Gamma-glutamyltransferase fractions in the clinical pathology of liver

Specialisation School in Clinical Pathology

Disciplinary Scientific Sector MED/05

γ-GLUTAMYLTRANSFERASE FRACTIONS

IN THE CLINICAL PATHOLOGY

OF LIVER.

Supervisors:

Prof. Aldo Paolicchi

Prof. Alfonso Pompella

Candidate:

Dr. Maria Franzini

Academic Year 2012 - 2013

I

1.1.1 Generalities and tissue distribution ... 9

1.1.2 GGT gene family, protein synthesis and glycosylation ... 10

1.1.3 Reaction Mechanism ... 13

1.1.4 Physiological function of membrane GGT ... 14

1.2 Plasma GGT: analytical and structural characteristics ... 18

1.2.1 Pre-analytical variables ... 18

1.2.2 Enzymatic assay for plasma GGT ... 18

1.2.3 Reference values for plasma GGT ... 19

1.2.4 GGT plasma fractions ... 20

1.2.5 Fractional GGT analysis ... 23

1.3 Total plasma GGT activity and liver ... 27

1.3.1 Factors influencing plasma GGT levels ... 28

1.3.2 Plasma GGT and HCV-related hepatitis ... 29

1.3.3 Plasma GGT and alcohol intake... 30

1.3.4 Hepatic GGT expression vs. plasma GGT activity ... 31

1.3.5 Molecular pathways of GGT induction ... 33

1.4 Total plasma GGT activity: epidemiological associations ... 34

1.4.1 Long-term survival and cardiovascular risk ... 34

1.4.2 Plasma GGT and metabolic diseases: type 2 diabetes... 36

1.4.3 GGT and metabolic diseases: metabolic syndrome... 39

1.4.4 Plasma GGT, hepatic insulin resistance and fatty liver ... 41

1.4.5 GGT deficiency... 44

2. Correlates and reference limits of plasma GGT fractions from the FHS ... 45

3. Accuracy of b-GGT fraction for the diagnosis of non-alcoholic fatty liver disease ... 56

4. High-sensitivity GGT fraction pattern in alcohol addicts and abstainers ... 63

5. Circulating GGT fractions in liver cirrhosis ... 68

A

ALP Alkaline phosphatase

ALT Ala-aminotransferase

AST Asp-aminotransferase

BMI Body mass index

CHC Chronic viral hepatitis C DBP Diastolic blood pressure FLI Fatty liver index

γGluAMC γ-glutamyl-7-amido-4-methylcoumarin GGT γ-glutamyltransferase GSH glutathione GSNO S-nitrosoglutathione HDL High-density lipoprotein HR Heart rate

hsCRP High sensitive C-reactive protein

HSU (GGT) Heavy subunit

IFG Impaired fasting glucose LDH Lactate dehydrogenase LDL Low density lipoprotein

LSU (GGT) Light subunit

LT Leukotrienes

MetS Metabolic syndrome

NAFLD Non-alcoholic fatty liver disease SBP systolic blood pressure

T2D Type 2 diabetes

TG Triglycerides

WC Waist circumference

K

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

A

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 people and 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 GGT, while in CHC and alcoholics occurred through a prominent increase of s-GGT suggests that the s-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.

1.

I

Activity of the enzyme γ-glutamyltransferase (GGT; EC 2.3.2.2) in serum or plasma is commonly measured in clinical laboratories as a sensitive but not very specific liver function test, in fact its elevation is observed in case of cholestasis, steatosis, steatohepatitis, viral hepatitis, hepatocellular carcinoma, alcohol abuse. The measurement of GGT using automated analysers is quick, cheap, and precise. However, the application of the test is based mostly on empirical evidence rather than on any deep understanding of the pathophysiological basis of the abnormalities of GGT in liver disease or in other conditions.

GGT measurement was introduced into clinical laboratories in the late „60th [Szczeklik et al., 1961; Lum and Gambino 1972; Sheehan and Haythorn, 1979] and over that time a large amount of information on factors influencing its activity in serum has accumulated. In fact serum 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]. Also pre-existing ischemic heart disease, diabetes mellitus, menopause and use of antihypertensive medication, lipid lowering drugs and oral contraceptives show a positive association with GGT level, while an inverse association has been observed with coffee consumption, physical activity, and lung function (FEV1) [Whitfield, 2001].

In addition, in the last 30 years, several large epidemiological studies conducted in unselected populations have demonstrated that serum 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]. Serum 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., 2009] and in patients with prior coronary artery disease [Emdin et al., 2001]. Accordingly, elevation of serum 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].

Theories have been put forward about GGT normal function within the body and its role in numerous pathological conditions. Within the last few years there have been significant advances in the understanding of GGT‟s physiological roles and their consequences at cellular level.

1.1

B

γ-

.

1.1.1 Generalities and tissue distribution.

GGT is an evolutionary conserved enzyme that specifically catalyses the cleavage of the γ-glutamyl bond of glutathione (GSH) and the transfer of the γ-γ-glutamyl group to water (hydrolysis), amino acids, or peptides (transpeptidation) [Meister 1995; Keillor et al. 2005]. Mammalian GGT is a dimeric glycoprotein consisting of a heavy (HSU 55-62 KDa, 380 aminoacids) and a light subunit (LSU 20-30 KDa, 189 aminoacids) linked by non-covalent bonds. In the N-terminal portion of the heavy subunit there is a hydrophobic domain which allows the enzyme to be anchored to cell plasma membrane; in particular, both subunits are exposed in the extracellular environment [Finidori et al., 1984]. GGT catalytic site is localized in the light subunit, thus the enzyme acts on extracellular substrates [Tate and Meister, 1977; Ikeda et al., 1995].

GGT has a central role in GSH metabolism and in the γ-glutamyl cycle, which includes synthesis and degradation of GSH [Meister, 1995]. In this tripeptide glutamic acid and cysteine are linked by a particular peptide bond in which the carboxylic group on γ-carbon of glutamic acid binds the amino group on α-carbon of cysteine. The γ-glutamyl bond makes GSH resistant to peptidase, but not to GGT which is able to hydrolyze it or to transfer the glutamic acid to an acceptor (amino acid or dipeptide). GSH is the most abundant substrate for GGT, but it is not the only one: actually all γ-glutamyl-compound are substrate for the enzyme, e.g.: GSH conjugates of xenobiotics, leukotrien C4 [Whitfield, 2001], S-nitrosoglutathione [Hogg et al., 1997]

GGT is widely distributed, being found in bacteria [Sakai et al., 1996], plants [Martin and Slovin, 2000], as well as in all members of the animal kingdom. The distribution of immunoreactive GGT in normal human tissues was studied by Hanigan and Frierson [1996]. They showed that GGT is present in the plasma membrane of virtually all cells, but it is principally localized in epithelial tissues with secretory or absorptive functions. The highest activity is present in the kidney, where GGT is localized to the luminal surface of the proximal tubule cells, while it is virtually absent in distal tubules and glomeruli. In liver, GGT is concentrated in biliary epithelial cells and bile canaliculi. In pancreas the major GGT activity is in the acinar cells. A strong immunoreactivity is observed also in endothelial cells lining the capillaries in the brain (e.g. choroid plexus), ciliary body and spinal cord. GGT positive are also cells of sweat and submandibular glands, galactophorous duct, bronchial epithelium, epididymis, seminal vesicle and prostate.

On the other hand a number of cell types, without in secretory or absorptive functions, shows significant GGT activity. GGT is expressed in almost all blood cells: in granulocytic and lymphocytic cell lineages is a surface marker reflecting differentiation in normal and neoplastic cells [Novogrodsky et al., 1976; Khalaf and Hayhoe, 1987; Grisk et al., 1993; Sener and Yardimci, 2005]; in platelets GGT is present in the secretory granules [Bolodeoku et al., 1997] as well as in neutrophils which release GGT activity upon activation [Corti et al., 2012]. Furthermore, GGT is normally found in serum as result of organ release, its level thus reflecting the quantitative modification of its production and release in the blood [Huseby and Ingebretsen, 1993].

1.1.2 GGT gene family, protein synthesis and glycosylation

In human genome is present a multigene family for GGT including at least seven different genes [Courtay et al., 1994]. Most of the latter are localized on chromosome 22 in the region 22q11.1-q11.2 close to the BCR and IG-λ loci [Bulle et al., 1987; Collins et al., 1997]. Correlated sequences have been identified on chromosomes 18, 19, 20 [Figlewicz et al., 1993]. Clones representing all of these seven possible genes were present in a human genomic library constructed with DNA from a karyotypically normal lymphoblast cell line. This excluded the possibility that these seven types of genomic clones were alleles of a single highly polymorphic gene [Courtay et al., 1994]. It is very likely that the multiple human GGT genes are the products of duplications that occurred in 22q11, an unstable DNA region associated with birth defects [Scambler, 1993].

Among the duplicated GGT genes, only the type I gene (ggt1) is ubiquitously expressed and codes for a complete and functional proteins. Of the other six genes, at least four are transcripted into mRNA possibly coding for peptides that largely consist of either large or small subunits, but these proteins have not been characterized [Courtay et al., 1994; Chikhi et al., 1999]. For a comprehensive review on the human GGT gene family see Heisterkamp et al. [2008].

GGT is an N-teminal nucleophile hydrolase. In 1995, Brannigan and collaborators recognized a new protein structural superfamily called N-teminal nucleophile hydrolase (Ntn-hydrolase). Although the amino acid sequence homology is almost completely absent, this superfamily is characterized by a four-layered catalytically active αββα-core structure; all the known Ntn-hydrolases are translated into a single propetide autocatalytically processed to yield an active enzyme, contain a N-terminally located catalytic nucleophile, and catalyse amide bond hydrolysis. Members of Ntn-hydrolase include penicillin G acylase, the proteasome, aspartylglucosaminidase, glutamine PRPP aminotransferase (GAT) and L-aminopeptidase-D-Ala-esterase/amidase [Oinonen and Rouvinen, 2000].

Having all the characteristic mentioned above, GGT can be considered a member of Ntn-hydrolase superfamily [Suzuki and Kumagai, 2002], in fact:

1) the analysis of the crystal structure E. coli [Okada et al., 2006] and human [West et al., 2013] GGT confirmed the presence of the αββα-core structure

2) GGT mRNA is translated into a single propetide [Barouki et al., 1984] of 569 amino acids cleaved to yield a stable heterodimer with the amphipathic HSU (380 amino acids) and the LSU (189 amino acids) [Sakamuro et al., 1988]. Propetide cleavage occurs while GGT is still within Endoplasmic Reticulum [Kinlough et al., 2005] through an autocatalytic process as assessed for GGT from E. coli [Suzuki and Kumagai, 2002], rat [Kinlough et al., 2005], H. pylori [Boanca et al., 2006], and human [West et al. 2011]

3) The N-terminal Thr, the first amino acid of LSU, is the catalytic nucleophile [Inoue et al., 2000; Kinlough et al., 2005; West et al. 2011].

4) GGT cleaves the γ-glutamyl amide bond to liberate Cys-Gly.

Role of glycosylation on the autocatalytic cleavage of human GGT (Figure 1.1). GGT exhibits a heterogeneous pattern of glycosylation that varies in a tissue-specific manner, yet the role that N-glycosylation plays in modulating the functional maturation and kinetic behaviour of the enzyme has to be fully ascertained. According to the primary sequence, human GGT possesses seven potential N-glycosylation sites (sequon Asn-X-Ser/Thr, X≠Pro): six in the large subunit (Asn95, Asn120, Asn230, Asn266, Asn297, Asn344) and one in the small subunit (Asn511) [Sakamuro et al., 1988; Castonguay et al., 2007]. All these sites have been confirmed to be glycosylated on human kidney and liver tissue and glycans structure for these two tissue specific GGTs has been defined. Interestingly the liver synthesizes an array of N-glycans on the GGT polypeptide that are completely distinct from those that are synthesized by the kidney [West et al., 2011].

Although variations in the compositional features of the glycans themselves have been shown to modulate the reaction kinetics of the mature heterodimer, little is known as regard the impact of glycosylation on the structural integrity and autocatalytic cleavage of the nascent enzyme [Meredith, 1991]. A first insight comes from studies of West and collaborators (2011) performed on wild-type or mutated human GGT expressed in the HEK293 cell line (human embryonic kidney). These authors showed that the loss of a single N-glycosylation site is functionally tolerated, although the Asn95Gln mutation resulted in a marked decrease in the auto-proteolysis efficiency of the propeptide. However, each of the single site mutants exhibited decreased thermal stability relative to wild-type GGT. Combined mutagenesis of all N-glycosylation sites resulted in the accumulation of the inactive propeptide form of the enzyme. Furthermore it has been showed that co-translational glycosylation is the critical event governing the proper folding and subsequent cleavage of the nascent propeptide into an active enzyme. In fact the enzymatic deglycosylation of the mature

wild-type GGT does not substantially impact either the kinetic behaviour or thermal stability of the fully processed human enzyme [West et al., 2011].

Figure 1.1 N-glycosylation sites on human GGT

A) Schematic representation of the human GGT propeptide and its seven N-glycosylation sites. The positions of the transmembrane domain (TM), N-glycosylation consensus sites (N), and propeptide cleavage site are indicated [West et al., 2011].

B) Ribbon diagram of human GGT (large subunit: blue; small subunit: green). Glycosylated asparagine residues are highlighted with CPK atom colouring, and their positions in the amino acid sequence are indicated with white numbers. The position of the substrate channel and catalytic threonine (T381, red sticks) are shown. The homology model is based on the crystal structure of soluble GGT from E. coli. The first 34 aminoacids of human GGT contain the transmembrane domain and do not have homology to E. coli GGT. The position of amino acid 35 (aa35) is shown, which defines the orientation of the enzyme on the cell surface [West et al. 2010].

1.1.3 Reaction Mechanism

GGT catalyses the cleavage of the γ-glutamyl linkage of γ-glutamyl compounds, such as GSH and its S-conjugated, including leukotriene C4, S-nitrosoglutathione (GSNO), and GSH adducts of xenobiotics formed by the action of GSH-S-transferases [Whitfield, 2001]. In a second step, GGT catalyses the transfer of the γ-glutamyl moiety to other aminoacids or dipeptides [Keillor et al., 2005]. In the most widely accepted reaction mechanism, the highly conserved Thr (381 in human enzyme), which is also responsible for autoprocessing, attacks the C=O group of the γ -glutamyl-compound to form a γ-glutamyl-enzyme intermediate. The intermediate then reacts with water, to release glutamate in a hydrolysis reaction, or with an acceptor, to give a transpeptidation reaction forming new γ-glutamyl compounds (Figure 1.2). The simplest acceptor substrates include single amino acids (Cis, Met, Gln, Glu) and glycine dipeptides (CisGly, MetGly, GlnGly, CysGly, GlyGly) [Allison, 1985].

Figure 1.2 Proposed mechanism for catalysis of γ-glutamyl peptide cleavage by GGT.

The hydroxyl group of the N-terminal Thr residue of the small subunit attacks the γ-glutamyl peptide bond of GSH and leads to the formation of a tetrahedral transition state, whose collapse leads to the formation of a tetrahedral intermediate (γ-glutamyl enzyme complex). The intermediate is stabilized through interactions with two conserved glycines, and concomitant expulsion of the leaving group. A second Thr in the sequence of the small subunit can increase the reactivity of catalytic Thr. [Castellano and Merlino, 2012].

1.1.4 Physiological function of membrane GGT

GGT enzymatic activity is so specific for the cleavage of the γ-glutamyl peptide bond that only the latter is critical for the interaction between the enzyme and the substrate, in fact all γ-glutamyl-compounds are potential substrates for GGT. The main physiological substrates of GGT are the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), the leukotriene C4, the glutathione conjugates produced by GSH-transferase, and the S-nitrosoglutathione.

Antioxidant function: glutathione synthesis.

GSH is deeply involved in the maintenance of normal cellular redox status [Pompella et al., 2003]. Intracellular GSH level depends on the equilibrium existing between its consumption and synthesis, the latter is regulated by the availability of the three amino acid precursor (Glu, Cys, Gly). GGT catalyses the first step in the degradation of extracellular GSH, thus making possible the uptake of the three separate amino acids by cells (Figure 1.3).

GSH is considered the main form of storage and transport of Cys, the latter, in fact, is extremely unstable and it rapidly auto-oxidizes to cystine, producing potentially toxic oxygen free radicals [Lu, 1999]. Releasing CysGly from GSH, GGT allows the efficient utilization of GSH as Cys storage. Since cystine is the preferred acceptor for GGT transpeptidation reaction [Meister, 1988; Meister, 1995], Cys can be taken up by cells also through γ-glutamylcystine (Figure 1.3). Cys is the limiting substrate for GSH synthesis, but it is especially important for protein synthesis, being an essential aminoacids [Lu, 1999; Zhang et al., 2005].

GGT-dependent pro-oxidant reactions.

Stark et al. [1993] first proposed that GGT-dependent catabolism of GSH can drive pro-oxidant reactions in particular in presence of ferric iron. GGT was thus shown to stimulate lipid peroxidation (LPO) in several systems involving GSH as substrate, Fe(III) complexes as redox catalysts, GlyGly as transpeptidation acceptor and having as targets: linoleic acid [Stark et al., 1993], isolated LDL [Paolicchi et al., 1999] or cells expressing GGT (e.g. hepatic pre-neoplastic lesions or isolated hepatocytes and HepG2 cells) [Pompella et al., 1996; Paolicchi et al. 1997].

The mechanism of GGT-dependent LPO is based on CysGly, the sulfhydryl group of which is predominantly dissociated in the anion form at pH 7.4. CysGly thiolate anion, turning into thiyl radical, can redox-couple with Fe(III) thus triggering production of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide (H2O2) in first place (Figure 1.3). ROS and thiyl radicals are

then responsible of LPO [Zalit et al., 1996]. GGT-dependent pro-oxidant reactions were observed also in presence of physiological sources of iron, i.e. transferrin and ferritin, that means these reactions can take place in vivo and, moreover, that reducing power of CysGly, originated by GGT,

is sufficient to effect the reductive release of redox-active iron from its storage protein [Stark et al., 1993; Drozdz et al., 1998; Corti et al., 2004]. Thus GSH catabolism by GGT could represent a mechanism to increase locally the availability of free iron [Paolicchi et al., 2002].

The effects of GGT-dependent pro-oxidant reactions on the extracellular environment and cell metabolism have been reviewed by Dominici S. and collaborators [2005]; in the light of these modifications, the role of GGT in cancer have been reviewed by Corti and collaborators [2010].

S-Nitrosoglutathione metabolism.

The nitric oxide (NO) is an important mediator of many biological functions, the most important are: the relaxation of smooth muscular cells (vasodilator function), inhibition of platelet aggregation, it is a neurotransmitter, and as a radical compound can behave either as an oxidant or anti-oxidant. The NO can also react with thiol groups of proteins (albumin, hemoglobin) or with low molecular weight thiols (GSH, CysGly, Cys, homocysteine) forming nitrosothiols (RSNO) that represent a form of stabilization and transport of NO [Giustarini et al., 2003]. In particular nitrosoglutathione (GSNO) has many pharmacological activities comparable to those of free NO [Zeng et al., 1991; Rassaf et al., 2002].

Figure 1.3 Membrane GGT activity and GSH catabolism.

A) γ-glutamyl cycle: degradation of extracellular GSH and its intracellular re-synthesis of GSH. GGT also promotes the recovery of Cys both by degrading GSH and using L-Cis as acceptor of the transpeptidation reaction.

B) GGT-dependent pro-oxidant reactions in presence of FeIII. The main targets of CysGly thiyl radical and of reactive oxygen species are both cell surface molecules (proteins and membrane lipids) and intracellular proteins sensitive to the redox state of the cell (e.g. NFkB, AP1) [Franzini et al., 2009].

The GSNO is a substrate for GGT that catalyses its conversion into S-nitrosocystenylglycine (CG-SNO), the latter spontaneously dissociates into Cys-Gly and NO in the presence of transition metal ions [Hogg et al., 1997]. So, if the GSNO allows the transport of NO, the activity of GGT may allow its use by promoting the dissociation of the GSNO. A possible target could be just the blood vessels, since the GGT is expressed by endothelial cells. In fact in in vitro model it has been shown that endothelial GGT activity mediates the vasorelaxant effect of GSNO in rat aorta under physiological conditions [Dahboul et al., 2012].

Detoxifying role: mercapturic acid formation.

GGT participates to detoxification processes having a role in the synthesis of mercapturic acids which derive from GSH conjugates. Being a nucleophile, GSH can directly reacts with electrophile compound, otherwise GSH conjugates are also formed by the cytosolic enzyme GSH S-transferase. Anyway, GSH-adducts are actively secreted from the cell where enter the pathway of mercapturic acid formation, after the removal of Glu from GSH by GGT activity. The metabolism of GSH conjugates to mercapturic acids begins either in the biliary tree, intestine, or kidney and they are eliminated in bile and urine [Hinchman et al., 1998; Kearns and Hall, 1998].

Leukotriene metabolism.

Leukotrienes (LT) belong to a class of arachidonic acid-derived lipid inflammatory mediators produced by lipoxygenase pathways. They include the cysteinyl-LTs LTC4, LTD4, and LTE4, representing biologically active constituents of the long-known “slow-reacting substance of anaphylaxis” and the dihydroxyeicosatetraenoate LTB4. Leukotriene C4 is conjugated to a GSH molecule and it is converted into leukotriene D4 by GGT activity. The cleavage of Gly from LTD4 yields LTE4 [Lewis et al., 1990]. Leukotrienes, C4 and D4 included, bind to specific receptors on smooth muscle cells causing prolonged bronchoconstriction [Anderson et al., 1982; Bernstrom and Hammarstrom, 1982].

Cysteinyl-LTs metabolism has been recently investigated in three patients with GGT deficiency [Mayatepek et al., 2004]. Patients displayed an abnormal profile of LTs in urine and in plasma with increased concentrations of LTC4 and absence of LTD4 as well as LTE4, whereas LTB4 synthesis was not affected. GGT deficiency thus can be regarded as an inborn error of cysteinyl-LT synthesis. LTC4 synthesis deficiency has been found to be associated with a fatal developmental syndrome, including severe muscular hypotonia, psychomotor retardation, failure to thrive, and microcephaly [Mayatepek et al., 1998]. Variable neurological disorders are associated with GGT deficiency too; therefore Mayatepek et al. [2004] proposed that LT metabolic defect, either excessive LTC4 or more likely lack of LTD4 and LTE4, may contribute to some or even all of the observed symptoms.

GGT and the cytokine-like function.

Recently a new and exciting function has been proposed for GGT. Niida et al. [2004], in fact, reported that the addition of GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. The same result was obtained after inhibition of the enzymatic activity by acivicin, but not in presence of an antibody which recognized GGT without affecting the enzymatic activity. Furthermore, it was shown that both native and inactive GGT stimulated the expression of the receptor activator of NF-kB ligand (RANKL) mRNA and protein from bone marrow stromal cells. Thus GGT seems to possess a cytokine-like biological function independently of its enzymatic activity. A subsequent study showed that urinary excretion of GGT changes in parallel with established biochemical markers of bone resorption (i.e. deoxypyridinoline and type I collagen N-telopeptide) both in animal models and human subjects [Asaba et al., 2006].

Structural study of the human GGT crystal showed the presence of two intramolecular disulfide bonds between Cys-50 and Cys-74 and between Cys-192 and Cys-196. The Cys-50/Cys-74 disulfide bond is inaccessible to the solvent because it was buried 10 Å beneath the surface of the large subunit. The Cys-192/Cys-196 disulfide bond, instead, is partly exposed to the solvent at the molecular surface [West et al., 2013].

Interestingly, the Cys192 and 196 are arranged in a CX3C motif that is associated with several biological meaning:

1) it is a signature motif for one class of chemokines [Bazan et al., 1997], the only known member of which is fractalkine/neuroactin [Stievano et al., 2004].

2) it is an essential coordinate for copper binding in several yeast proteins [Balatri et al., 2003]. 3) it is essential for the functioning of the vaccinia virus protein A2.5L, a thiol oxidoreductase that controls disulfides formation in viral membrane proteins [Senkevich et al., 2002].

1.2

P

GGT:

1.2.1 Pre-analytical variables

The enzyme can be determined almost indifferently in lithium-heparin plasma and serum [Arbeitsgruppe et al. 2002], but the preferred type of sample, even for the execution of the reference procedure, is serum. The comparative analysis of serum and lithium-heparin plasma from 77 patients showed a mean value of the ratio plasma/serum close to 0.9 and a non-symmetrical distribution of the values of this ratio [Dominici R et al., 2004]. In practice GGT activity is measured on lithium-heparin plasma. The use of other anticoagulants (EDTA, citrate) is not recommended.

While in the blood at room temperature the enzyme is not very stable, and a significant lowering of its activity can be observed within 24h, in plasma and serum the enzyme is stable for several years at -20 ° C, and for 7 days at 4-8 °C or at 20-25 °C [Heins et al., 1995].

Numerous biological pre-analytical variables can influence GGT activity levels in blood [Whitfield, 2001]. Among them pregnancy, birth, ethnicity, tobacco smoking, use of oral contraceptives can be mentioned. Age and gender also have a marked effect, which will be discussed in the next paragraph.

1.2.2 Enzymatic assay for plasma GGT

Compared to other enzymes measured in blood for medical purposes, GGT occupies a "lucky" position, since a single type of assay has been set up for its measurement, which is the same currently used in all automated platform.

In general terms, the reaction catalysed by GGT has the format:

γ-glutamyl-x + acceptor  γ-glutamyl-acceptor + x

As "donor" of γ-glutamyl (γ-glutamyl-x) different synthetic substrates have been employed [Fossati et al., 1986], among them the γ-glutamyl-p-nitroanilide encountered a particular favour. It behaves as a chromogenic substrate allowing a direct and continuous spectrophotometric measurement of the reaction trend. In the automated assay is preferentially used its carboxylate form, L-γ-glutamyl-3-carboxy-4-nitroanilide, which is much more soluble, thus allowing to work at concentration close to the saturating one. As "acceptor" of gamma-glutamyl, among the different amino acids and peptides, the choice has been directed towards the glycylglycine, which has also good buffering capacity at pH values optimal for the enzyme activity.

The International Federation of Clinical Chemistry (IFCC) has defined the reference analytical procedures for serum GGT first in 1983 [Shaw et al., 1983] and then in 2002 , whose characteristics are resumed in Table 1.1.

Table 1.1 Characteristics of the reference (standard) method for serum GGT determination

according to IFCC [Schumann et al., 2002]

Components Concentrations* Assay conditions

GlyGly 150 mmol/L T: 37.0 ± 0.1 °C. λ: 410 ± 1 nm

pH 7.70 ± 0.05 Δλ: 2 nm

L-γ-glutamyl-3-carboxy-4-nitroanilide 6 mmol/L Optical path: 10.00 ± 0.01 nm Volume ratio sample/reaction mixture 0.0909 (1/11) Incubation time: 180 s

Delay time: 60 s

Measurement range: 180 s N° of readings: 6

* In the final reaction mixture

This method can be applied to the different models of automatic analyser, thus the measured values can be easily traceable to the reference method. Anyway, being impossible to guarantee the absolute accuracy of some instrumental variables (with particular reference to the wavelength), it is advisable the use of commutable standardised calibrators [Cattozzo et al., 2008].

Data from the External quality assessment program of Regione Lombardia (Italy) performed in 2007 (12 serum with different GGT concentrations) showed that the inter-laboratory variability was about 8% for GGT levels higher than 100 U/L and 13.6% for 28 U/L; the variability was lower (4-7%) when considering only the laboratories that used the same automated platform. These data suggest the an important component of this variability is due to the calibration, this component can be decreased by the use of standardized calibrators [Franzini et al., 2009].

1.2.3 Reference values for plasma GGT

The reference values of serum GGT are modulated by different pre-analytical variables, including age and gender, thus they should be formulated separately for the two genders and for age classes, but a single reference range is more commonly used by laboratories.

Reference values for several plasma analytes have been derived by retrospective analysis of a large database (n=61246), those for GGT resulted 1-34 U/L for women and 1-45 U/L for men [Grossi et al. 2005]. From this database the variation of the upper reference limit (URL) for GGT according to age groups (2 to 87 years of age, 5-year classes of amplitude) and gender has been calculated (Figure 1.4), which is very similar to that already reported [Whitfield, 2001].

On a practical level, variations depending on the age, such as those observed for GGT, are not easily manageable. In addition, the division of the whole age range in classes of uniform width is not considered the best approach to calculate the reference values, rather partitions based on the statistical detections of sub-Gaussian populations within the overall population should be identified [Gellerstedt, 2006; Gellerstedt, 2007]. Alternatively, age can be considered a continuous variable (such as it is) and the equation of the regression curve that best fits the experimental points (URL/age) can be used to calculate the URL for each age.

Infants levels overcome 6–7 times the upper limit of the adult reference range, which declines to adult levels around age 7 months [Cabrera-Abreu, Green, 2002].

1.2.4 GGT plasma fractions

As is the case for the concentrations of all blood components, even for GGT the homeostatic concentration in blood is the result of a dynamic equilibrium between the release into and the clearance from plasma. Despite the frequent clinical and diagnostic use, knowledge on the tissue origin of plasma GGT as well as on the mechanisms of secretion and removal from the circulation are fragmentary.

Plasma GGT is believed to be of hepatic origin as suggested by its physico-chemical characteristics and kinetics, in fact circulating and liver GGT have the same molecular weight, same

Figure 1.4 Variation of the upper reference limit for serum GGT according to age.

URL for serum GGT in men (black diamond) and women (white circle). The interpolating curves have to the following equations [Franzini et al., 2009]:

Women y = 0.5248x + 11.564; r2 _{= 0.9646}

Huseby, 1981] and similar enzyme kinetic [Shaw et al., 1978]. Parameters which are rather distinct from the enzymes purified from kidney, pancreas or urine [Shaw et al., 1978; Shaw et al., 1980; Huseby, 1981].

Plasma GGT can be divided into two fractions, a hydrophilic and a hydrophobic one, which differ in charge, size, and density [Huseby, 1978; Huseby, 1982; Selvaraj et al., 1984]. The hydrophobic fraction is constituted by the enzyme GGT associated with molecular complexes whose structure has to be defined in detail yet. The hydrophilic component includes a soluble form of the GGT enzyme lacking the lipophilic N-terminal peptide. The hydrophilic portion of the plasma GGT represents about 60-70% of the total activity in normal subjects, but only 15-30% in patients with liver disease, because of the increase in the hydrophobic component [Huseby, 1982; Selvaraj et al., 1984]. The first studies conducted to determine the nature of plasma GGT carriers seemed to suggest that the enzyme was associated with circulating lipoproteins (VLDL, LDL, HDL, and chylomicrons) through the N-terminal peptide of the heavy chain, responsible for the normal enzyme insertion in the plasma membrane [Huseby, 1982; Watanabe et al., 1984; Wenham et al., 1984].

The development of a new chromatographic method for the separation of the circulating complexes with GGT activity (described in the next section) [Franzini et al., 2008] has allowed deepening further the matter [Fornaciari et al., 2014]. The results of this study demonstrated that none of the three GGT fractions could be completely identified with the corresponding class of lipoprotein sharing the same molecular weight. Indeed, excluding s-GGT, all the other GGT fractions has distinctive features as compared to lipoproteins, and they can be physically separated from them. These results are against what suggested in early studies, i.e. that circulating lipoproteins are GGT carriers and that GGT is too lipophilic to circulate in a free form [Huseby, 1982]. Furthermore, it was also observed that plasma f-GGT fraction has a molecular weight corresponding to the free GGT protein, thus demonstrating that part of plasma GGT do not require any carrier, such as lipoproteins [Huseby, 1982] or albumin [Pompili et al., 2003], to be transported in blood.

In this study [Fornaciari et al., 2014] it has been showed that plasma b-GGT fraction is constituted of membrane microvesicles (microparticles and exosomes), b-GGT dimension and density (30-80 nm; 1.06-1.21 g/ml) being compatible with those of membrane microvesicles (40-100 nm; 1.15-1.27 g/ml) [Cocucci et al., 2009]. According to this interpretation, b-GGT was also shown to be sensitive to deoxycholic acid action, i.e. a detergent that can disrupt membrane microvesicles [Cocucci et al., 2009], and immunogold analysis confirmed the association between GGT protein and exosomes (Figure 1.5). The vesicular nature of b-GGT explain why all cell lines expressing

GGT on plasma membrane release soluble GGT as a 2000 kDa molecular complex, identical to the plasma b-GGT [Franzini et al., 2009b].

More complicated seems to be the biogenesis of the other GGT fractions (m-GGT, s-GGT and f-GGT) that might arise from progressive modifications of the b-GGT fraction. In this perspective it has been demonstrated that b-GGT is sensitive to protease papain only after a pre-treatment with deoxycholic acid, this possibly suggesting a protective role of b-GGT phospholipid bilayer on papain cleavage site. On the other hand, the fact that deoxycholic acid is able to convert b-GGT fraction into micelles of s-GGT size suggests that these latter might be constituted of micelles of bile acids, and that this conversion would take place in the extracellular compartment, after b-GGT secretion. Similarly to s-GGT, also m-GGT fraction appeared to be sensitive to papain but insensitive to deoxycholic acid: these characteristics, together with its physical properties (size, density), suggest that m-GGT could be constituted of micelles of bile acids with sizes greater than s-GGT ones. Finally, f-s-GGT corresponds to the free, soluble form of the enzyme, lacking the N-terminal anchoring peptide, which is present in all other fractions; f-GGT might originate directly from both m-GGT and s-GGT fraction as a consequence of a proteolytic cleavage [Fornaciari et al., 2014].

The clearance mechanisms of plasma GGT have been studied in vivo in animal models (rabbit and rat) using the hydrophobic component of circulating GGT separated from sera of patients having total GGT levels higher than > 200U/L or the enzyme purified from human liver. The latter enzyme preparation corresponded to the soluble (hydrophilic) component of serum GGT, in fact the protocol to purify GGT from tissues involves the use of proteases, such as papain, to hydrolyses the N-terminal trans-membrane peptide. From these studies it has been showed that the hydrophilic

Figure 1.5 GGT localization on exosomes by transmission electron microscopy.

Exosomes separated from plasma by ultracentrifugation were incubated with a primary antibody directed against GGT and with a secondary 15 nm gold particle-conjugated antibody. After fixation with glutaraldehyde, samples were negatively stained with uranyl acetate and examined under a transmission electron microscope [Fornaciari et al., 2014].

GGT has a half-life in blood of 9 hours against 20 of the hydrophobic [Grøstad and Huseby, 1990]. The clearance kinetics of the hydrophilic GGT showed a biphasic pattern with a fast first phase (half-life 15-20 min) followed by a slow one (65% removal after 2 hours). The first step, but not the second, strictly depended on the degree of protein sialylation, in fact the desialylated enzyme had even a shorter half-life, less than 10 min [Huseby et al., 1992]. Still using the hydrophilic GGT of hepatic origin, labelled with 125_{I, it was shown that the still active protein is selectively removed from}

the asialoglycoprotein receptor of hepatocytes [Huseby et al., 1992; Huseby et al., 1993; Mortensen and Huseby, 1997]. These studies suggest also that the clearance pathway for the hydrophobic and hydrophilic GGT components might be different, depending on the carrier characteristics.

1.2.5 Fractional GGT analysis

With the aim to improve the specificity of the GGT analysis, several methods for the separation and quantification of circulating enzyme fractions have been set up [Nemesanszky and Lott, 1985]. The first methods were mainly based on electrophoretic techniques, such as on cellulose acetate [Sacchetti et al., 1988], agarose [Nemesanszky and Lott, 1985; Bellini et al., 1997] or polyacrylamide [Yao et al., 1998].

The most widely used method in the 90s for the study of plasma GGT fractions in the clinical setting was the one based on cellulose acetate electrophoresis and the use of the synthetic substrate γ-glutamyl-7-amido-4-methylcoumarin (γGluAMC) for the visualization of GGT activity bands [Sacchetti et al., 1988]. With this method it has been possible to reveal between six and eleven GGT bands [Sacchetti et al. 1988a; Sacchetti et al. 1988b; Sacchetti et al. 1989]. α1- and α2-GGT bands were always present, dep-, γ- and β-GGT were mainly present in liver cholestatic diseases, while alb-GGT was mainly present in patients with primary or metastatic liver cancer and/or cirrhosis. In combination with alpha-fetoprotein levels higher than 20ng/mL, alb-GGT discriminated patients with liver cancer from those with cirrhosis with 84% sensitivity, 61% specificity and 74% accuracy [Pompili et al., 2003].

In other two studies, aimed to discriminate patients with cirrhotic or hepatocellular carcinoma, function based on seven plasma components has been set up (alpha-fetoprotein, alkaline phosphatase, lactate dehydrogenase 5, total GGT, aspartate aminotransferase, copper and the sum of dep-, γ- and β-GGT). This algorithm allowed the correct classification of 93% of cases in a cohort composed of 135 patients with cirrhosis and 77 with hepatocellular carcinoma. In this study alb-GGT was not considered [Castaldo et al., 1996; Castaldo et al., 1999].

From all these studies it was found that the GGT fractional analysis might add specificity to the determination of the total activity, however, the low sensitivity and reproducibility of the electrophoretic methods have prevented the effective diagnostic use.

A high performance gel filtration chromatography method for GGT fraction analysis. I have set up, and patented (Paolicchi et al., US8486650-B2) a new method for the separation and quantification of plasma GGT fractions [Franzini et al., 2008]. According to this technique, the determination of fractional GGT activity is performed with a FPLC system (fast protein liquid chromatography) using a column for molecular exclusion chromatography (Superose 6HR 10/300 GL, GE Healthcare) which allows the separation of the plasma components solely on the base of their molecular mass in the range between 5000 and 5 kDa. GGT activity was revealed thank to an on-line post-column enzymatic reaction. Through a T-connection, eluate is continuously mixed to a solution containing the fluorescent substrate γGluAMC, specific for the GGT enzyme. The mixing of the eluate and the substrate occurs at 37 °C in a 2.4 ml loop, which correspond to an incubation of about 4 minutes (Figure 1.5). The presence of the GGT enzyme in the eluate is revealed by the fluorescent reaction product aminomethylcoumarin (AMC) according to the reaction (where GlyGly is the acceptor or transpeptidation reaction):

γ-Glu-AMC + GlyGly → γ-Glu-GlyGly + AMC

The product AMC is detected by a fluorometer detector operating at a excitation wavelength of 380 nm and emission of 440 nm. The reaction conditions are such that the detection limit of the activity is 0.5 U/L; the area under AMC peaks is proportional to the fractional activity of GGT and the total area under the chromatogram, between 10 and 25 mL of elution volume, is proportional to total GGT activity.

Total plasma GGT activity, expressed in U/L, is calculated on the basis of a calibration curve obtained analysing plasma samples with known total GGT activity. Total and fractional GGT area is calculated with the aid of a computer program (MATLAB Version 7 MathWorks, Inc.) to resolve overlapping peaks (Figure 1.6). The curve fitting of the four GGT-chromatographic peaks is conducted with a nonlinear least-squares minimization algorithm using four exponentially modified Gaussian (EMG) curves; upper and lower bounds for the curve amplitude, width, position and asymmetry were set for each peak.

The main disadvantage of this method is the time required for a determination, 50 min, but the post-column enzyme reaction makes the determination of the plasma GGT fractions faster, more sensitive and reproducible than with the previous methods.

Using this new chromatographic methods I have characterised the distribution of plasma GGT fractions in 200 healthy subjects, blood donors (100 men and 100 women) [Franzini et al., 2008a]. In all subjects I have identified four fractions named: big-GGT GGT (b-GGT, 2000 kDa), medium-GGT (m-medium-GGT, 940 kDa), small-medium-GGT (s-medium-GGT, 140 kDa) and free-medium-GGT (f-medium-GGT, 70 kDa). The molecular weight of each fraction is independent from the associated levels of GGT activity, that suggests the existence of a specific interaction between the enzyme and the different carriers rather than a random adsorption in the bloodstream, so it is possible that specific pathways of secretion and removal exist for each fraction. Analysing the distribution of the four fractions as a function of the total GGT activity, I have found that, in both genders, f-GGT is the most represented fraction at low values of total GGT activity (<20 U/L), while the increase in the latter depends primarily on the s-and b-GGT fractions (Figure 1.7).

Another aspect that distinguishes f-GGT fraction from those with high molecular weight is the shape of the frequency distribution in the population, that is Gaussian for f-GGT and asymmetric to the right, as for the total GGT, for the other three [Franzini et al., 2008a]. The reference values for the fractions, calculated in the 200 blood donors population are reported in Table 1.2.

Figure 1.6 High performance gel filtration chromatography for GGT fraction analysis.

A) Schematic representation of the instrumental components used for fractional GGT separation and quantification. B) Representative elution profile of plasma GGT activity in a sample of plasma obtained from a healthy subjects. The four Gaussian curves corresponding to GGT fractions are highlighted with coloured lines [Franzini et al., 2008; Franzini et al., 2008a].

In the same population, I investigated the determinants of plasma GGT fractions finding out that established cardiovascular risk factors (diastolic arterial pressure, C-reactive protein, triglycerides, LDL-cholesterol) correlated primarily with plasma b-GGT [Franzini et al., 2010].

The study of plasma GGT fractions continues to be of considerable interest both for a better diagnostic and prognostic use of this enzyme, and to improve the understanding of the pathogenesis of diseases related to its increase. Anyway this will be possible only using a reproducible and standardised method for measuring plasma GGT fractions.

Table 1.2 Total and fractional plasma GGT activities in healthy humans [Franzini et al., 2008a].

Men (n = 100) Women (n = 100)

Median 5th _{– 95}th _{perc. Median 5}th _{– 95}th _{perc. P}

Total GGT* 25.3 12.3 – 60.5 14.4 8.4 – 30.9 < 0.001

b-GGT* 2.4 0.7 – 10.7 1.1 0.4 – 5.2 < 0.0001

m-GGT* 1.0 0.2 – 3.3 0.5 0.2 – 1.2 < 0.0001

s-GGT* 9.2 2.8 – 33.7 3.9 1.5 – 11.6 < 0.0001

f-GGT 13.2 8.3 – 19.6 8.9 5.9 – 12.5 < 0.0001

Data are expressed as U/l. *Student‟s t-test performed on ln-transformed data.

Figure 1.7 Elaboration of the elution profiles of plasma GGT fractions in healthy subjects.

A) men (n = 100), B) women (n = 100). The chromatograms represent the GGT activity elution profile of the 25th _{(dashed line), 50}th _{(solid line), 75}th _{(dotted line) percentile of the}

1.3

T

GGT

GGT was investigated and adopted as a liver function test or liver enzyme in the 1960s and 1970s [Szczeklik et al., 1961; Lum and Gambino 1972; Betro et al. 1973; Betro et al., 1973a; Sheehan and Haythorn, 1979]. Among plasma analytes of liver origin, GGT is the most sensitive test (Table 1.3), being abnormal in most patients with liver diseases such as cholestasis, hepatosteatosis, hepatocellular carcinoma, viral or alcoholic hepatitis.

Table 1.3 Predictive values of various liver function tests with respect to diagnosis

of liver disease (n=181) [Sheehan and Haythorn, 1979]

Test PPV NPV Sensitivity GGT 79 95 93 Asp-aminotransferase 86 85 74 Ala-aminotransferase 78 81 68 Alkaline phosphatase 77 80 68 Direct bilirubin 97 75 50 Total bilirubin 93 73 47

PPV: positive predictive value; NPV: negative predictive value.

In fact, GGT above the reference category is a sensitive predictor of hepatobiliary-related death (including both cancer and non-cancer causes, Figure 1.8); however the diagnostic use of GGT test is limited by its lack of specificity, given the wide range of diseases or other conditions (pancreatitis, diabetes, obesity, excessive alcohol intake, use of enzyme-inducing drugs) that can also cause high serum GGT.

Figure 1.8 Hepatobiliary and

hepatoma mortality according to plasma GGT levels.

GGT was classified separately for women and men according to Ruttmann et al. [2005] as:

1 normal low (<9U/L W, <14 U/L M), 2 normal high (9-17, 14-27 U/L),

3 moderately elevated (18-26, 28-41 U/L), 4 increased (27-35, 42-55 U/L),

5 highly elevated (≥36, ≥56 U/L). The lowest category served as reference. [from Kazemi-Shirazi et al., 2007]

1.3.1 Factors influencing plasma GGT levels

Plasma levels of GGT activity are affected by many factors, such as alcohol intake, body fat content, plasma lipid/lipoproteins, and glucose levels, thus many factors might confound the correct interpretation of plasma GGT. Elevated GGT levels have also been associated with use of many drugs, including barbiturates, antibiotics, histamine receptor blockers, antifungal agents, antidepressants, testosterone [Whitfield, 2001] and oral contraceptives [Schiele et al., 1998]. Smoking can also increase GGT levels. On the other hand, ursodeoxycholic acid and clofibrate decrease GGT levels [Santos et al., 2003; Mabile et al., 2003].

The main factors influencing plasma GGT levels are listed in the Table 1.4 (see also Whitfield 2001).

Table 1.4 Factors influencing plasma GGT activity [Franzini et al., 2009].

Positive correlation Negative correlation

Gender Age

Menopause Pregnancy

Body mass index Physical activity

Waist circumference

Alcohol consumption Coffee

Smoke Forced expiratory volume FEV-1

Blood pressure Cardiac frequency Total cholesterol LDL cholesterol HDL cholesterol Triglycerides Fasting glucose Insulin Uric acid Ferritin Type II diabetes Hypertension Drugs: Drugs:

Oral contraceptives Fibrates

Anti-hypertensive Ursodeoxycholic acid

Analgesic

Inflammation and oxidative stress indexes: C-reactive protein

Fibrinogen F2-isoprostans

Urine 8-Hydroxydeoxyguanosine

Increased plasma GGT levels have also been associated with pancreatitis, type II diabetes [Lee DH et al., 2003b], cardiovascular disease [Hashimoto et al., 2001; Lee DH et al. 2003], stroke, and hypertension [Liu CF et al. 2012]. However, the reasons of these correlations are not well known. In