Paraoxonase 1 L55M, Q192R and paraoxonase 2 S311C alleles in atherothrombosis.

Increased oxidative stress is known to play a role in the pathogenesis of atherosclerosis, and polymorphisms in genes encoding for enzymes involved in modulation of oxidant stress, such as paraoxonases (PONs), provide a potentially powerful approach to study the risk of disease susceptibility. Aim of our study is to investigate the possible association among PONs polymorphisms, clinical and metabolic factors, and atherothrombotic events in an Italian population. We evaluated in 105 subjects, with or without atherosclerotic risk factors, the presence of PON1 L55M, PON1 Q192R, and PON2 S311C genetic variants, as well as lipid profile, the concentration of aminothiols (blood reduced glutathione, plasma total glutathione, homocysteine, cysteine, cysteinyl glycine), and malondialdehyde as markers of lipid peroxidation. Clinical, biochemical, and genetic variables were correlated with a history of atherothrombosis. Previous atherothrombotic events were found in 42 patients (40 %): myocardial infarction in 24, stroke or transient ischemic attack in 18. By multiple logistic regression analysis, hypertension (OR = 5.538; 95 % CI 2.202-13.902, P < 0.001), HDL-cholesterol concentration (OR = 0.947; 95 % CI 0.910-0.985, P = 0.007), and the presence of C allele in PON2 gene (OR = 3.595; 95 % CI 1.247-10.361, P = 0.018) were independently associated with atherothrombotic events. Our study sheds light on the role of PON2 as a possible cofactor in determining the risk of events together with the well-known risk markers HDL-cholesterol and hypertension.

[Endothelial dysfunction and oxidative stress in sepsis]. / Disfunzione endoteliale e stress ossidativo nella sepsi.

Central to the pathophysiology of sepsis and septic shock is an alteration in endothelial cell function and oxidative stress. Highly complex, integrated responses that include the activation of a number of cell types, inflammatory mediators and the hemostatic system are involved in endothelial dysfunction. On the other hand, the imbalance between the excessive production of reactive oxygen species and/or inadequate antioxidative defenses characterizes the oxidative stress. The overview of all these mechanisms suggests clinical biochemical markers as a possible therapeutic target together with correct intervention timing.

Super-normal 201Tl retention in hibernating myocardium: an ex-vivo study using the failing human heart.

OBJECTIVE: Although the relationship between delayed 201Tl distribution and blood flow in acutely ischemic and infarcted myocardium has been widely explored in the experimental setting, its behaviour in chronically hypoperfused dysfunctioning human myocardium has not yet been evaluated. METHODS: In tissue samples of excised failing hearts taken from ischemic (IHD) patients and idiopathic dilated cardiomyopathy (IDC) controls, we evaluated the relationship between delayed 201Tl retention (4 h redistribution), blood flow (assessed by means of 99mTc-labelled human albumin microspheres injected during transplantation) and biochemically-assessed fibrosis. 201Tl activity was expressed as the percent of the activity in the region with highest flow and the least fibrosis. RESULTS: Fibrosis and 201Tl activity were inversely related (r = -0.62, P = 0.0001). In IDC controls, low flows corresponded to uniformly preserved 201Tl retention. In IHD, 46 segments with flows < or = 0.60 ml.min-1.g-1 and 20 segments with flows > 0.60 ml.min-1.g1 showed matching delayed 201Tl retention and flow values; in the remaining 27, there was a disproportionately high tracer accumulation in comparison with flow (flow/201Tl mismatch). Despite significantly less fibrosis and lower flows, the mismatch segments showed significantly greater. 201Tl activity than the segments with concordantly high tracer retention and flow values. Conversely, at equivalent flow rates, the mismatch regions had less fibrosis than the areas with concordantly depressed 201Tl activity and perfusion. CONCLUSIONS: This super-normal 201Tl retention in hibernating myocardium may indicate a mechanism of cell adaptation to chronic hypoperfusion.

High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive disease. Assessment of total plasma concentration of homocysteine (tHcys) requires accurate and reproducible measurements. The aim of this study was to test a rapid isocratic HPLC method for tHcys analysis with an internal standard (I.S.) of alpha-mercaptopropionylglycine (MPG), 2-mercaptoethylamine (ME), or N-acetylcysteine (NAC) or without I.S., and to verify whether the use of an I.S. improves the precision. The method without I.S. showed an excellent linearity (y = 1.59x - 0.15, r = 1), recovery (100%) and a within-assay relative standard deviation (R.S.D.) of 1.2%. Instead, in our hands, the presence of I.S.s decreased the reproducibility (within-assay R.S.D. ranged from 4.5 to 6.5%) and lengthened the chromatogram by up to four to five times. In conclusion, HPLC measurement of plasma tHcys without I.S. improves accuracy with respect to determination with I.S.; moreover, this approach allows to routinely process larger amounts of plasma samples.

A new HPLC micromethod to measure total plasma homocysteine in newborn.

Total plasma homocysteine (tHcy) in children may be an useful biochemical marker for genetic risk of premature cardiovascular disease. We reported a rapid, isocratic HPLC method able to process very small amount of newborn plasma samples. A blood sample from heel capillary circulation was collected, using a heparinized capillary glass tube. Plasma sample from 1 to 10 microl was derivatized with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate after reduction with tri-n-butylphosphine and analyzed on Discovery C18 column, with a solution of acetonitrile-dihydrogenphosphate 0.1 M (8:92 v/v pH*2.1). This assay ensures a good recovery (95%), precision (CV 4.5%) and linearity (y=2.41x + 0.31, r=1). Due to its simplicity and reliability, our method is suitable for routine analysis of tHcy and other aminothiols (Cys, Cys-Gly, GSH) assessed for clinical and research purposes. With this HPLC method we have assayed tHcy levels in 1400 apparently healthy newborn babies (tHcy mean value=4.9+/-2.7 microM). In conclusion, this accurate and linear HPLC method allows measurement of tHcy in newborn during the routinary capillary blood collection in the fourth living day without any other invasive procedure.

Homocysteine, vitamin B12, and serum and erythrocyte folate in peritoneal dialysis and hemodialysis patients.

BACKGROUND: Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. High levels of plasma Hcy have been observed in end-stage renal disease patients. Few studies have compared peritoneal dialysis (PD) and hemodialysis (HD) patients and few data are available on erythrocyte folate (ery-F) levels in dialysis patients. OBJECTIVES: To evaluate plasma Hcy concentrations, vitamin B12 (B12), and folate status in dialysis patients; to analyze the possible causes of high Hcy levels; to follow up changes in folate and B12 concentrations after 6 months. DESIGN: A cross-sectional observational study. SETTING: Nephrology division and laboratory of hematology in a university and clinical research hospital. PATIENTS: The study included 82 patients treated with PD for 37 + 37 months and 70 patients treated with HD for 136 + 95 months. LABORATORY METHODS: Plasma Hcy was measured by the immunoenzymatic IMx Hcy FPIA method (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, U.S.A.), serum folate (s-F) and ery-F by the Stratus folate fluorometric enzyme-linked assay, and B12 by the Stratus vitamin B12 fluorometric enzyme-linked assay (DADE-Behring, Newark, DE, U.S.A.). RESULTS: Ninety-six percent of PD and 97% of HD patients had Hcy levels above the cutoff (13.5 micromol/L). Homocysteine level was higher in HD than in PD patients, while the prevalence of hyperhomocysteinemia was similar with the two techniques. Erythrocyte folate was significantly higher in PD (1333 +/- 519 pmol/L) than in HD (1049 +/-511 pmol/L, p < 0.01). Statistically significant correlations were observed between Hcy and B12, s-F, ery-F, and dialysis duration. Multivariate analysis showed a strong correlation between s-F and Hcy. After 6 months there were no differences in Hcy, B12, s-F, and ery-F levels. CONCLUSIONS: Plasma Hcy levels were high in more than 95% of our dialysis patients, with no relation to the type of dialysis. Vitamin B12 and folate were normal in the majority of our patients. However, serum folate was the major determinant of Hcy levels. Such a relation between Hcy and folate suggests that levels of folate within the reference interval are inadequate for dialysis patients.

Neopterin levels are independently associated with cardiac remodeling in patients with chronic heart failure.

OBJECTIVES: Neopterin, a marker of inflammation and monocyte activation, is found increased in patients with heart failure (HF). This study investigates whether neopterin levels correlate with left ventricular (LV) remodeling and brain natriuretic peptide (BNP), a marker of cardiac stress, in chronic HF (CHF) patients with different severity of disease. DESIGN AND METHODS: The relationship between neopterin and LV dimensions, NT-proBNP, and pro-inflammatory cytokines were studied in 98 CHF patients, while nineteen healthy subjects were enrolled as controls. Nineteen (19%) patients were in NYHA class I, 38 (39%) in NYHA class II, 27 (28%) in NYHA class III, and 14 (14%) in NYHA class IV. RESULTS: Neopterin levels were higher in CHF patients than in age- and gender-matched healthy controls, and related with indexed LV end-diastolic volume (LVEDVi). Prospectively CHF patients were separated into tertiles of low, medium and high neopterin levels. Among patients, male gender, LVEDVi, diuretic treatment, NYHA class I, NT-proBNP and IL-8 levels were significant determinants of urine neopterin levels by bivariate analysis. Neopterin levels were associated only to LV remodeling, as assessed by LVEDVi, and IL-8 levels, a crucial monocyte chemoattractant, by multivariate ordinal regression analysis. CONCLUSIONS: The relationship between elevated neopterin levels and LV enlargement in CHF patients suggests a crucial role of monocyte activation in the development of cardiac dysfunction in CHF patients. Assessment of neopterin levels is a potential biomarker to evaluate the progression of LV remodeling in CHF patients.

Effects of combined dietary supplementation on oxidative and inflammatory status in dyslipidemic subjects.

BACKGROUND AND AIM: Dyslipidemia is one of the main risk factors for atherosclerosis, usually the underlying cause of cardiovascular diseases which are the major cause of morbidity and mortality in developed countries. The aim of this study was to assess the effects and the advantages of a combined dietary supplementation with PUFA n-3, vitamin E, niacin and gamma-oryzanol on lipid profile, inflammatory status and oxidative balance. METHODS AND RESULTS: Fifty-seven dyslipidemic volunteers were randomly assigned to receive: placebo (group A, 19 subjects); PUFA n-3 and vitamin E (group B, 18 subjects); the same as B plus gamma-oryzanol and niacin (group C, 20 subjects). Lipid profile, reactive oxygen species (ROS), total antioxidant capacity (TAC), vitamin E, interleukin 1-beta (IL1-beta), tumor necrosis factor (TNF-alpha) and thromboxane B2 (TXB2) were determined at baseline (T0) and after four months (T1). All dyslipidemic subjects showed, at baseline, oxidative stress and, after four months, all biochemical markers improved significantly in groups treated with dietary supplementation. Particularly in group C all lipid patterns improved significantly. CONCLUSIONS: Our findings demonstrate that the strategy of combining different compounds, which protect each other and act together at different levels of the lipid chain production, improves lipid profile, inflammatory and oxidative status, allowing us to reduce the dose of each compound under the threshold of its side effects.

Coronary blood flow, metabolism, and function in dysfunctional viable myocardium before and early after surgical revascularisation.

OBJECTIVES: To assess the link between perfusion, metabolism, and function in viable myocardium before and early after surgical revascularisation. DESIGN: Myocardial blood flow (MBF, thermodilution technique), metabolism (lactate, glucose, and free fatty acid extraction and fluxes), and function (transoesophageal echocardiography) were assessed in patients with critical stenosis of the left anterior descending coronary artery (LAD) before and 30 minutes after surgical revascularisation. SETTING: Tertiary cardiac centre. PATIENTS: 23 patients (mean (SEM) age 57 (1.7) years with LAD stenosis: 17 had dysfunctional viable myocardium in the LAD territory, as shown by thallium-201 rest redistribution and dobutamine stress echocardiography (group 1), and six had normally contracting myocardium (group 2). RESULTS: LAD MBF was lower in group 1 than in group 2 (58 (7) v 113 (21) ml/min, p < 0.001) before revascularisation and improved postoperatively in group 1 (129 (133) ml/min, p < 0.001) but not in group 2 (105 (20) ml/min, p = 0.26). Group 1 also had functional improvement in the LAD territory at intraoperative echocardiography (mean regional wall motion score from 2.6 (0.85) to 1.5 (0.98), p < 0.01). Oxidative metabolism, with lactate and free fatty acid extraction, was found preoperatively and postoperatively in both groups; however, lactate and free fatty acid uptake increased after revascularisation only in group 1. CONCLUSIONS: MBF is reduced and oxidative metabolism is preserved at rest in dysfunctional but viable myocardium. Surgical revascularisation yields immediate perfusion and functional improvement, and increases the uptake of lactate and free fatty acids.

Lysyl oxidase expression and collagen cross-linking during chronic adriamycin nephropathy.

Collagen cross-linking induced by lysyl oxidase has been implicated in liver and lung fibrosis. To define the role of this process in kidney fibrosis, we investigated the renal expression of lysyl oxidase and the content in collagen cross-links at various stages of chronic Adriamycin nephropathy in Sprague-Dawley rats. Lysyl oxidase expression was determined by RT-PCR; collagen pyridinium residues, indicating lysyl oxidase induced cross-links, were evaluated by HPLC. These parameters followed a synergic albeit asynchronous outcome: (a) lysyl oxidase mRNA levels in total kidney, glomeruli and medulla from Adriamycin-treated rats increased up to 3 times compared to controls between week 8 and 12, then returning within the normal range; (b) the pyridinium residue content did not show any significant difference between Adriamycin-treated and control rats, until diffuse interstitial fibrosis developed (16 weeks), showing at this time a 2- to 3-fold increment. Lysyl oxidase was expressed by several renal cell lines and in tubular-epithelial cells it was up-regulated in vitro by TGF beta-1, a recognized fibrogenetic factor in Adriamycin nephropathy. Our observations demonstrated that an increased expression of lysyl oxidase in the kidney precedes the development of diffuse fibrotic lesions and that, at this stage, collagenic structures contain highly cross-linked components, the final product of lysyl oxidase activity. The evidence of lysyl oxidase up-regulation in tubular epithelial cells by the same factor implicated in Adriamycin toxicity in the kidney suggests a common pathogenetic mechanism. Collagen cross-link formation by lysyl oxidase may be implicated in the pathogenesis of irreversible, fibrotic renal lesions.