Irene Fornaciari

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.

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.

Cultured human cells release soluble γ-glutamyltransferase complexes corresponding to the plasma b-GGT

Serum γ-glutamyltransferase (GGT) is thought to derive from the liver, but its values predict morbidity and mortality for several diseases, such as cardiac infarction, stroke, diabetes, renal failure and cancer. We assessed total GGT and its fractions in the culture supernatants of human cell lines (melanoma, prostate cancer, bronchial epithelium) by gel filtration chromatography. We also compared the GGT elution profile in plasma and the corresponding very-low-density lipoprotein (VLDL) fraction. All the cell lines tested released soluble GGT whose activity increased in parallel with the cell growth. Released GGT presented a molecular weight of 2000 kDa, identical to the b-GGT fraction of human plasma and corresponding to that of VLDL. But ultracentrifugation studies showed that b-GGT had a higher density than VLDL. The b-GGT present in human plasma can be produced by tissues other than the liver, thus explaining the increase of serum GGT observed in diseases of other organs.

High-sensitivity gamma-glutamyltransferase fraction pattern in alcohol addicts and abstainers

BACKGROUND Four fractions of gamma-glutamyltransferase (GGT) with different molecular weight (b-, m-, s-, and f-GGT) are present in human plasma. Differential GGT fraction pattern is found in non-alcoholic liver disease (NAFLD) and chronic viral hepatitis, characterized by normal or decreased b-GGT/s-GGT (b/s) ratio, respectively. METHODS Chromatographic fractional GGT analysis was performed on plasma obtained from 51 subjects: 27 alcoholics (mean (SD), age 45 (9) years; 23 males; 14 positive for viral infection), 24 abstinents from at least 1 month (43 (12) years; 20 males; 6 positive for viral infection). Twenty-seven blood donors matched for age and gender (44 (9) years; 23 males) were selected as controls. RESULTS All fractions were significantly increased in alcoholics (P<0.001), s-GGT showing the largest increase, while only m-GGT and s-GGT were elevated in abstainers (P<0.01), in comparison with controls. The b/s ratio was significantly lower in both alcoholics and abstainers than in controls (median (25th-75th perc.): 0.10 (0.07-0.15), 0.16 (0.10-0.24), 0.35 (0.29-0.53), respectively, P<0.001). Viral infection did not significantly changes absolute values of individual GGT fractions in alcoholics, but the b/s ratio was significantly lower in virus positive than in virus negative subjects (0.08 (0.05-0.12), 0.14 (0.09-0.20), respectively, P<0.01). CONCLUSIONS The fraction pattern analysis might increase the specificity of GGT as biomarker of alcohol abuse, especially concerning the differential diagnosis between alcoholism and NAFLD, a common cause of elevated GGT level in the general population.

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.

β3-adrenergic receptor activity modulates melanoma cell proliferation and survival through nitric oxide signaling

We have recently shown in B16F10 melanoma cells that blockade of β3-adrenergic receptors (β3-ARs) reduces cell proliferation and induces apoptosis, likely through the involvement of nitric oxide (NO) signaling. Here, we tested the hypothesis that the effects of β3-AR blockade on melanoma cells are mainly mediated by a decrease in the activity of the NO pathway, possibly due to reduced expression of inducible NO synthase (iNOS). B16F10 cells were used. Nitrite production, iNOS expression, cell proliferation, and apoptosis were evaluated. β3-AR blockade with L-748,337 reduced basal nitrite production, while β3-AR stimulation with BRL37344 increased it. The effects of β3-AR blockade were prevented by NOS activation, while the effects of β3-AR activation were prevented by NOS inhibition. Treatments increasing nitrite production also increased iNOS expression, while treatments decreasing nitrite production reduced iNOS expression. Among the different NOS isoforms, experiments using L-748,337 or BRL37344 with activators or inhibitors targeting specific NOS isoforms demonstrated a prominent role of iNOS in nitrite production. β3-AR blockade decreased cell proliferation and induced apoptosis, while β3-AR activation had the opposite effects. The effects of β3-AR blockade/activation were prevented by iNOS activation/inhibition, respectively. Taken together, these results demonstrate that iNOS-produced NO is a downstream effector of β3-ARs and that the beneficial effects of β3-AR blockade on melanoma B16F10 cell proliferation and apoptosis are functionally linked to reduced iNOS expression and NO production. Although it is difficult to extrapolate these data to the clinical setting, the targeted inhibition of the β3-AR-NO axis may offer a new therapeutic perspective to treat melanomas.

Acetyl-11-keto-β-boswellic acid reduces retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

Retinal diseases characterized by pathologic retinal angiogenesis are the leading causes of blindness worldwide. Although therapies directed toward vascular endothelial growth factor (VEGF) represent a significant step forward in the treatment of proliferative retinopathies, further improvements are needed. In the last few years, an intense research activity has focused around the use of herbal and traditional natural medicines as an alternative for slowing down the progression of proliferative retinopathies. In the present study, we investigated the antiangiogenic effects of acetyl-11-keto-β-boswellic acid (AKBA), one of the active principles derived from the plant Boswellia serrata, used in Ayurvedic systems of medicine. We studied the antiangiogenic properties of AKBA using the mouse model of oxygen-induced retinopathy (OIR), which mimics the neovascular response seen in human retinopathy of prematurity. We first evaluated the effects of subcutaneously administered AKBA on the expression/activity of proteins which are known to play a role in the OIR model. In the retina, AKBA increased expression and activity of Src homology region 2 domain-containing phosphatase 1 and reduced the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) as well as VEGF expression and VEGF receptor (VEGFR)-2 phosphorylation. Likely as a result of these effects, AKBA significantly reduced retinal neovascularization in OIR mice without affecting retinal cell survival and retinal function. Using retinal explants cultured in hypoxia and an activator of STAT3 phosphorylation, we showed that the AKBA-induced inhibition of VEGFR-2 phosphorylation is likely to be mediated by a mechanism depending on an SHP-1/STAT3/VEGF axis. In the OIR model, neovascularization results from the activation of retinal endothelial cells, therefore we evaluated whether AKBA affected the angiogenic response of human retinal microvascular endothelial cells (HRMECs). We observed that AKBA reduced proliferation, migration and tube formation in HRMECs stimulated with exogenous VEGF, while it reduced migration and tube formation in untreated HRMECs. Taken together, our results demonstrate the antiangiogenic effects of AKBA in a model of pathologic neovascularization, providing a rationale for further investigation of AKBA as a promising therapeutic agent to reduce the impact of proliferative retinopathies.

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.