Maria Franzini

γ-Glutamyltransferase activity in human atherosclerotic plaques—Biochemical similarities with the circulating enzyme

BACKGROUND AND PURPOSE Serum gamma-glutamyltransferase (GGT) activity has been identified as a predictor of complications of atherosclerosis, with a prognostic value for cardiovascular diseases and stroke. Human atherosclerotic lesions contain active GGT, which can give rise to pro-oxidant molecular species; thus a direct contribution of GGT to atherosclerosis progression is conceivable. The relationship between plaque and serum GGT is however unclear. METHODS AND RESULTS Human carotid plaques obtained from 18 consecutive patients undergoing carotid endoarteriectomy were analyzed, of which 6 were used for anion exchange and gel filtration chromatography/western blot studies, 7 for beta-lipoprotein precipitation, and 5 for RNA extraction and determination of low molecular weight thiols. Mean GGT activity in crude plaque homogenates was 60.9+/-21.5 (S.D.) mU/g tissue. The characteristics of GGT activity were compared in plaque homogenates and in serum obtained from controls (healthy blood donors). The methods employed (anion exchange and gel chromatography, western blot) showed the presence in plaque homogenates of two distinct complexes containing GGT activity, one of which comparable with plasma LDL/GGT complexes. Accordingly, precipitation of beta-lipoproteins from plaque homogenates resulted in removal of GGT activity. RT-PCR indicated in plaques the presence of GGT mRNA transcribed from GGT-I gene. Analysis of plaque extracts also revealed the presence of enzyme product cysteinyl-glycine both as free and protein-bound form, confirming that GGT-dependent pro-oxidant reactions may occur within the plaque environment. CONCLUSIONS The results obtained suggest the presence in plaques of a serum-like GGT protein, indicating that a direct contribution of serum GGT to enzyme activity found within atherosclerotic lesions is possible. Data also indicate the occurrence of GGT-mediated redox reactions within plaque environment, which might influence plaque progression.

State of the art of BNP and NT-proBNP immunoassays: The CardioOrmoCheck study

To evaluate differences in analytical performance and clinical results of BNP and NT-proBNP immunoassays, a proficiency testing program, called CardioOrmoCheck study, has been organized since 2005 under the patronage of the Study Group of the Cardiovascular Biomarkers of the Italian Society of Clinical Biochemistry (SIBIOC). On average more than 100 Italian laboratories were involved in the annual 2005-2011 cycles. In total, 72 study samples were distributed and measured by participant laboratories for a total of 6706 results. A great difference in between-method variability was found between BNP (43.0 CV%) and NT-proBNP (8.7 CV%) immunoassays. However, with the only exception of the POCT method for BNP assay, all immunoassay methods showed an imprecision ≤ 10 CV% at the cut-off levels (i.e. 100 ng/L for BNP and 400 ng/L for NT-proBNP assay, respectively). Furthermore, CardioOrmoCheck study demonstrated that the most popular BNP immunoassays are affected by large systematic differences (on average more than 2 folds between TRIAGE Beckman-Coulter and ADVIA Centaur Siemens methods), while the agreement between NT-proBNP methods was better. CardioOrmoCheck study demonstrates that there are marked differences in analytical performance and measured values in particular among commercial methods for BNP assay. These findings suggest that it may be not reasonable to recommend identical cut-off or decision values for all BNP immunoassays.

Antioxidant capacity and protein oxidation in cerebrospinal fluid of amyotrophic lateral sclerosis

BackgroundThe causes of Amyotrophic Lateral Sclerosis (ALS) are unknown. A bulk of evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis.MethodsWe assessed, in cerebrospinal fluid (CSF) and in plasma of 49 ALS patients and 8 controls, the amount of oxidized proteins (AOPP, advanced oxidation protein products), the total antioxidant capacity (FRA, the ferric reducing ability), and, in CSF, two oxidation products, the 4-hydroxynonenal and the sum of nitrites plus nitrates.ResultsThe FRA was decreased (p = 0.003) in CSF, and AOPP were increased in both CSF (p = 0.0039) and plasma (p = 0.001) of ALS patients. The content of AOPP was differently represented in CSF of ALS clinical subsets, resulting in increase in the common and pseudopolyneuropathic forms (p < 0.001) and nearly undetectable in the bulbar form, as in controls. The sum of nitrites plus nitrates and 4-hydroxynonenal were unchanged in ALS patients compared with controls.ConclusionOur results, while confirming the occurrence of oxidative stress in ALS, indicate how its effects can be stratified and therefore implicated differently in the pathogenesis of different clinical forms of ALS.

γ-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteine-containing tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by gamma-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its -SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug.

Accuracy of b-GGT fraction for the diagnosis of non-alcoholic fatty liver disease

Serum gamma‐glutamyltransferase (GGT) activity is a sensitive but non‐specific marker of non‐alcoholic fatty liver disease (NAFLD). Recently, four GGT fractions (big‐, medium‐, small‐, free‐GGT) were described in humans.

Cardiovascular risk factors and gamma-glutamyltransferase fractions in healthy individuals

Abstract Background: Serum γ-glutamyltransferase activity (GGT), even when within its normal reference range, catalyzes low density lipoprotein oxidation in vitro and predicts cardiovascular events. Of the four GGT fractions (b-GGT, m-GGT, s-GGT, and f-GGT) recently identified in blood, only b-GGT is found within atherosclerotic plaques. Our goal was to identify the determinants of the GGT fractions (b-, m-, s-, and f-GGT) and their association with established cardiovascular risk factors in healthy subjects. Methods: Multiple linear regression analysis was applied to estimate the association of fractional GGT with gender, age, body mass index, arterial pressure (AP), plasma glucose, alanine aminotransferase (ALT), high and low density lipoproteins (LDL-C) cholesterol (HDL-C), triglycerides (TG) and C-reactive protein (CRP) in 200 healthy subjects. Results: All GGT fractions were associated with ALT; b-GGT with AP, TG, and CRP; m-GGT with LDL-C, TG and CRP; s-GGT with TG and CRP, and f-GGT only with LDL-C, whereas gender was associated with s-GGT and f-GGT only. Conclusions: In healthy individuals, cardiovascular risk factors are associated with high molecular weight GGT fractions, namely with b-GGT, the only form present within the plaque. This finding adds to the present knowledge concerning the relevance of GGT, within its reference range, for atherosclerosis-related events. Clin Chem Lab Med 2010;48:713–7.

Cardiac biomarker testing in the clinical laboratory:Where do we stand? General overview of the methodology with special emphasis on natriuretic peptides.

Diagnosis of heart failure (HF) is not based on a single test, but on a combination of history, physical examination and appropriate investigations. For these reasons, the accuracy of diagnosis by clinical means alone is often inadequate, especially in the early, asymptomatic stages of the HF. Thus, there is an increasing interest in the development of new cardiovascular biomarkers and, consequently, a great number of laboratory tests have recently been proposed for their assay. The aim of this article is to provide a general overview on the biomarkers, recommended by international guidelines, for the diagnosis, risk stratification, and follow-up of patients with HF. Cardiac natriuretic peptides and in particular the B-type related peptides, which are considered to be the first line biomarker for HF by international guidelines, will be discussed with special emphasis.

Correlates and reference limits of plasma gamma-glutamyltransferase fractions from the Framingham Heart Study

BACKGROUND We assessed GGT fractions correlates and their reference values in the Offspring Cohort of the Framingham Heart Study. METHODS Correlates of GGT fractions were assessed by multivariable regression analysis in 3203 individuals [47% men, mean age (SD): 59 (10) years]. GGT fractions reference values were established by empirical quantile analysis in a reference group of 432 healthy subjects [45% men, 57 (10) years]. RESULTS Fractional GGT levels were higher in men than in women (P<0.0001). In both sexes, fractions were associated with: triglycerides were associated with b-GGT, alcohol consumption with m-, s- and f-GGT. C-reactive protein with m- and s-GGT, while plasminogen activator inhibitor-1 with b- and f-GGT. Body mass index, blood pressure, glucose and triglycerides correlated with b- and f-GGT. In comparison with the reference group [b-GGT/s-GGT median (Q1-Q3): 0.51 (0.35-0.79)U/L], subjects affected by cardiovascular disease or diabetes showed no change of b/s ratio [0.52 (0.34-0.79)U/L, 0.57 (0.40-0.83)U/L, respectively]. The b/s ratio was higher in presence of metabolic syndrome [0.61 (0.42-0.87)U/L, P<0.0001], while lower in heavy alcohol consumers [0.41 (0.28-0.64)U/L, P<0.0001]. CONCLUSIONS Metabolic and cardiovascular risk markers are important correlates of GGT fractions, in particular of b-GGT.

The calculation of the cardiac troponin T 99th percentile of the reference population is affected by age, gender, and population selection: A multicenter study in Italy

BACKGROUND The aim of this study is to determine the 99th upper-reference limit (URL) for cardiac troponin T (cTnT) in Italian apparently healthy subjects. METHODS The reference population was selected from 5 cities: Bolzano (n=290), Milano (CAMELIA-Study, n=287), Montignoso (MEHLP-Study, n=306), Pisa (n=182), and Reggio Calabria (MAREA-Study, n=535). Subjects having cardiac/systemic acute/chronic diseases were excluded. Participants to MEHLP project underwent cardiac imaging investigation. High-sensitive cTnT was measured with Cobas-e411 (Roche Diagnostics). RESULTS We enrolled 1600 healthy subjects [54.6% males; age range 10-90years; mean (SD): 36.4 (21.2) years], including 34.6% aged <20years, 54.5% between 20 and 64years, and 10.9% over 65years. In the youngest the 99th URL was 10.9ng/L in males and 6.8ng/L in females; in adults 23.2ng/L and 10.2ng/L; and in elderly 36.8ng/L and 28.6ng/L. After the exclusion of outliers the 99th URL values were significantly decreased (P<0.05) in particular those of the oldest (13.8ng/L and 14ng/L). MEHLP participants were divided in healthy and asymptomatic, according to known cardiovascular risk factors (HDL, LDL, glucose, C-reactive protein): the 99th URL of cTnT values of these subgroups was significantly different (19.5 vs. 22.7, P<0.05). CONCLUSIONS 99th URL of cTnT values was strongly affected by age, gender, selection of subjects and the statistical evaluation of outliers.

Contribution by Polymorphonucleate Granulocytes to Elevated Gamma-Glutamyltransferase in Cystic Fibrosis Sputum

Background Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail. Principal Findings The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils. Conclusions In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.