**2. Glutaminase genetic study**

It has been described that there are two forms of mitochondrial glutaminase in the body, liver type glutaminase (L-GA) and kidney type glutaminase (K-GA) or extrahepatic (located in other organs such as the intestine). In humans, increased glutaminase activity is localized to the duodenum. There are indirect data suggesting that glutaminase activity in the enterocyte is increased in cirrhotic patients, as demonstrated by the fact that after oral administration of glutamine there is a rapid increase in blood ammonia level in cirrhotic individuals but not in healthy controls (Romero-Gomez, Grande et al. 2002).

to urea by a healthy liver increases to toxic levels. In this situation, the enzyme glutamine synthetase (GS) plays a pivotal role in ammonia detoxification, effectively removing ammonia during the conversion of glutamate to glutamine (Rose, Michalak et al. 1999). Glutamine deamidation by intestinal GA seems to be the main source of ammonia in patients with cirrhosis (Olde Damink, Jalan et al. 2002), and hyperammonaemia and hepatic encephalopathy can appear without the participation of gut bacteria (Weber and Veach

The following data support the hypothesis that a genetic factor is implicated in the development of overt hepatic encephalopathy: Glutaminase activity has been linked to hepatic encephalopathy and ammonia production; 40% of persons with cirrhosis and minimal hepatic encephalopathy do not develop overt hepatic encephalopathy in long-term follow-up (Romero-Gomez, Boza et al. 2001); patients with cirrhosis who have the same degree of liver dysfunction and the same precipitating factor (for example, variceal bleeding) may or may not develop overt hepatic encephalopathy; and at least 2 different polymorphisms in the promoter region of the glutaminase gene influence protein activity by

In this chapter we analyze the studies supporting the inhibition of glutaminase based in the identification of mutations in the glutaminase gene to facilitate selection of patients for close monitoring and evaluation for expedited transplantation. Firstly, we have identified a variant in the promoter region of the glutaminase gene that increases glutaminase activity and is associated with the development of HE. Following a simple blood test to identify these patients with the variant, it would be possible to offer a treatment with glutaminase inhibitors. Based on these and another studies we have developed a new molecule, THDP17

An alternative treatment to HE is currently being investigated: Ornithine phenylacetate (OP). OP is a novel drug that is targeted at reducing ammonia concentration in patients with liver disease and therefore a potential treatment for HE (Jalan, Wright et al. 2007). The mechanism by which OP directly reduces ammonia levels in cirrhosis is by normalization of gut glutaminase activity and concomitant increasing muscle glutamine synthesis activity, subsequently trapping the increased glutamine with phenylacetate,and increasing ammonia

These studies support glutaminase such as focus for new treatments of HE and the identification of a genetic variation greatly facilitates the selection of patients for close

It has been described that there are two forms of mitochondrial glutaminase in the body, liver type glutaminase (L-GA) and kidney type glutaminase (K-GA) or extrahepatic (located in other organs such as the intestine). In humans, increased glutaminase activity is localized to the duodenum. There are indirect data suggesting that glutaminase activity in the enterocyte is increased in cirrhotic patients, as demonstrated by the fact that after oral administration of glutamine there is a rapid increase in blood ammonia level in cirrhotic

individuals but not in healthy controls (Romero-Gomez, Grande et al. 2002).

increasing or decreasing glutaminase activity (Taylor L 2001).

that inhibits glutaminase in CACO-2 cell cultures (intestinal cells).

excretion as phenylacetylglutamine in the urine.

**2. Glutaminase genetic study** 

monitoring and evaluation for expedited transplantation.

1979).

Hyperammonemia is noticably marked in patients with liver cirrhosis with poor liver function, but this increase in ammonia production following a glutamine challenge returns to normal after liver transplantation and normalization of liver function. The specific activity of glutaminase in the enterocyte is a crucial point in the stability of nitrogen metabolism in patients with liver cirrhosis. It has been shown that glutaminase activity is increased in cirrhotic subjects compared to controls and that this activity is related to the presence of encephalopathy and the degree of hepatic dysfunction (Romero-Gomez, Ramos-Guerrero et al. 2004). Thus, also the accumulation of glutamine in the astrocyte is responsible, in large part on the toxicity induced by ammonia (Albrecht and Norenberg 2006).

The cDNA of the human renal-type glutaminase (HK-GA) was cloned in 1998 and subsequently further cDNAs have been isolated encoding for three isoforms of HK-GA, which were designated as K-GA (which is predominantly expressed in kidney, intestine and brain but not liver), M-GA (which is expressed only in cardiac and skeletal muscle), and C-GA (which is expressed primarily in cardiac muscle and pancreas but not in brain or liver). The K-GA isoform is localized to the kidney and has 669 amino acids, C-GA, a protein of 598 amino acids and differs from the K-GA in the carboxyl terminus and the M-GA is a protein of 169 amino acids, which is identical to C-GAP up to amino acid 161 only varying for the c-terminal 8residues. After sequencing the genomic DNA, it was proposed that the three isoforms were the product of alternative splicings of the same gene. Recently described two haplotypes of this gene: TACG and CACG, which place a lower activity of glutaminase, which results in lower intestinal production of ammonia and improved liver function and lower risk of developing hepatic encephalopathy (Romero-Gomez 2005).

We assessed whether alterations in the glutaminase gene could explain, at least in part, the risk for overt hepatic encephalopathy in patients with cirrhosis. We have described a variation in the promoter region of the glutaminase gene that is associated with development of HE in patients with cirrhosis (Romero-Gomez, Jover et al. 2010).

This study included subjects from outpatient clinics from 6 Spanish hospitals: 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants. Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study.

The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child–Turcotte–Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; *P=*0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; *P=*0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; *P=*0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; *P=*0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity. In Figure 1 is shown that patients with long microsatellite showed higher risk of overt hepatic encephalopathy.

Fig. 1. Actuarial curve showing patients free of bouts of overt hepatic encephalopathy according to the microsatellite in the promoter region. Patients with long microsatellite showed higher risk of overt hepatic encephalopathy (log-rank: 7.74; p<0.01)
