Vanna Fierabracci

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.

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.

Oral tungstate treatment improves only transiently alteration of glucose metabolism in a new rat model of type 2 diabetes.

It has been shown that tungstate is an effective hypoglycemic agent in several animal models of diabetes. In this study, we examined the effectiveness of oral tungstate treatment in a new experimental diabetic syndrome, induced by streptozotocin (STZ) and nicotinamide in adult rats, that shares several features with human type 2 diabetes. Sodium tungstate was administered in the drinking water (2 mg/mL) of control and diabetic rats for 15, 30, 60, and 90 d. Glucose metabolism was explored in vivo by intravenous glucose tolerance test. Insulin secretion and action were assessed in vitro in the isolated perfused pancreas and isolated adipocytes, respectively. Two weeks of tungstate treatment did not modify the moderate hypergly cemia of diabetic rats but reduced their intolerance to glucose, owing to an enhancement of postloading insulin secretion. However, this effect was transient, since it declined after 30 d and vanished after 60 and 90 d of tungstate administration, whereas a trend toward a reduction in basal hyperglycemia was observed on prolonged treatment. Oral tungstate was unable to modify glucose-stimulated insulin secretion in the isolated perfused pancreas, as well as muscle glycogen levels, hepatic glucose metabolism, and insulin-stimulated lipogenesis in isolated adipocytes. Nevertheless, the decreased insulin content of pancreatic islets of diabetic rats was partially restored on prolonged tungstate treatment. In conclusion, in the STZ-nicotinamide model of diabetes, tungstate was unable to permanently correct the alterations in glucose metabolism, despite some indirect evidence of a trophic effect on β-cells. The ineffectiveness of tungstate could be related to the absence, in this diabetic syndrome, of relevant metabolic alterations in the liver, which thus appear to constitute the major target of tungstate action.

Gamma-Glutamyltransferase Fractions in Human Plasma and Bile: Characteristic and Biogenesis

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile. Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation. Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063–1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063–1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions. These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme.

The regional distribution of adenosine-regulating enzymes in the left and right ventricle walls of control and hypertrophic heart.

SummaryThe transmural distribution of the adenosine-generating enzyme 5′-nucleotidase (5′N) and of the adenosine-degrading enzymes adenosine deaminase (ADA), AMP deaminase (AMP-D) and adenosine kinase (Ado-K) were determined across the walls of left and right ventricles of control and hypertrophic rat hearts.The enzyme distribution across the left ventricle wall (but not across the right wall) of normal hearts was not uniform: 5′N activity shows its highest levels in the subepicardial and in the subendocardial regions, whereas all the other enzyme activities show their lowest levels. A similar pattern of transmural distribution was also detected in other mammalian species (ox and pig).In the experimental cardiac hypertrophy, caused by two different types of chronic cardiac overload, the levels and the profiles of transmural distribution of 5′N and ADA enzyme activities may significantly change across the rat left ventricle wall.

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species.

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.

Monocytes/macrophages activation contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques

BackgroundGamma-glutamyltransferase (GGT) is a well-established independent risk factor for cardiovascular mortality related to atherosclerotic disease. Four GGT fractions have been identified in plasma, but only b-GGT fraction accumulates in atherosclerotic plaques, and correlates with other histological markers of vulnerability. The present study was aimed to evaluate whether macrophagic lineage cells may provide a source of b-GGT within the atherosclerotic plaque.MethodsGGT expression and release were studied in human monocytes isolated from peripheral blood of healthy donors. The growth factors GM-CSF and M-CSF were used to induce differentiation into M1-like and M2-like macrophages, respectively. Plaque GGT was investigated in tissue samples obtained from patients undergoing carotid endoarterectomy.ResultsWe found that M1-like macrophages express higher levels of GGT as compared to M2-like, and that both monocytes and M1-like macrophages—but not M2-like—are able to release the b-GGT fraction upon activation with pro-inflammatory stimuli. Western blot analysis of b-GGT extracted from plaques confirmed the presence of a GGT immunoreactive peptide coincident with that of macrophages.ConclusionsOur data indicate that macrophages characterized by a pro-inflammatory phenotype may contribute to intra-plaque accumulation of b-GGT, which in turn may play a role in the progression of atherosclerosis by modulating inflammatory processes and favouring plaque instability.

Circulating gamma-glutamyltransferase fractions in cirrhosis

Four gamma‐gultamyltransferases (GGT) fractions (b‐, m‐, s‐, and f‐GGT) have been identified in human plasma and their concentrations and ratios vary in different pathological conditions. To assess the behaviour of fractional GGT in cirrhotic patients evaluated for liver transplantation.

Serum gamma-glutamyltransferase fractions in Myotonic Dystrophy type I: Differences with healthy subjects and patients with liver disease

OBJECTIVES Elevation of serum gamma-glutamyltransferase (GGT), in absence of a clinically significant liver damage, is often found in Myotonic Dystrophy type-1 (DM1). In this study we investigated if a specific GGT fraction pattern is present in DM1. DESIGNS AND METHODS We compared total and fractional GGT values (b-, m-, s-, f-GGT) among patients with DM1 or liver disease (LD) and healthy subjects (HS). RESULTS The increase of GGT in DM1 and LD, vs HS, was mainly due to s-GGT (median: 32.7; 66.7; and 7.9 U/L, respectively), and b-GGT (8.5; 18.9; and 2.1 U/L). The subset of DM1 patients matched with HS with corresponding serum GGT showed higher b-GGT (6.0 vs 4.2 U/L). CONCLUSIONS DM1 patients with normal total GGT values showed an alteration of the production and release in the blood of GGT fractions. Since increased s-GGT is also found in LD, a sub-clinical liver damage likely occurs in DM1 subjects apparently free of liver disease.

Helicobacter, gamma-glutamyltransferase and cancer: Further intriguing connections

Virulence of Helicobacter pylori, Helicobacter suis and other bacteria appears to be partly mediated through a release of gamma-glutamyltransferase (GGT), an enzyme activity capable of promoting biochemical reactions ultimately resulting in damage to gastric epithelium and suppression of immune response. Recently published studies show that secretion of bacterial GGT occurs in the form of exosome-like vesicles. Very similar GGT-rich exosomes have been described to originate from human cancer cells, and the hypothesis is thus forwarded that in the resistant and invasive phenotype of malignant cells such vesicular/exosomal GGT may play roles akin to those described for Helicobacter infection, thus providing a significant contribution to the establishment of cancer metastases.