• gamma-glutamyltransferase fractions
• gel filtration chromatography
• diagnostic accuracy
• biomarker
• liver parenchyma
• cirrhosis
• alcohol addict
• non-alcoholic fatty liver disease; viral hepatitis

Total plasma γ-glutamyltransferase (GGT) activity is currently considered a sensitive but non-specific diagnostic marker of hepato-biliary disorders and of alcohol abuse [Whitfield, 2001].
Plasma GGT activity is affected by genetic factors, with heritability estimated between 0.3 and 0.5 [Whitfield et al., 2002; Lin et al., 2009] but it has many other correlates within its normal range. GGT shows a positive association with alcohol consumption and smoking habit, heart rate (HR), systolic (SBP) and diastolic (DBP) blood pressure, obesity indexes, such as waist circumference and body mass index (BMI), and with serum level of glucose, triglycerides, total and LDL cholesterol and uric acid [Whitfield, 2001].
Several large epidemiological studies have demonstrated that plasma GGT elevation is, an independent predictor of all-cause mortality [Brenner et al., 1997], and mortality due to either hepatic or neoplastic diseases [Kazemi-Shirazi et al., 2007]. Circulating total GGT activity has been also associated with an increased risk for arterial hypertension, diabetes, and metabolic syndrome [Lee DH et al.,2003; Lee DS et al., 2007; Fraser et al., 2009]. Plasma GGT levels within the upper normal range (25-40 U/L) were found to be associated with increased risk of cardiovascular events, independently of established cardiovascular risk factors, both in unselected populations (including the community-based Framingham Heart Study) [Ruttmann et al., 2005; Lee DH et al., 2006; Meisinger et al., 2006; Lee DS et al., 2007; Fraser et al., 2007] and in patients with prior coronary artery disease [Emdin et al., 2001]. Accordingly, elevation of plasma GGT concentrations was associated with an increase in the SCORE risk function [Ulmer et al., 2005], and GGT was also found to incrementally add to Framingham Risk Score function [Kim et al., 2011].
Recently, I have set up a reproducible chromatographic method [Franzini et al., 2008], disclosing that total plasma GGT activity corresponds, in healthy subjects, to four distinct fractions showing distinct physico-chemical properties [Fornaciari et al., 2014]. These fractions consist in three GGT-containing molecular complexes b-, m-, s-, with molecular weight >2000, 940, 140 kDa, respectively, and the free enzyme, f-GGT (70 kDa). f-GGT is the most abundant fraction, while b-GGT correlates with the level of serum triglycerides, LDL-cholesterol, C-reactive protein (CRP), and DBP [Franzini et al., 2010]. Interestingly, the active enzyme found inside the atherosclerotic plaque was shown to correspond to the b-GGT fraction [Franzini et al., 2009a].

The INTRODUCTION gives a review of what is known about the GGT protein structure and enzymatic function, then it focuses on plasma GGT reporting about its physico-chemical properties and its clinical correlations. In the following chapters, the main results obtained and published during the Specialization in Clinical Pathology are reported.

The overall aim of this research is to investigate if plasma GGT fraction pattern is associated with specific structural and functional derangements of liver parenchyma, and if each fraction is associated with a distinct diagnostic meaning.
The specific aims and main results are summarized below.

CHAPTER 2 – “CORRELATES AND REFERENCE LIMITS OF PLASMA γ-GLUTAMYLTRANSFERASE FRACTIONS FROM THE FRAMINGHAM HEART STUDY”.
Aims To establish the reference values of GGT fractions, to assess their correlates in a large reference sample of healthy subjects from the Offspring Cohort of the Framingham Heart Study, and to study the clinical correlates in the larger community sample.
Methods Correlates of GGT fractions were assessed by multivariable regression analysis in 3203 individuals [47% men, mean age (SD): 59 (10) yrs.]. GGT fractions reference values were established by empirical quantile analysis in a reference group of 432 healthy subjects [45% men, 57 (10) years].
Results The reference values for each of the four GGT fractions were established in a subgroup of healthy subjects (n= 432). In both sexes, 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.

CHAPTER 3 – “ACCURACY OF b-GGT FRACTION FOR THE DIAGNOSIS OF NON-ALCOHOLIC FATTY LIVER DISEASE”.
Aim To establish if a specific GGT fraction pattern might be associated with different liver diseases, such as non-alcoholic fatty liver diseases (NAFLD) and chronic viral hepatitis C (CHC).
Methods GGT fractions were determined in patients with NAFLD (n=90), and compared with those in control subjects (n=70), and chronic viral hepatitis C (CHC, n=45) age and gender matched.
Results Total GGT was elevated in NAFLD as compared to controls (median, 25°-75°percentile: 39.4, 20.0–82.0 U/L vs. 18.4, 13.2–24.9 U/L respectively, P<0.001). All fractions were higher in NAFLD than in controls (P<0.001). The b-GGT showed the highest diagnostic accuracy for NAFLD diagnosis (ROC-AUC: 0.85; cut-off 2.6 U/L, sensitivity 74%, specificity 81%). Also subjects with CHC showed increased GGT (41.5, 21.9–84.5 U/L, p<0.001 vs. controls, P=n.s. vs. NAFLD), as well as m-, s-, and f-GGT, while b-GGT did not show any significant increase (P=n.s. vs. HS, P<0.001 vs. NAFLD). In subjects with CHC, s-GGT showed the best diagnostic value (ROC-AUC: 0.853; cut-off 14.1 U/L, sensitivity 73%, specificity 90%). Plasma GGT did not show any value in the differential diagnosis between NAFLD and CHC (ROC-AUC 0.507, P=n.s.), while b-GGT/s-GGT ratio showed the highest diagnostic accuracy for distinguishing NAFLD and CHC (ROC-AUC: 0.93; cut-off value 0.16, sensitivity 82%, specificity 90%).
Conclusions b-GGT increases in NAFLD, but not in CHC. GGT fractions analysis might help in improving the sensitivity and specificity of the diagnosis of NAFLD and other liver dysfunction.

CHAPTER 4 – “HIGH-SENSITIVITY γ-GLUTAMYLTRANSFERASE FRACTION PATTERN IN ALCOHOL ADDICTS AND ABSTAINERS”.
Aims To describe the fractional GGT pattern in current and previous alcohol addicts.
Methods Chromatographic fractional GGT analysis was performed on plasma obtained from 51 subjects: 27 alcoholics [mean (SD), age 45 (9) yrs.; 23 males; 14 positive for viral infection], 24 abstinent from at least 1 month [43 (12) yrs.; 20 males; 6 positive for viral infection]. Twenty-seven blood donors matched for age and gender [44 (9) yrs.; 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 percentile): 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 general population.

CHAPTER 5 – “CIRCULATING γ-GLUTAMYLTRANSFERASE FRACTIONS IN LIVER CIRRHOSIS”.
Aim To assess the behaviour of fractional GGT in cirrhotic patients evaluated for liver transplantation, and to assess their correlation with routine biomarkers of liver function.
Methods This was a single-centre, cross-sectional study; GGT fractions were determined by gel-filtration chromatography.
Results 264 cirrhotic patients (215 males; median age 54.5 years) were included and compared against a group of 200 healthy individuals (100 males; median age 41.5). Median (25th-75th percentile) total and fractional GGT were higher in cirrhotics, with s-GGT showing the greatest increase [36.6 U/L (21.0-81.4) vs. 5.6 U/L (3.2-10.2), (p<0.0001)], while the median b-GGT/s-GGT ratio was lower in cirrhotics than in healthy controls [0.06 (0.04-0.10)] vs. 0.28 (0.20-0.40), p<0.0001]. The ratio showed higher diagnostic accuracy (ROC-AUC, 95% CI: 0.951, 0.927-0.969) then either s-GGT (0.924, 0.897-0.947; p<0.05) or total GGT (0.900, 0.869-0.925; p<0.001). The diagnostic accuracy of the ratio was maintained (0.940, 0.907-0.963) in cirrhotic patients (n=113) with total GGT values within the reference range. Interestingly in all cirrhotic patients the s-GGT fraction consisted of two components, with one (s2-GGT) showing a significant positive correlation with serum AST, ALT, LDH, ALP and bilirubin, and negative with albumin. The b-GGT fraction showed a positive correlation with albumin, fibrinogen, and platelet counts, and negative with INR, bilirubin and LDH.
Conclusions The ratio performs as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of aetiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range.


Overall conclusions The analysis of Framingham Offspring cohort [Chapter 2] showed that the correlates of plasma activity vary for each GGT fraction. Markers of metabolic syndrome (BMI, DBP, glucose, triglycerides) showed the highest positive correlation with the b- and f-GGT fractions. These results agree with the finding that b-GGT fraction holds the best specificity and sensitivity for the diagnosis of non-alcoholic fatty liver disease (NAFLD) [Chapter 3]. Alcohol consumption showed a prominent association with the m- and s-GGT fractions: in fact, fractional GGT profile of alcohol addicts was characterized by a greatest increase in m- and s-GGT levels vs. other fractions [Chapter 4]. Besides, I observed the elevation of s-GGT fraction also in patients with chronic viral hepatitis C (CHC) [Chapter 3], this suggesting the s-GGT fraction as a marker of hepatocellular damage.
In the Framingham Offspring cohort, comparison of total GGT values between the subsets of healthy subjects and of subjects affected by cardiovascular disease, metabolic syndrome, diabetes, or characterized by heavy alcohol consumption confirmed that total plasma GGT activity is a sensitive but nonspecific marker of disease. On the other hand, each subset was characterized by a specific fractional GGT pattern, better described by the b/s ratio: heavy alcohol intake was characterized by the highest values of s-GGT and the lowest b/s ratio, while individuals with metabolic syndrome and diabetes had the highest values of both b-GGT and b/s ratio. These data were confirmed on selected populations of patients affect by NAFLD, CHC [see Chapter 3], or in alcohol addicts [see Chapter 4].
GGT fraction analysis in NAFLD patients showed a GGT fraction pattern characterized by increased levels of all GGT fractions. Patients with CHC, and similar levels of total plasma GGT as NAFLD patients, showed instead a GGT fraction pattern characterised by the prominent increase of s-GGT, but not of b-GGT. That suggested that the b/s ratio could be key aspect of the disease-associated GGT fraction pattern. Indeed the b/s ratio showed the highest specificity for distinguishing between CHC and NAFLD, as well as for the diagnosis of CHC as compared to total GGT and all individual fractions.
The elevation of total GGT in alcohol addicts [Chapter 4] was associated with a prominent s-GGT increase, and a lesser increase of all three other fractions, thus the b/s ratio resulted significantly lower than in controls. This pattern corresponded to that found in subjects with CHC, but not in subjects with NAFLD who were characterized by b/s ratio values comparable to those of healthy subjects [Chapter 3]. Interestingly, despite similar absolute values of total GGT and its fractions, the decrease in b/s ratio was more marked in alcohol addicts proved to be positive for viral infection, suggesting that b/s ratio is a distinct and potentially quantitative biomarker of hepatocellular damage in alcoholism, even in the presence of viral infection. Abstinence from alcohol resulted in lowering of total GGT values: b-GGT and f-GGT fractions returned to normal values, while m-GGT and s-GGT levels remained persistently high, in addition to a lower b/s ratio than in controls. This finding confirms b/s ratio as a sensitive biomarker of persistent liver damage, independently from total GGT level. In addition, the differential decrease in the GGT fractions in alcohol abstainers suggests that GGT fraction analysis might perform better than total GGT, as for monitoring abstinence.
The fact that in NAFLD the increase of plasma GGT occurred through a proportional increase of b-GGT and s-GGT, while in CHC and alcoholics occurred through a prominent increase of s-GGT suggests that the GGT fraction pattern specificity might depend on its ability to reflect the different extents of inflammatory, structural and functional derangement in liver disease.
To verify if and how the structural and functional derangement of liver parenchyma could affect plasma GGT fractions, I analysed plasma samples from patients with end-stage liver disease in waiting list for liver transplant [Chapter 5]. Collected data confirmed that liver parenchymal architecture influence the synthesis and release of GGT fractions, and showed that the b/s ratio actually performed as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of aetiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range. With regard to the individual GGT fractions, this study underlined that the b-GGT was correlated with liver function, as suggested by the positive association with serum albumin, fibrinogen, platelet counts, and the negative association with INR. On the opposite, the s-GGT - and its component s2-GGT in particular - behaved as a marker of cell injury and cholestasis, showing a positive association with AST, ALT, LDH, ALP, and bilirubin, and a negative association with serum albumin.

Although an obvious consideration is that the diagnosis of CHC relies on virological tests, rather than on serum enzymes, and that fractional GGT analysis does not add any clinical information on patients evaluated for liver transplantations, these findings show that understanding the nature, properties, and pathophysiological of GGT fractions might allow a better understanding of the pathogenesis of the disease associated with increased GGT. Furthermore, for the first time fractional GGT analysis open the perspective of a positive diagnosis of NAFLD that might be helpful as a screening test or to perform large population studies on the prevalence of NAFLD and related diseases.