Günter Fauler

Simvastatin Versus Ezetimibe Pleiotropic and Lipid-Lowering Effects on Endothelial Function in Humans

Background—Statins may exert important pleiotropic effects, ie, improve endothelial function, independently of their impact on LDL cholesterol. In humans, however, pleiotropic effects of statins have never been unequivocally demonstrated because prolonged statin treatment always results in reduced LDL cholesterol levels. We therefore tested the hypothesis that similar reductions in LDL cholesterol with simvastatin and ezetimibe, a novel cholesterol absorption inhibitor, result in different effects on endothelial function. Methods and Results—Twenty patients with chronic heart failure were randomized to 4 weeks of simvastatin (10 mg/d) or ezetimibe (10 mg/d) treatment. Flow-dependent dilation (FDD) of the radial artery was determined by high-resolution ultrasound before and after intra-arterial vitamin C to determine the portion of FDD inhibited by radicals (ΔFDD-VC). Activity of extracellular superoxide dismutase, a major vascular antioxidant enzyme system, was determined after release from the endothelium by a heparin bolus injection. Endothelial progenitor cells were analyzed with an in vitro assay. Simvastatin and ezetimibe treatment reduced LDL cholesterol to a similar extent (15.6% versus 15.4%; P=NS), whereas changes in mevalonate, the product of HMG-CoA-reductase, differed between groups (Δmevalonate-simvastatin, −1.04±0.62 versus Δmevalonate-ezetimibe, 1.79±0.94 ng/mL; P<0.05 between groups). Importantly, FDD was markedly improved after simvastatin (10.5±0.6% versus 5.1±0.7%; P<0.01) but not after ezetimibe treatment (5.6±0.5% versus 5.8±0.6%; P=NS). ΔFDD-VC was substantially reduced after simvastatin but not after ezetimibe treatment. Extracellular superoxide dismutase activity was increased by >100% (P<0.05) after simvastatin but not ezetimibe treatment. Simvastatin treatment increased the number of functionally active endothelial progenitor cells, whereas ezetimibe had no effect. Conclusions—Four weeks of simvastatin treatment improves endothelial function independently of LDL cholesterol lowering, at least in part by reducing oxidant stress. Simvastatin may thereby exert important pleiotropic effects in humans.

Alterations in Lipid Metabolism Mediate Inflammation, Fibrosis, and Proliferation in a Mouse Model of Chronic Cholestatic Liver Injury

BACKGROUND & AIMS The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. METHODS We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. RESULTS Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. CONCLUSIONS Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.

Atorvastatin in patients with primary biliary cirrhosis and incomplete biochemical response to ursodeoxycholic acid

Statin therapy may target both hypercholesterolemia and cholestasis in primary biliary cirrhosis (PBC). However, little is known about the efficacy and safety of statins in PBC. The aim of this single‐center study was therefore to prospectively examine the effects of atorvastatin on serum markers of cholestasis, aminotransferases, and lipid and bile acid metabolism as well as inflammatory and immunological markers in patients with PBC. Fifteen patients with early‐stage PBC and an incomplete biochemical response to ursodeoxycholic acid (UDCA) therapy (defined as alkaline phosphatase 1.5‐fold above the upper limit of normal after 1 year) were treated with atorvastatin 10 mg/day, 20 mg/day, and 40 mg/day for 4 weeks, respectively. Serum levels of alkaline phosphatase increased during atorvastatin 20 mg and 40 mg (P < 0.05), whereas leucine aminopeptidase and γ‐glutamyltransferase remained unchanged. No statistical differences in overall serum ALT, AST, bilirubin, and IgM levels were observed. However, atorvastatin was discontinued in 1 out of 15 patients because of ALT 2‐fold above baseline, and 2 patients showed ALT elevations 3‐fold above the upper limit of normal at the end of the atorvastatin treatment period. Serum total cholesterol and low‐density lipoprotein cholesterol levels decreased by 35% and 49%, respectively (P < 0.001). Precursors of cholesterol biosynthesis (lanosterol, desmosterol, lathosterol) showed a similar pattern. No changes in serum bile acid levels and composition were observed during treatment. Conclusion: Atorvastatin does not improve cholestasis in PBC patients with an incomplete biochemical response to UDCA but effectively reduces serum cholesterol levels. (HEPATOLOGY 2007.)

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

OBJECTIVE Primary 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. METHODS Nineteen 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. RESULTS Concentrations 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. CONCLUSION Treatment 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.

Involvement of Rho and p38 MAPK in endothelin-1-induced expression of PGHS-2 mRNA in osteoblast-like cells

Prostaglandins (PGs) play an important role in bone remodeling because eicosanoids are local mediators of bone metabolism, which can induce physiological and pathological responses of bone tissue. Biosynthesis of PGs is catalyzed by constitutively expressed PG endoperoxide G/H synthase (PGHS) 1 and by the inducible isoform PGHS‐2. In MC3T3‐E1 osteoblast‐like cells, expression of PGHS‐2 was shown by mechanical forces, cytokines, growth factors, and hormones. Recently, endothelin (ET) 1‐stimulated PGHS‐2 mRNA expression was described, leading to a burst in prostaglandin E2 (PGE2) production. In this study, we investigated ET‐1‐induced signal transduction pathway(s) involved in the PGHS‐2 mRNA production. Time course of PGHS‐2 mRNA expression reaching the maximum within 45 minutes is in good agreement with the concept of an immediate early gene product. Inhibition of phospholipase C (PLC), phospholipase D (PLD), phosphatidylinositol‐3 kinase (PI‐3‐kinase), and protein kinase C (PKC) had no influence on PGHS‐2 synthesis. Using specific blockers of tyrosine kinases indicated involvement of p38 MAPK but not p42/44 MAPK. By preloading cells with exoenzyme C3, we were able to show requirement of the Rho family of G proteins for p38 MAPK phosphorylation and PGHS‐2 mRNA synthesis, whereas pertussis toxin (PTX) and cholera toxin (CTX) had no remarkable effect.

Chemistry of the oil gland secretion of Collohmannia gigantea (Acari: Oribatida).

The chemistry of the lemon-scented oil gland secretion ofCollohmannia gigantea, a middle-derivative mixonomatanoribatid mite, was investigated by gas chromatography – massspectrometry.Gas chromatographic profiles of whole body extracts of C.gigantea revealed two distinct chromatographic zones, the firstcontaining a set of six volatile compounds, comprising the lemon-scentedmonoterpene aldehydes neral and geranial, the scented monoterpene ester nerylformate, a distinctly scented aromatic aldehyde(2-hydroxy-6-methyl-benzaldehyde= 2,6-HMBD), and the two non-scented hydrocarbons, tridecane and pentadecane.All six components appeared to be present in steady relative proportions inscenting mites only, indicating their unity within the scented secretion. Incontrast, the components of the second chromatographic zone were less volatileand found in both, scenting and non-scenting mites. Chemically, they representaset of fatty acids of already known cuticular origin.The secretion bouquet ofthe first chromatographic zone was linked with oil glands by histochemicalmeans: large amounts of aldehydes were present only in oil gland reservoirs,notin any other region of the mite body. While chemical profiles of oil glandsecretions of several dozen astigmatid mites are known, only one other oribatidoil gland composition, from a desmonomatan species, has been elucidated, beingalmost the same as that of C. gigantea. Moreover, allcomponents of these two secretions are widely distributed amongst astigmatidmite species and may also be common in a restricted set of middle-derivativeoribatids. These findings are consistent with the idea of astigmatid miteoriginfrom a mixonomatan-desmonomatan group.

Ursodeoxycholic acid in patients with chronic heart failure: a double-blind, randomized, placebo-controlled, crossover trial.

OBJECTIVES This study sought to assess the effects of ursodeoxycholic acid (UDCA) on endothelial function and inflammatory markers in patients with chronic heart failure (CHF). BACKGROUND Endothelial dysfunction is commonly observed in patients with CHF, and it contributes to the limitation in exercise capacity that accompanies this condition. Bacterial lipopolysaccharide may trigger proinflammatory cytokine release and promote further endothelial dysfunction. UDCA, a bile acid used in the treatment of cholestatic liver disease, has anti-inflammatory and cytoprotective properties and may contribute to the formation of mixed micelles around lipopolysaccharide. These properties may help to improve peripheral blood flow in patients with CHF. METHODS We performed a prospective, single-center, double-blind, randomized, placebo-controlled crossover study of UDCA in 17 clinically stable male patients with CHF (New York Heart Association functional class II/III, left ventricular ejection fraction <45%). Patients received in random order 500 mg UDCA twice daily for 4 weeks and placebo for another 4 weeks. The primary endpoint was post-ischemic peak peripheral arm blood flow as assessed by strain-gauge plethysmography. RESULTS Sixteen patients completed the study. UDCA was well tolerated in all patients. Compared with placebo, UDCA improved peak post-ischemic blood flow in the arm (+18%, p = 0.038), and a trend for improved peak post-ischemic blood flow in the leg was found (+17%, p = 0.079). Liver function improved: compared with placebo, levels of γ-glutamyl transferase, aspartate transaminase, and soluble tumor necrosis factor α receptor 1 were lower after treatment with UDCA than after placebo (all p < 0.05). There was no change in 6-min walk test or New York Heart Association functional class, and levels of tumor necrosis factor α and interleukin-6 were unchanged or increased compared with placebo. CONCLUSIONS UDCA is well tolerated in patients with CHF. UDCA improves peripheral blood flow and is associated with improved markers of liver function.

Negative ion chemical ionization for the determination of methylphenidate in human plasma by stable isotope dilution gas chromatography/mass spectrometry

A sensitive and specific method for the determination of methylphenidate in human plasma is presented. Methylphenidate was extracted from plasma by solvent extraction with hexane at pH 9.3 and derivatized to its heptafluorobutyrate derivative. The derivative was measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostically useful fragment ion at m/z 369 was obtained at high relative abundance. (18)O(2)-labelled methylphenidate was used as an internal standard and its rapid and facile preparation from the unlabeled compound is described. Calibration graphs were linear within the range 0.14-18.25 ng ml(-1). The inter-assay precision was 8.7% (0.14 ng ml(-1)) and 3.1% (4.56 ng ml(-1)) and the intra-assay variability was 1.3% (0.14 ng ml(-1)) and 0.4% (4.56 ng ml(-1)). Accuracy determinations showed deviations of +0.7% (0.14 ng ml(-1)) and -2.5% (4.56 ng ml(-1)). The method is rugged, rapid and robust and has been applied to the batch analysis of methylphenidate during pharmacokinetic profiling of the drug.

Quantitative analysis of morphine in human plasma by gas chromatography-negative ion chemical ionization mass spectrometry

A sensitive and specific method for the quantitative determination of morphine in human plasma is presented. Morphine was extracted from plasma by solvent extraction with ethyl acetate and derivatized to its heptafluorobutyrate derivative. The derivatives were measured by gas chromatography-negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 637 is obtained at high relative abundance. Deuterated morphine was used as an internal standard. Calibration graphs were linear within a range of 0.78 ng/ml and 50 ng/ml. Inter-assay precision was 2.3% (2.85 ng/ml) and 1.4% (14.25 ng/ml), intra-assay variability was found to be 1.5% (3.71 ng/ml) and 0.5% (14.54 ng/ml). Accuracy showed deviations of -9.3% (2.85 ng/ml) and -4.2% (14.25 ng/ml). The method is rugged and robust and has been applied to the batch analysis of morphine during pharmacokinetic profiling of the drug.

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase–hydrogen peroxide–chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood–brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting.