysis of the randomized placebo-controlled 4D Study that had evaluated the efficacy and safety of atorvastatin in 1255 patients with type 2 diabetes mellitus who were on maintenance hemodialysis. Here we determined the relationship between atorvastatin treatment, C-reactive protein, and the outcome of patients who had pre-specified and adjudicated endpoints of all-cause mortality, composite vascular endpoint, myocardial infarction, sudden death, and stroke. Atorvastatin had no significant effect on the risk of composite vascular endpoint or death relative to placebo in any quartile of baseline C-reactive protein. These baseline levels were not significantly different between the treated and placebo group and remained stable at 6 months on atorvastatin but significantly increased in those patients on placebo. All of the patients with baseline C-reactive protein in the fourth quartile had a significantly increased risk of deaths and in composite vascular endpoint compared to patients in the first quartile. The mean value of two consecutive C-reactive protein measurements was associated with significant increases in the risk of sudden death, stroke, all-cause mortality and composite vascular endpoint. Our results show that C-reactive protein was highly predictive of outcome, but atorvastatin treatment was not associated with reduced relative risks in the composite vascular endpoint or mortality in patients on hemodialysis with or without inflammation.

Changes in adiponectin and the risk of sudden death, stroke, myocardial infarction, and mortality in hemodialysis patients

Adiponectin is an adipocyte-specific cytokine that has a protective role in the development of cardiovascular morbidities. As chronic kidney disease progresses, adiponectin levels increase and cardiovascular risk profiles change. Here we determined the association of baseline and longitudinal changes in adiponectin with different cardiovascular outcomes in 1255 type 2 diabetic hemodialysis patients in the German Diabetes and Dialysis Study. Within 4 years of follow-up, the hazard ratios to reach pre-specified, adjudicated end points were determined. The increased risk of cardiovascular events observed with high adiponectin levels at baseline was associated with high risks of sudden death and stroke but not of myocardial infarction. Adiponectin was negatively correlated with C-reactive protein and positively correlated with NT-pro-BNP, the latter significantly attenuating the associations with adverse outcome. Increased longitudinal levels of adiponectin during follow-up were associated with higher risks of adverse cardiovascular outcomes and death; associations weakened by a confounding effect of increased NT-pro-BNP. Our study suggests that high basal and increasing adiponectin levels in the dialysis population largely reflect a consequence of disease circumstances. Most likely, this rise is a counter-regulatory response to worsening health in keeping with adiponectins potential to counteract inflammation.

Change in N-terminal-pro-B-type-natriuretic-peptide and the risk of sudden death, stroke, myocardial infarction, and all-cause mortality in diabetic dialysis patients

Aims N-terminal-pro-B-type-natriuretic-peptide (NT-pro-BNP) concentrations are altered in renal failure. This study examined the effect of baseline and change from baseline NT-pro-BNP on cardiovascular outcome and mortality in haemodialysis patients. Methods and results On the basis of the German Diabetes and Dialysis Study, which evaluated atorvastatin in 1255 type 2 diabetes mellitus (T2DM) haemodialysis patients (median follow-up 4 years), the impact of NT-pro-BNP on pre-specified, adjudicated endpoints was investigated: sudden death (SD; n = 160), stroke (n = 99), myocardial infarction (MI; n = 200), cardiovascular events (CVEs: cardiac death, MI, stroke; n = 465), all-cause mortality (n = 612). Patients with baseline NT-pro-BNP ≥9252 pg/mL (fourth quartile) exhibited a more than four-fold risk of stroke [hazard ratio (HR) 4.1; 95% confidence interval (CI) 2.0–8.4] and a more than two-fold risk of SD (HR 2.0; 95% CI 1.2–3.3), CVE (HR 2.0; 95% CI 1.5–2.7), and mortality (HR 2.1; 95% CI 1.6–2.7) compared with patients with baseline NT-pro-BNP ≤ 1433 pg/mL (first quartile). Change in NT-pro-BNP was strongly associated with SD, CVE, and mortality. Doubling of NT-pro-BNP increased the risk of death by 46% (95% CI 1.1–2.0). Neither baseline nor change in NT-pro-BNP was significantly associated with MI. Conclusion Increasing NT-pro-BNP is a risk factor for SD, CVE, and mortality in haemodialysis patients with T2DM. Whether NT-pro-BNP-guided treatment improves outcome needs to be evaluated prospectively.

Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

BackgroundTherapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated.From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards.MethodsPlasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes.ResultsLow quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards.ConclusionsThe authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.

Genetic variants of the promoter of the heme oxygenase-1 gene and their influence on cardiovascular disease (The Ludwigshafen Risk and Cardiovascular Health Study)

BackgroundHeme oxygenase-1 is an inducible cytoprotective enzyme which handles oxidative stress by generating anti-oxidant bilirubin and vasodilating carbon monoxide. A (GT)n dinucleotide repeat and a -413A>T single nucleotide polymorphism have been reported in the promoter region of HMOX1 to both influence the occurrence of coronary artery disease and myocardial infarction. We sought to validate these observations in persons scheduled for coronary angiography.MethodsWe included 3219 subjects in the current analysis, 2526 with CAD including a subgroup of CAD and MI (n = 1339) and 693 controls. Coronary status was determined by coronary angiography. Risk factors and biochemical parameters (bilirubin, iron, LDL-C, HDL-C, and triglycerides) were determined by standard procedures. The dinucleotide repeat was analysed by PCR and subsequent sizing by capillary electrophoresis, the -413A>T polymorphism by PCR and RFLP.ResultsIn the LURIC study the allele frequency for the -413A>T polymorphism is A = 0,589 and T = 0,411. The (GT)n repeats spread between 14 and 39 repeats with 22 (19.9%) and 29 (47.1%) as the two most common alleles. We found neither an association of the genotypes or allelic frequencies with any of the biochemical parameters nor with CAD or previous MI.ConclusionAlthough an association of these polymorphisms with the appearance of CAD and MI have been published before, our results strongly argue against a relevant role of the (GT)n repeat or the -413A>T SNP in the HMOX1 promoter in CAD or MI.

Low-dose atorvastatin improves dyslipidemia and vascular function in patients with primary biliary cirrhosis after one year of treatment.

OBJECTIVEnPrimary biliary cirrhosis (PBC) is frequently associated with hypercholesterolemia and with an increased cardiovascular morbidity and mortality. Statins lower serum cholesterol levels and may thus improve the cardiovascular risk in PBC patients. The aim of our study was to prospectively examine the efficacy of low-dose atorvastatin on cholestasis as well as cardiovascular risk markers such as dyslipidemia and vascular function in patients with PBC.nnnMETHODSnNineteen patients with early-stage (biopsy proven and AMA positive) PBC and low-density lipoprotein cholesterol (LDL-C) above 130mg/dL were included in this single-center study and treated with atorvastatin 10mg per day for one year.nnnRESULTSnConcentrations of total cholesterol, LDL-C, LDL triglycerides, oxLDL, IgG and sVCAM-1 decreased significantly after 48 weeks of atorvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery as an indicator of vascular function significantly increased, while carotid artery intima-media thickness and vascular wall stiffness did not progress under treatment. No statistical differences in liver enzymes were observed except a transient increase of alkaline phosphatase.nnnCONCLUSIONnTreatment with low-dose atorvastatin is safe in early-stage PBC, effectively reduces total cholesterol, LDL-C, LDL triglycerides, oxLDL and sVCAM-1 and improves vascular function as reflected by FMD, without affecting cholestasis progression. Therefore, statin therapy should be considered in PBC patients with additional risk factors for cardiovascular disease.

Genetic variants and haplotypes of lipoprotein associated phospholipase A2 and their influence on cardiovascular disease (The Ludwigshafen Risk and Cardiovascular Health Study)

Summary.u2002 Background:u2002 There is increasing evidence that lipoprotein‐associated phospholipase A2 (LpPLA2) is associated with cardiovascular disease. However, it is still unclear whether LpPLA2 is simply a marker or has a causal role as either a pro‐ or anti‐atherogenic factor. Methods:u2002 We analyzed the association of five polymorphisms (−1357G>A, −403T>C, Arg92His, Ile198Thr, Ala379Val) and related haplotypes at the PLA2G7 locus with angiographic coronary artery disease (CAD), plasma LpPLA2 activity, and long‐term survival in 3234 patients scheduled for coronary angiography. Results:u2002 The promoter variant −403C and His92 were associated with a decrease and Val379 with an increase in plasma LpPLA2 activity. Both coding variants revealed a clear gene‐dose effect. Interestingly, the rare Thr198 allele, which was not associated with any change in plasma LpPLA2 activity, was more frequent in subjects without CAD (Pu2003=u20030.009), with an adjusted odds ratio for CAD of 0.69 (95% CI: 0.49–0.96; Pu2003=u20030.029). None of the analyzed variants showed any robust association with all‐cause or cardiovascular mortality. Conclusion:u2002 Irrespective of the significant association between some variants with plasma LpPLA2 activity, it is still unclear whether these polymorphisms or haplotypes are associated with the risk and outcome of cardiovascular disease in Caucasians.

Ezetimibe alone and in combination lowers the concentration of small, dense low-density lipoproteins in type 2 diabetes mellitus

OBJECTIVEnThe effectiveness of the cholesterol absorption inhibitor ezetimibe on LDL subfractions and ultimately on the atherosclerotic risk profile remains controversial. We thus determined the concentration of atherogenic small, dense LDL (sdLDL) in patients with type 2 diabetes and an elevated cardiovascular risk profile.nnnRESEARCH DESIGN AND METHODSnMulticenter, randomized, open-label 6-week study investigating the effect of ezetimibe 10mg (E), simvastatin 20mg (S) and the combination of ezetimibe-/simvastatin 10/20mg (C) on the concentration of sdLDL separated from fresh plasma by gradient ultracentrifugation in patients with type 2 diabetes (NCT01384058).nnnRESULTSnFifty-six patients were screened for sdLDL, 41 were randomized, and 40 patients (12 E, 14 S and 14 C) completed the study. Total and LDL cholesterol fell by 14% (p=0.004) and 15% (p=0.006) with E, 22% (p<0.001) and 32% (p<0.001) with S, and 32% (p<0.001) and 44% (p<0.001) with C, respectively. E reduced the concentration of sdLDL by 20% (p=0.043) whereas S and C reduced sdLDL by 24% (p=0.020) and 33% (p=0.003), respectively, and non-sdLDL by 28% (p=0.004) and 42% (p<0.001), respectively. However, the further drop in sdLDL by adding E to S was not significant.nnnCONCLUSIONnEzetimibe alone and in combination with simvastatin reduced the concentration of atherogenic sdLDL in patients with type 2 diabetes.

Association of LDL cholesterol and inflammation with cardiovascular events and mortality in hemodialysis patients with type 2 diabetes mellitus.

BACKGROUNDnIn the general population, C-reactive protein (CRP) in addition to low-density lipoprotein (LDL) cholesterol level is useful in predicting cardiovascular events. In hemodialysis patients, the additive value is unknown. The association between LDL cholesterol level and outcome previously was suggested to be inverse and confounded by inflammation.nnnSTUDY DESIGNnProspective cohort study.nnnSETTING & PARTICIPANTSn1,255 hemodialysis patients with type 2 diabetes mellitus randomly assigned to atorvastatin versus placebo in the German Diabetes Dialysis Study.nnnPREDICTORSnBaseline LDL cholesterol level.nnnOUTCOMES & MEASUREMENTSnCombined vascular end point (cardiac death, myocardial infarction, and stroke), mortality, myocardial infarction, sudden death, and stroke.nnnRESULTSnDuring 4 years, 465 combined vascular events, 612 deaths, 160 sudden deaths, 200 myocardial infarctions, and 99 strokes occurred. Median LDL cholesterol level was 123 mg/dL. LDL cholesterol level (millimoles per liter and quartiles) was not predictive of outcome. This was analyzed further in patients with and without inflammation. In patients with inflammation (CRP level > 5 mg/L), the adjusted relative risk of combined vascular events was 29% greater compared with those without inflammation and a low LDL cholesterol level (LDL cholesterol < or = 123 mg/dL). This was irrespective of whether LDL cholesterol level was low or high (hazard ratio [HR] for LDL < 123 mg/dL [HR (for LDL< or =123 mg/dL)], 1.29, with 95% confidence interval [CI], 0.98 to 1.70; HR(LDL>123 mg/dL), 1.29, with 95% CI, 0.99 to 1.69). Similar results were found for all-cause death (HR(LDL< or =123 mg/dL), 1.47 [95% CI, 1.16 to 1.86]; HR(LDL>123 mg/dL), 1.48 [95% CI, 1.16 to 1.88]), sudden death (HR(LDL< or =123 mg/dL), 1.98 [95% CI, 1.23 to 3.20]; HR(LDL>123 mg/dL), 1.66 [95% CI, 1.01 to 2.75]), and myocardial infarction (HR(LDL< or =123 mg/dL), 1.74 [95% CI, 1.14 to 2.66]; HR(LDL>123 mg/dL), 1.54 [95% CI, 0.99 to 2.38]). In patients without inflammation, the respective risks did not differ significantly between patients with varying LDL cholesterol levels. However, there was a trend toward an increased risk of myocardial infarction (HR(LDL>123 mg/dL), 1.45 [95% CI, 0.95 to 2.21]) in patients with high compared with low LDL cholesterol levels. P values for the interaction between CRP and LDL cholesterol levels were 0.9 (composite vascular end point), 0.5 (mortality), 0.9 (sudden death), 0.09 (stroke), and 0.2 (myocardial infarction).nnnLIMITATIONSnSelected patient cohort, post hoc analysis.nnnCONCLUSIONnBecause CRP level more than LDL cholesterol level determined outcome, the value of regular LDL cholesterol measurements in long-term hemodialysis patients with type 2 diabetes needs reassessment.