Anne Burke

Alcohol-induced generation of lipid peroxidation products in humans

To address the hypothesis that elevated blood alcohol increases systemic oxidant stress, we measured urinary excretion of isoprostanes (iPs), free radical-catalyzed products of arachidonic acid. Ten healthy volunteers received acute doses of alcohol (Everclear-R) or placebo under randomized, controlled, double-blind conditions. Urinary iPF2a-III increased in a time- and dosage-dependent manner after dosing with alcohol, with the peak urinary iPF2a-III excretion correlating with the rise in blood alcohol. To determine whether oxidant stress was associated with alcohol-induced liver disease (ALD), we then studied the excretion of iP in individuals with a documented history of alcohol-induced hepatitis or alcohol-induced chronic liver disease (AC). Both urinary iPF2a-III and urinary iPF2a-VI were markedly increased in patients with acute alcoholic hepatitis. In general, urinary iPF2a-III was significantly elevated in cirrhotic patients, relative to controls, but excretion was more pronounced when cirrhosis was induced by alcohol than by hepatitis C. Excretion of iPF2a-VI, as well as 4-hydroxynonenal and the iPF2a-III metabolite, 2,3-dinor-5, 6-dihydro-iPF2a-III, was also increased in AC. Vitamin C, but not aspirin, reduced urinary iPs in AC. Thus, vasoactive iPs, which serve as indices of oxidant stress, are elevated in the urine in both acute and chronic ALD. Increased generation of iPs by alcohol in healthy volunteers is consistent with the hypothesis that oxidant stress precedes and contributes to the evolution of ALD.

Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid production in inflammation

To examine the role of cyclooxygenase (COX) isozymes in prostaglandin formation and oxidant stress in inflammation, we administered to volunteer subjects placebo or bolus injections of lipopolysaccharide (LPS), which caused a dose-dependent increase in temperature, heart rate, and plasma cortisol. LPS caused also dose-dependent elevations in urinary excretion of 2,3-dinor 6-keto PGF(1alpha) (PGI-M) and 11-dehydro thromboxane B(2) (Tx-M). Platelet COX-1 inhibition by chronic administration of low-dose aspirin before LPS did not alter the symptomatic and febrile responses to LPS, but the increment in urinary PGI-M and Tx-M were both partially depressed. Pretreatment with ibuprofen, a nonspecific COX inhibitor, attenuated the febrile and systemic response to LPS and inhibited prostanoid biosynthesis. Both celecoxib, a selective COX-2 inhibitor, and ibuprofen attenuated the pyrexial, but not the chronotropic, response to LPS. Experimental endotoxemia caused differential expression of the COX isozymes in monocytes and polymorphonuclear leucocytes ex vivo. LPS also increased urinary iPF(2alpha)-III, iPF(2alpha)-VI, and 8,12-iso-iPF(2alpha)-VI, isoprostane (iP) indices of lipid peroxidation, and none of the drugs blunted this response. These studies indicate that (a) although COX-2 predominates, both COX isozymes are induced and contribute to the prostaglandin response to LPS in humans; (b) COX activation contributes undetectably to lipid peroxidation induced by LPS; and (c) COX-2, but not COX-1, contributes to the constitutional response to LPS in humans.

Oxidative stress and smoking-induced vascular injury

The relative importance of mechanisms relevant to smoking-induced vascular injury is poorly understood. Cigarette smoke is a source of free radicals but also results in cellular activation and consequent generation of free radicals in vivo. Here we consider several approaches to estimating the consequences of free radical generation in vivo, using measurements of modified lipids, proteins, and DNA. Smoking appears to result in elevation of several biomarkers of oxidant stress, some in a dose-related fashion. There is also some evidence that disordered endothelial function in smokers may be partly attributable to oxidant stress. Other effects of smoking on hemostatic activation, sympathoadrenal function, and lipoprotein structure and function may also be modulated by smoking-induced oxidant stress. The emergence and application of rational quantitatively reliable indexes of oxidant stress in vivo is likely to elucidate the relative contribution of oxidant stress to smoking-induced vascular injury.

Non‐Alcoholic Fatty Liver Disease, Non‐Alcoholic Steatohepatitis and Orthotopic Liver Transplantation

Obesity, non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH) are becoming increasingly common medical problems in the developed world, often in the setting of the metabolic or insulin resistance syndrome (IRS). It is predicted that by the year 2025 > 25 million Americans may have NASH‐related liver disease. NASH and NAFLD also affect the donor population. The use of steatotic donor livers for liver transplantation (LT) is associated with an increased risk of primary nonfunction (PNF) in the allograft. There is particular reluctance to use steatotic livers for living donor LT. There is indirect evidence to suggest that patients undergoing LT for cirrhosis resulting from NASH may have poorer outcome, despite careful selection of LT candidates. Indeed it is likely that many potential LT candidates with NASH are excluded from LT due to co‐morbid conditions related to IRS. The post‐LT patient is at risk of several components of IRS, such as diabetes mellitus, hypertension, hyperlipidaemia and obesity and there is increasing recognition of de novo and recurrent NAFLD and NASH after LT. Thus NAFLD and NASH affect all aspects of LT including donors, patients in evaluation and the LT recipient.

The influence of pravastatin and atorvastatin on markers of oxidative stress in hypercholesterolemic humans.

OBJECTIVES The aim of this study was to determine the effects of pravastatin and atorvastatin on markers of oxidative stress in plasma. BACKGROUND Hydroxymethylglutaryl coenzyme A reductase inhibitors reduce low-density lipoprotein cholesterol (LDL-C) and cardiovascular risk, but their effects on circulating biomarkers of oxidative stress are not well-defined. METHODS Hypercholesterolemic subjects (n = 120, ages 21 to 80 years with LDL-C 130 to 220 mg/dl) were randomized in a double-blind, parallel design to pravastatin 40 mg/day (prava40), atorvastatin 10 mg/day (atorva10), atorvastatin 80 mg/day (atorva80), or placebo. At baseline and 16 weeks, urinary isoprostanes (8, 12-iso-iPF(2 alpha)-VI isoform), plasma lipoprotein-associated phospholipase A2 (Lp-PLA2), Mercodia oxidized LDL (OxLDL) with antibody 4E6, oxidized phospholipids/apolipoprotein B-100 particle (OxPL/apoB) with antibody E06, immunoglobulin (Ig)G/IgM autoantibodies to malondialdehyde (MDA)-LDL, and apolipoprotein B (apoB)-immune complexes (IC) were measured. RESULTS After 16 weeks, there were no significant changes in urinary 8, 12-iso-iPF(2 alpha)-VI. The Lp-PLA2 and OxLDL were reduced in statin-treated groups, but after adjusting for apoB, only prava40 led to a reduction in Lp-PLA2 (-15%, p = 0.008) and atorva10 to a decrease in OxLDL (-12.9%, p = 0.01). The OxPL/apoB increased 25.8% (p < 0.01) with prava40 and 20.2% (p < 0.05) with atorva80. There were no changes in MDA-LDL autoantibodies, but significant decreases in IC were noted. CONCLUSIONS This study suggests that 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.

A critical review of candidacy for orthotopic liver transplantation in alcoholic liver disease.

The majority of candidates with end-stage alcoholic liver disease (ESALD) in the United States who are eligible for referral for liver transplantation (LT) are not being referred. There is a lack of firm consensus for the duration of abstinence from alcohol as well as what constitutes good psychosocial criteria for listing for LT. Evidence shows that the general public and the practicing physicians outside the transplant community perceive that patients with a history of alcohol abuse will make poor transplant candidates. However, physicians in the transplant community perceive selected patients with ESALD as good candidates. When considering patients for listing for LT, 3 months of alcohol abstinence may be more ideal than 6 months. Patients with a lack of social support, active smoking, psychotic or personality disorders, or a pattern of nonadherence should be listed only with reservation. Those who have a diagnosis of alcohol abuse as opposed to alcohol dependence may make better transplant candidates. Patients who have regular appointments with a psychiatrist or psychologist in addictions treatment training also seem to do more favorably.

Fibrinogen β-Chain Tyrosine Nitration Is a Prothrombotic Risk Factor

Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the β-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob “B” mimetic peptides Gly-His-Arg-Proam and Ala-His-Arg-Proam suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.

A prospective analysis of oxidative stress and liver transplantation.

Background. Oxidative stress (OS) is a potential mechanism of injury in many deleterious complications of orthotopic liver transplantation (OLT). Therefore, we evaluated OS prospectively in a cohort of 50 adult patients before, during, and after OLT. Methods. Urine, collected preoperatively, perioperatively, and at intervals for the first postoperative year, was analyzed for dinor-dihydro-isoprostane F2&agr;-III (dinor-dihydro iPF2&agr;-III), a well-characterized in vivo indicator of OS. Clinical events were extracted from the clinical records. Results. One-year patient and graft survival were 86% and 80%, respectively. There were nine episodes of acute cellular rejection (ACR). Twenty patients each experienced at least one adverse event. Pretransplantation urinary dinor-dihydro iPF2&agr;-III levels were elevated, compared to healthy volunteers, and rose significantly following reperfusion of the grafted liver. Levels fell sharply following OLT but never reached those of control subjects. Urinary dinor-dihydro iPF2&agr;-III levels steadily increased thereafter, reaching preoperative levels within 12 months of transplantation. There was no significant difference in dinor-dihydro iPF2&agr;-III excretion, with respect to hepatitis C virus (HCV) status, the development of ACR, or the presence or absence of a composite of predesignated adverse events. Conclusions. OLT recipients exhibit enhanced lipid peroxidation in vivo, which is augmented during intra-operative liver reperfusion. Although OS declines following OLT, it redevelops gradually, albeit without association with clinical events such as acute cellular rejection, organ failure, or infection of the allograft by HCV. We conclude that OS increases in the first year after liver transplantation, in a time-dependent fashion, but independent of clinical events affecting the allograft.