Isabella Fermo

Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study

BACKGROUND The plasma concentration of asymmetrical dimethylarginine (ADMA), an inhibitor of nitric-oxide synthase, which has been linked to endothelial dysfunction and atherosclerosis in the general population, is raised in patients with end-stage renal disease and could contribute to the high cardiovascular risk in patients with chronic renal failure. We investigated the relation between cardiovascular risk factors and plasma ADMA concentration in a cohort of haemodialysis patients (n=225), and tested the predictive power of ADMA for mortality and cardiovascular outcomes. METHODS Patients had standard dialysis three times a week. We accurately recorded cardiovascular events over a mean follow-up of 33.4 months (SD 14.6); these events were reviewed by a panel of physicians. We identified correlates of plasma ADMA by univariate and multivariate analyses. FINDINGS On univariate analysis, ADMA concentration in plasma was directly related to concentrations of fibrinogen and L-arginine in plasma, duration of dialysis treatment, and serum cholesterol concentration, and was inversely related to serum albumin concentration. On multivariate analysis, only plasma fibrinogen (p=0.0001) and serum albumin (p=0.04) concentrations were independently related to plasma ADMA concentration (multiple r=0.44, p=0.0001). 83 patients died, 53 (64%) by cardiovascular causes. In a Coxs proportional-hazards model, plasma ADMA ranked as the second factor predicting overall mortality (hazard ratio 1.26, 95% Cl 1.11-1.41, p=0.0001) and cardiovascular events (1.17, 1.04-1.33, p=0.008). INTERPRETATION In haemodialysis patients, plasma ADMA is a strong and independent predictor of overall mortality and cardiovascular outcome. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular disease in chronic renal failure.

Inflammation is associated with carotid atherosclerosis in dialysis patients

Objective To investigate the relationship between inflammatory processes and atherosclerosis in uraemic patients on chronic dialysis. Design A cross-sectional study in 138 dialysis patients (92 on haemodialysis and 46 on continuous ambulatory peritoneal dialysis). Methods Serum C-reactive protein (CRP), IgG anti-Chlamydia pneumoniae antibodies, lipoprotein (a), fibrinogen and plasma homocysteine as well as the intima–media thickness and the number of atherosclerotic plaques of the carotid arteries (by Echo-Colour-Doppler) were measured in each patient. Results One hundred and eight patients had at least one plaque and 26 had more than six plaques. Serum CRP was above the upper limit of the normal range (5 mg/l) in 85 of 138 patients (62%). IgG anti-Chlamydia pneumoniae antibodies were detectable in 64% of patients (high level in 24%, intermediate in 33% and low in 7%) and undetectable in the remaining 36% of patients. In a multiple regression model age (β = 0.35), serum CRP (β = 0.23), plasma homocysteine (β = 0.19), duration of dialysis (β = 0.19) and pulse pressure (β = 0.18) were independent predictors of intima–media thickness (R = 0.54, P < 0.0001). Similarly, age (β = 0.33), serum CRP (β = 0.29), plasma homocysteine (β = 0.20) and serum albumin (β = −0.18) were independent correlates of the number of atherosclerotic plaques (R = 0.55, P < 0.0001). Furthermore, in smokers, the interaction serum CRP–IgG anti–Chlamydia pneumoniae antibodies was the stronger independent predictor (β = 0.43, P = 0.0001) of the number of atherosclerotic plaques while no such relationship (P = 0.73) was found in non-smokers. Conclusions In patients on chronic dialysis treatment CRP is independently associated to carotid atherosclerosis and appears at least in part to be explained by IgG anti-Chlamydia pneumoniae antibodies level. These data lend support to the hypothesis that inflammation plays a role in the pathogenesis of atherosclerosis in these patients.

Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease

Homocysteine, a thiol-containing amino acid, is the branch point between the transsulfuration and the transmethylation pathways of methionine [1]. In the last decade, investigators have studied the role of homocysteine metabolism in the pathogenesis of occlusive vascular disease. Homocystinuria, a disorder caused by homozygous cystathionine -synthase (E.C. 4.2.1.22) deficiency or defects in the vitamin B12 or folate-dependent remethylation of homocysteine to methionine and characterized by fasting plasma homocysteine levels of 200 mol/L or greater, is frequently associated with severe vascular disease during infancy or childhood. More than half of homocystinuric patients have a first thromboembolic event before the age of 30 years, and premature atherosclerosis is a common development [2]. Although the relation of hyperhomocysteinemia to the occurrence of vascular disorders is still unclear, it is of increasing interest that less severe enzymatic abnormalities of the methionine metabolic pathway may predispose to the development of premature vascular disease. Moderate hyperhomocysteinemia, which is characterized by elevations of fasting plasma homocysteine levels as high as 100 mol/L or by abnormally high levels of plasma homocysteine after methionine load [3], has been observed with increasing frequency in young patients with arterial occlusive conditions such as myocardial infarction and cerebrovascular and peripheral occlusive diseases [4-7]. However, although venous thromboembolism accounts for 50% of the vascular complications of homocystinuria, few data are available on the relation between moderate hyperhomocysteinemia and venous thromboembolic disease [8, 9]. The term thrombophilia refers to an increased tendency to thrombosis sustained by an ongoing stimulus to thrombogenesis or by a defect of the natural anticoagulant or fibrinolytic mechanisms [10]. Genetic factors play an important role in thrombophilia because thrombosis is often familial or is associated with congenital deficiencies of the protein C anticoagulant pathway, antithrombin III, heparin cofactor II, or plasminogen. The main clinical features of patients with inherited thrombophilia are recurrent thrombosis, thrombosis at a young age, idiopathic thrombosis, thrombosis after trivial provocation, and thrombosis in an unusual site [10]. The aim of our study was to evaluate the prevalence of moderate hyperhomocysteinemia and of established disorders of inherited thrombophilia in a series of consecutive patients with early-onset venous or arterial occlusive disease. Methods Patients We studied consecutive unrelated patients referred to our coagulation service from November 1992 to October 1994. Patients were enrolled in the study if they were younger than 45 years at the time of the occlusive event (n = 142) or if they had thrombosis at unusual sites (central retinal vein or artery, cerebral veins), provided that local risk factors were absent. Exclusion criteria were the presence of overt cancer, liver disease, acquired coagulation abnormalities (for example, lupus anticoagulants, anti-protein S antibodies, and inhibitors of fibrin polymerization), or risk factors known to disturb methionine metabolism (diabetes, hypertension, hyperlipidemia and renal failure). Thirty-two patients referred during the study period were excluded on the basis of these criteria. At the first study visit, patients were asked [by means of a standard questionnaire] about their personal and family history of thrombosis and whether they had predisposing factors for thrombosis. Diagnoses were confirmed by objective methods as follows: 1) Peripheral arterial occlusive disease was confirmed by Doppler ultrasonography and angiography; 2) cerebrovascular disease was confirmed by angiography and computed tomography or nuclear magnetic resonance imaging; 3) arterial or venous retinal occlusion was confirmed by retinal fluorescein angiography; 4) deep venous thrombosis was confirmed by ultrasonography or phlebography; 5) caval or pelvic venous thrombosis was confirmed by color flow Doppler imaging; 6) pulmonary embolism was confirmed by scintigraphy or angiography; and 7) myocardial infarction was confirmed by electrocardiography. The control group consisted of 60 apparently healthy persons. These controls were either subjects, students, or members of the hospital staff (30 women and 30 men; mean age SD, 35.8 11.8 years) who were not taking any medications and who were recruited during the study period. Patients who were receiving antiplatelet drugs were not excluded from the investigation. All patients had blood drawn at least 3 months after the occlusive event if they were not receiving heparin or oral anticoagulant treatment. If patients were receiving anticoagulant treatment, laboratory evaluation was done 6 months after the event and at least 1 week after interruption of oral anticoagulant treatment. Therapies known to induce variations in homocysteine levels were not given to any patient before testing. All study participants gave informed consent. Laboratory Studies Venous blood samples were obtained in vacutainer tubes (Becton Dickinson, Rutherford, New Jersey) containing 0.129 M sodium citrate (9:1, v/v) from an antecubital vein and centrifuged within 30 minutes at 2000 g for 10 minutes at room temperature. Plasma aliquots were snap-frozen with methanol and dry ice and stored at 70 C. Assays were done within 3 months of blood collection. Total plasma homocysteine, which corresponds to the sum of free homocysteine, cysteine-homocysteine mixed disulfide, and protein-bound forms, was obtained after cleavage and reduction reactions with sodium borohydride followed by iodoacetic acid treatment. Homocysteine and O-phthaldialdehyde were purchased from Fluka (Buchs, Switzerland). All chemicals and solvents were obtained from BDH (Poole, United Kingdom). High-performance liquid chromatographic analysis was done using the O-phthaldialdehyde precolumn derivatization method [11]. Plasma vitamin B12 and folate levels were determined by using chemiluminometric immunoassays (Ciba Corning ACS, Medfield, Massachusetts). Because heterozygosity for cystathionine -synthase deficiency in patients who have normal fasting plasma homocysteine levels may be detected by an abnormal increment in the total plasma homocysteine level after administration of an oral methionine load [1], all patients older than 14 years were asked to receive oral methionine and have their plasma homocysteine levels measured. Eighty-seven patients (55%) gave their consent. After an overnight fast, a venous blood sample was drawn at 0800 h and L-methionine (0.1 g/kg body weight) was orally administered in 200 mL of fruit juice. Methionine intake was followed by a simple Italian coffee breakfast, and 5 hours later by a regular lunch. In a previous study [12], no diurnal variation in total plasma homocysteine levels in relation to food intake had been observed. The blood specimen was taken 8 hours after methionine intake. In 12 healthy persons, the transient increase in plasma homocysteine level peaked 6 to 8 hours after methionine administration. Increments in plasma homocysteine levels after methionine load were measured in controls. The activity of antithrombin III (Coatest Antithrombin, Chromogenix, Stockholm, Sweden) and plasminogen (Coatest Plasminogen, Chromogenix) were tested by amydolytic methods adapted to the ACL 300 (Instrumentation Laboratories, Milan, Italy). The activity of heparin cofactor II was tested as previously described [13], and protein C and protein S anticoagulant activity [14, 15] and antigens [14, 16] were measured by previously described methods. The anticoagulant response to activated protein C was tested in an activated partial thromboplastin time-based assay using a commercial reagent (Actin FSL, Baxter, Miami, Florida) and human purified protein C activated by the thrombin-thrombomodulin complex [17]. Because this assay was introduced in our laboratory in March 1993, patients who were enrolled in the study before this date were reinvestigated for activated protein C resistance. Family Studies When an abnormality in the laboratory variables was detected, a second blood sample was taken to confirm the observation. In addition, whenever possible, the relatives of the propositus were studied to confirm inheritance of the abnormality. First-degree relatives and siblings of 12 patients with mild hyperhomocysteinemia were studied both while fasting and after methionine intake. For 8 of these families, both parents of the proband were available for study. First-degree relatives and siblings of 11 probands were evaluated for activated protein C resistance and deficiency of protein S, protein C, and plasminogen. Statistical Analysis Results are expressed as mean SD or as median and range. Hyperhomocysteinemia was defined by a persistent elevation of fasting plasma homocysteine levels or by plasma homocysteine levels after methionine load that exceeded the 95th percentile of the distribution in the control group in the presence of normal folate and vitamin B12 plasma levels [5, 18]. In children, hyperhomocysteinemia was diagnosed when fasting plasma homocysteine levels were greater than the 95th percentile of an age-matched control group (12 mol/L [12]). Deficiency of protein C, protein S, and plasminogen and resistance to activated protein C were confirmed by the observation of values persistently lower than the 5th percentile distribution of values in the control group. In the control group, all laboratory variables were normally distributed except total plasma homocysteine levels, which were log-transformed to approximate normal distribution. Homocysteine levels are lower in women than in men [12, 19]. Because sex-related differences in plasma homocysteine levels were also observed in our control group (P < 0.0001), separate 95th percentiles were used for men (fasting levels, 19.5 mol/L; lev

Acute Intravenous l-Arginine Infusion Decreases Endothelin-1 Levels and Improves Endothelial Function in Patients With Angina Pectoris and Normal Coronary Arteriograms Correlation With Asymmetric Dimethylarginine Levels

Background—We tested the hypothesis that asymmetric dimethylarginine (ADMA) levels could be elevated and influence endothelin-1 and nitric oxide release and action in patients with cardiac syndrome X (CSX). In addition, we evaluated whether an intravenous infusion of l-arginine would improve endothelial function in these subjects. Methods and Results—Nine patients with CSX and 14 control subjects underwent a continuous infusion of l-arginine (0.125 g/min) or saline for 120 minutes. Sixty minutes after l-arginine or saline infusions, an intravenous insulin bolus (0.1 U/kg) combined with a euglycemic clamp was performed. Basal ADMA and endothelin-1 levels were higher in patients with CSX than in controls. At the end of the first hour of infusion, compared with saline, l-arginine infusion increased basal forearm blood flow, nitrite and nitrate (NOx), and forearm cGMP release and decreased endothelin-1. After insulin bolus, during saline, insulin-induced NOx, endothelin-1, and forearm cGMP release was almost abolished. Conversely, l-arginine restored a physiological profile of all endothelial variables compared with control subjects. In control subjects, compared with saline infusion, l-arginine infusion did not modify any parameter. ADMA levels were positively correlated with basal endothelin-1 levels and negatively correlated with insulin-induced incremental levels of NOx and forearm cGMP release. Conclusions—Plasma ADMA levels are increased in patients with CSX, and they are correlated with increases in endothelin-1 and reductions in insulin-induced increments in plasma NOx and cGMP, effects that are reversed by intravenous l-arginine. These data suggest that increased ADMA levels play a role in the abnormal vascular reactivity that is observed in patients with CSX.

High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction.

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected. The recovery of diclofenac was 92%, the analysis lasted 7 min with a sensitivity of 5 ng/ml and intra- and inter-day RSDs of 3 and 8%, respectively. The pharmacokinetics of diclofenac after oral and rectal administration in 10 healthy volunteers are reported.

Genetic and clinical heterogeneity of ferroportin disease

Ferroportin is encoded by the SLC40A1 gene and mediates iron export from cells by interacting with hepcidin. SLC40A1 gene mutations are associated with an autosomal type of genetic iron overload described as haemochromatosis type 4, or HFE4 (Online Mendelian Inheritance in Man number 606069), or ferroportin disease. We report three families with this condition caused by novel SLC40A1 mutations. Denaturing high‐performance liquid chromatography was employed to scan for the SLC40A1 gene. A D181V (A846T) mutation in exon 6 of the ferroportin gene was detected in the affected members of an Italian family and shown to have a de novo origin in a maternal germinal line. This mutation was associated with both parenchymal and reticuloendothelial iron overload in the liver, and with reduced urinary hepcidin excretion. A G80V (G543T) mutation in exon 3 was found in the affected members of an Italian family with autosomal hyperferritinaemia,. Finally, a G267D (G1104A) mutation was identified in exon 7 in a family of Chinese descent whose members presented with isolated hyperferritinaemia. Ferroportin disease represents a protean genetic condition in which the different SLC40A1 mutations appear to be responsible for phenotypic variability. This condition should be considered not only in families with autosomal iron overload or hyperferritinaemia, but also in cases of unexplained hyperferritinaemia.

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).

High-performance liquid chromatographic method with fluorescence detection for the determination of total homocyst(e)ine in plasma

A high-performance liquid chromatographic method for the determination of total plasma homocyst(e)ine [H(e)] after reduction with sodium tetrahydroborate and precolumn derivatization with o-phthaldialdehyde is described. The analyses, carried out on a reversed-phase C18 column, were based on spectrofluorimetric detection. The sensitivity was 1 pmol per injection and the intra- and inter-assay relative standard deviations were 1.8% and 5%, respectively. The plasma H(e) concentration determined in 40 healthy volunteers (20-60 years old) was 12.4 +/- 2.9 microM (mean +/- S.D.), in good agreement with reference values.

Telomere Length in Peripheral Blood Mononuclear Cells Is Associated with Folate Status in Men

Human chromosomes are capped by telomeres, which consist of tandem repeats of DNA and associated proteins. The length of the telomeres is reduced with increasing cell divisions except when the enzyme telomerase is active, as in stem cells and germ cells. Telomere dysfunction has been associated with development of age-related pathologies, including cancer, cardiovascular disease, Alzheimers disease, and Parkinsons disease. DNA damage in the telomeric region causes attrition of telomeres. Because folate provides precursors for nucleotide synthesis and thus affects the integrity of DNA, including that of the telomeric region, folate status has the potential to influence telomere length. Telomere length is epigenetically regulated by DNA methylation, which in turn could be modulated by folate status. In this study, we determined whether folate status and the 677C > T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene are associated with the telomere length of peripheral blood mononuclear cells in healthy men. The results of our study showed that plasma concentration of folate was associated with telomere length of peripheral blood mononuclear cells in a nonlinear manner. When plasma folate concentration was above the median, there was a positive relationship between folate and telomere length. In contrast, there was an inverse relationship between folate and telomere length when plasma folate concentration was below the median. The MTHFR 677C > T polymorphism was weakly associated (P = 0.065) with increased telomere length at below-median folate status. We propose that folate status influences telomere length by affecting DNA integrity and the epigenetic regulation of telomere length through DNA methylation.

Determination of asymmetric and symmetric dimethylarginines in plasma of hyperhomocysteinemic subjects

Summary.The aim of this study was to investigate the possible relationship among dimethylarginines (asymmetric, ADMA; symmetric, SDMA) and homocysteine (Hcy) levels in subjects affected by chronic, mild to intermediate, hyperhomocysteinemia.ADMA and SDMA were assayed by an optimised HPLC method in 75 patients (Hcy = 20.8 μmol/L, 17.1–30.2; median and percentile range) and, for comparison, in 85 healthy subjects (Hcy = 8.0 μmol/L, 7.0–9.1). In controls, the cut-off values were set at 0.61 μmol/L for ADMA and 0.56 or 0.48 μmol/L for male and female SDMA, respectively. In patients, ADMA and SDMA levels were increased (p<0.001) with respect to controls, but no correlation with Hcy was observed. Hyperhomocysteinemic subjects showed a different behaviour in respect to ADMA and SDMA levels and this allowed their stratification in 3 subgroups characterized by ADMA and SDMA in the normal range, only SDMA, or both ADMA and SDMA over the cut-off values. A lack of correlation with Hcy was again observed, thus minimizing the direct role of Hcy on ADMA and SDMA metabolism and suggesting the need for further studies on this issue.