Reversal of hepatorenal syndrome type 1 with terlipressin plus albumin vs. placebo plus albumin in a pooled analysis of the OT-0401 and REVERSE randomised clinical studies.

BACKGROUND: The goal of hepatorenal syndrome type 1 (HRS-1) treatment is to improve renal function. Terlipressin, a synthetic vasopressin analogue, is a systemic vasoconstrictor used for the treatment of HRS-1, where it is available. AIM: To compare the efficacy of terlipressin plus albumin vs. placebo plus albumin in patients with HRS-1. METHODS: Pooled patient-level data from two large phase 3, randomised, placebo-controlled studies were analysed for HRS reversal [serum creatinine (SCr) value ≤133 µmol/L], 90-day survival, need for renal replacement therapy and predictors of HRS reversal. Patients received intravenous terlipressin 1-2 mg every 6 hours plus albumin or placebo plus albumin up to 14 days. RESULTS: The pooled analysis comprised 308 patients (terlipressin: n = 153; placebo: n = 155). HRS reversal was significantly more frequent with terlipressin vs. placebo (27% vs. 14%; P = 0.004). Terlipressin was associated with a more significant improvement in renal function from baseline until end of treatment, with a mean between-group difference in SCr concentration of -53.0 µmol/L (P < 0.0001). Lower SCr, lower mean arterial pressure and lower total bilirubin and absence of known precipitating factors for HRS were independent predictors of HRS reversal and longer survival in terlipressin-treated patients. CONCLUSIONS: Terlipressin plus albumin resulted in a significantly higher rate of HRS reversal vs. albumin alone in patients with HRS-1. Terlipressin treatment is associated with improved renal function. (ClinicalTrials.gov identifier: OT-0401, NCT00089570; REVERSE, NCT01143246).

Diagnostic value of ascitic fluid lactic dehydrogenase, protein, and WBC levels.

Three characteristics of an exudate, ie, an ascitic fluid lactic dehydrogenase (LDH) level of greater than 400 Sigma units (SU), an ascitic fluid-serum LDH ratio of greater than 0.6, and an ascitic fluid-serum protein ratio of greater than 0.5, were studied in a prospective fashion to determine their usefulness in the differential diagnosis of ascites. The ascitic fluid LDH level did not exceed 400 SU in any patient with uncomplicated chronic liver disease, whereas in patients with malignant, tuberculous, or pancreatic ascites it exceeded 500 SU in 12/19 patients. The finding of two of the three characteristics indicated a nonhepatic cause for the ascites whereas the absence of all three strongly suggested uncomplicated liver disease as the sole cause. The ascitic fluid WBC count was also useful in that values exceeded 500/cu mm in bacterial and tuberculous peritonitis whereas it was low (297 +/- 49/cu mm) in chronic liver disease.

Characterization of glutathione S-transferase in cultured human keratinocytes.

The glutathione S-transferase activity and isozymic composition of cultured human keratinocytes were characterized. Keratinocytes were grown in culture and harvested at different stages of differentiation. Glutathione S-transferase activity was found in the soluble cell fraction but not in the microsomal cell fraction. The glutathione S-transferase specific activity of the soluble cell fraction was found to increase as the keratinocytes differentiated in culture. All of the enzymatic activity was found to reside with a single isozymic form that was concluded to be the pi form of the enzyme based on substrate specificity, sensitivity to inhibitors, molecular weight, and reactivity towards antibodies raised to alpha, mu, and pi forms of the enzyme. It is concluded that all of the isozymic forms of glutathione S-transferase noted in whole skin, with the exception of pi, are of extra-keratinocyte origin.

Differentiation-induced enhancement of the ability of cultured human keratinocytes to suppress oxidative stress.

Human keratinocytes in culture were harvested at different stages of differentiation. Both the level of antioxidants and the response of cells to oxidative stress were measured as a function of growth and differentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxidase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level of antioxidants was maintained until the cells began to lose viability. Further, as the keratinocyte cultures began to differentiate, they became more resistant to the toxic effect of cumene hydroperoxide in terms of both of the rate of loss of cell mass and total glutathione and of the rate of decline in the activity of oxidation-sensitive enzymes. To determine how tightly the observed effects are linked to the calcium-dependent aspects of differentiation and to rule out effects related to time in culture, the cells were switched from 1.2 mM Ca++ to 0.03 mM Ca++ to suppress Ca(++)-dependent differentiation. After 4 d, these cells were then treated with 0.5 mM cumene hydroperoxide. The switch to 0.03 mM Ca++ blocked the normal increases in both glutathione peroxidase and glucose-6-phosphate dehydrogenase activities. Further, cells in 0.03 mM Ca++ had reduced resistance to cumene hydroperoxide relative to cells cultured for the same length of time in 1.2 mM Ca++. This indicates that there is a differentiation-associated, Ca(++)-specific increase in both the level of antioxidants and in tolerance to organic hydroperoxides.

Natural history of portal hypertension.

A rise in pressure in the portal vein is a frequent occurrence in patients with cirrhosis. One common manifestation affecting at least 50% of cirrhosis patients is the development of gastroesophageal varices and portal hypertensive gastropathy. Bleeding from gastric or esophageal varices will occur in approximately 1/4 of cirrhotic patients with an associated high mortality. Large esophageal varices that have red color signs and isolated gastric varices in the fundus of the stomach are most likely to hemorrhage. The greatest risk of bleeding is during the first year following the index endoscopy. Once varices have bled they are almost certain to rebleed in the absence of therapy. Similarly, severe portal hypertensive gastropathy is likely to cause chronic blood loss. Knowledge of the natural history of gastroesophageal varices allows for the development of effective treatment strategies.

Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis.

The glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of dimeric proteins that catalyze reactions between glutathione (GSH) and various electrophiles. A partial cDNA for human GST pi was obtained and the open reading frame completed. The completed cDNA was cloned, and GST pi protein was expressed in bacteria. Cloned enzyme was purified and had the same kinetic constants, molecular mass, pI value, and N-terminal sequence as placental GST pi except that some of the polypeptides had N-terminal methionines. A radiolabeled azido derivative of GSH, S-(p-azidophenacyl)-[3H]glutathione, was used to photoaffinity-label the active site of the cloned enzyme. Labeled enzyme did not bind to a GSH-agarose affinity column. Labeling was prevented in the presence of S-hexylglutathione, and noncovalently-bound azido affinity label was a competitive inhibitor towards 1-chloro-2,4-dinitrobenzene and GSH. These results suggest that the azido label was binding at the active site of the enzyme. Photoaffinity-labeled enzyme was trypsinized, and two labeled peptides were purified and sequenced. One peptide corresponded to residues 183-188, whereas the other corresponded to residues 183-186. These residues appear to form part of the hydrophobic (H-site) binding region of human GST pi that has not been shown previously. Cloned enzyme was subjected to radiation inactivation to assess the importance of subunit interactions in the maintenance of catalytic activity. The target size of enzymatic activity (23 kDa) was not significantly different from that of the protein monomer (24 kDa). Therefore, each subunit of human GST pi appears to be capable of independent catalytic activity.

Structural, functional and hybridization studies of the glutathione S-transferases of rat liver.

We have purified five forms of glutathione S-transferase from rat liver. One form was the glutathione S-transferase B (ligandin), which is composed of two non-identical subunits with molecular weights of 22,000 (Ya) and 25,000 (Yc). Two of the other transferases were Ya and Yc homodimers. The other two transferases were also homodimers, but their subunit, Yb, had a molecular weight of 24,000. The three proteins containing either Ya or Yc subunits had similar substrate specificities, and all three contained peroxidase activity. The greatest peroxidase activity was present in proteins containing the Yc subunit. Enzymes composed of Yb subunits had minimal peroxidase activity in addition to different substrate specificities. The Ya and Yc containing enzymes bound the ligands bilirubin, and indocyanine green with high affinity (KD less than 5 microM), although the KD values of the YcYc protein were consistently 4- to 12-fold greater than those of the other two transferases. Studies were performed to define the origins of the various isozymes. There was no evidence for conversion of Yc to either Ya or Yb during storage or under conditions favorable to proteolysis. Hybridization studies were performed under denaturing conditions (6 M guanidine-HCl), and a YaYc hybrid was formed from the YaYa and YcYc proteins. In addition, both YaYa and YcYc hybrids were formed from transferase B. The hybrids were functionally similar to the proteins isolated originally from the liver. Attempts to form a YaYb hybrid from the YbYb and YaYa transferases were unsuccessful. This result is consistent with the lack of this enzyme form in the liver. Glutathione S-transferase B and the Ya and Yc homodimers appeared to be hybrids of common subunits. These three transferases had very similar functional and structural characteristics and differed from the transferases that are composed of Yb subunits.

Do the soluble glutathione S-transferases have direct access to membrane-bound substrates?

The ability of the soluble glutathione S-transferases to bind the membrane (liposome) bound substrates 1-chloro-2,4-dinitrobenzene and sulfobromophthalein was determined. The transferases were found to have access only to substrates in the aqueous phase. They could not not bind membrane-bound substrates and, thus, enzymatic activities were reduced by the membrane partitioning of the substrates. The reduction in enzymatic activity was directly proportional to the lipid solubility of the substrate. The liposomes had no direct effect on the enzyme per se. [35S]Sulfobromophthalein and [14C]chlorodinitrobenzene bound to liposomes were found to have rapid rates of release into the aqueous phase. Rates of hydration of chlorodinitrobenzene from liposomes were rapid enough such that rates of catalysis (measured in a stopped-flow spectrophotometer) were affected only by the partition coefficient of substrate between lipid phase and water, and not by the rate of transfer of substrate from lipid to water phase.

Serum hyaluronic acid in patients with veno-occlusive disease following bone marrow transplantation.

The development of hepatic veno-occlusive disease following bone marrow transplantation is associated with high-dose combination cytoreductive therapy. Experimental models have suggested that drug-induced injury to hepatic sinusoidal endothelial cells is involved in the pathogenesis of this syndrome. Hyaluronic acid is a polysaccharide that is metabolized, almost exclusively, by hepatic sinusoidal endothelial cells. The aim of the present study was to evaluate serum hyaluronic acid as a marker for endothelial cell injury in patients with veno-occlusive disease following bone marrow transplantation. Hyaluronic acid was measured in sera from patients with and without veno-occlusive disease using an enzyme-linked protein binding assay. Mean peak serum hyaluronic acid levels were significantly greater in patients who had a diagnosis of VOD compared to those transplant patients who did not, 1173.4 +/- 982.9 vs 444.9 +/- 735.6 ng/ml (P = 0.01). Serial serum samples obtained from a separate cohort of patients also demonstrated that serum hyaluronic acid levels were higher in patients with moderate or severe veno-occlusive disease compared to those with none or mild disease at days 7, 17 and 25 following transplantation (greatest difference at day 25: 366 +/- 327 vs 126 +/- 151, P = 0.01). Serum hyaluronic acid levels are increased in veno-occlusive disease and increase over time in patients with severe disease. Further studies are required to determine if elevated serum hyaluronic acid levels are due to decreased clearance by injured hepatic sinusoidal endothelial cells or increased production from early hepatic fibrogenesis associated with the acute liver injury.

Covalent labeling of the nonsubstrate ligand-binding site of glutathione S-transferases with bilirubin-Woodward's reagent K.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase YaYc and the YaYa homodimer were purified from rat liver cytosol. An enol ester derivative of bilirubin (bilirubin-Woodward's reagent K) was prepared and used to label covalently the nonsubstrate ligand-binding site on these two proteins. There was a linear relationship between the amount of bilirubin-Woodward's reagent K added to the reaction mixture and the amount of labeling achieved up to a ratio of 2:1 (bilirubin-Woodward's reagent K: protein-YaYc). A maximum of 0.87 mol of label bound per mol of transferase YaYc. At higher molar ratios, the label appeared to also be binding at a second site on the enzyme. The label blocked the nonsubstrate ligand-binding site of the two transferases but not the catalytic site. The divalent reagent was shown to label equally the Ya and Yc subunits of transferase YaYc, suggesting that the single high affinity bilirubin-binding site present on this protein is formed by an interaction between the subunits rather than residing on a specific subunit. At low ratios of label to protein, bilirubin-Woodward's reagent K appears to label specifically the nonsubstrate ligand-binding site of two forms of glutathione S-transferase, and use of this label should allow for the localization of the nonsubstrate ligand-binding site in the primary amino acid sequence of the Ya and Yc subunits.

The glutathione S-transferases: an update.

Over the last 15 years, we have passed through an initial period in which multiple forms of GST in various organs and different species were identified and characterized. The focus of current research is to define the role of the numerous isozymes in cell function, to ascertain the relationship between structure and function of different isozymes and to determine how the expression of GST is regulated in different tissues. During these studies, it is expected that new roles for the GST will be proposed, and this family of multifunctional proteins will continue to hold the interest of numerous investigators for many years.

Excretion of glutathione conjugates by primary cultured rat hepatocytes.

Conjugation of xenobiotics with glutathione occurs commonly within the liver, and these glutathione conjugates are then preferentially excreted into bile. We have characterized this excretory process using primary cultured hepatocytes (24 h). 1-Chloro-2,4-dinitrobenzene rapidly entered the cells and formed a glutathione conjugate, S-(dinitrophenyl)glutathione, irrespective of the temperature of incubation. In contrast, the efflux of the glutathione conjugate was essentially absent in the cold but recovered rapidly upon rewarming of the cells. Therefore, initial rates of efflux of the conjugate at 37 degrees C were measured from cells preloaded biosynthetically at 10 degrees C. Efflux was a saturable process with respect to intracellular S-(dinitrophenyl)glutathione with an apparent Km of 0.58 +/- 0.12 mM and Vmax of 0.15 +/- 0.05 nmol/min/mg of protein. The excretion of S-(dinitrophenyl)glutathione had an energy of activation of 15.3 kcal/mol. The glutathione conjugate of p-nitrobenzylchloride when formed within the hepatocytes acted as a competitive inhibitor of S-(dinitrophenyl)glutathione efflux. Cultured hepatocytes, therefore, appeared to have a specific transport process for the excretion of glutathione conjugates. The addition of S-(dinitrophenyl)glutathione, but not GSH, GSSG, or methionine, to the medium caused a decrease in the rate of efflux of radiolabeled S-(dinitrophenyl)glutathione. The hepatocytes were able, however, to excrete the glutathione conjugate against an excess of extracellular S-(dinitrophenyl)glutathione. This observation suggested that extracellular S-(dinitrophenyl)glutathione, although capable of binding to the carrier, entered the hepatocytes quite slowly relative to rates of efflux. This carrier may function in a manner that would minimize the reuptake by hepatocytes of conjugates that have been excreted into the bile.

Characterization of the activation of rat liver glutathione S-transferases by nonsubstrate ligands.

In previous work (D.A. Vessey and T.D. Boyer, 1986, Biochem. Pharmacol., 35, 289-295) the activity of glutathione S-transferase form YcYc from rat liver was found to be stimulated by the herbicide 2,4,5-T. We have extended that work and examined the effect of over 40 structural analogs on the activity of YcYc. Over half of these compounds stimulated by 10 to 232% when added to assays at a concentration of 1 mM. The best activators all contained the "2,4,5-trichlorophenyl-" structure. While 2,4,5-T gave the greatest activation at 1 mM (2.3-fold), 2,4,5-trichlorobenzene sulfonate gave the greatest maximum activation (6.0-fold). Compounds that had no effect on activity did not affect activation by 2,4,5-T suggesting that they have a poor affinity for the enzyme. Two of the analogs tested (chloramine-T and 6-hydroxydopamine) proved to be good inhibitors and ethacrynic acid was an extremely potent inhibitor. Indomethacin activated at low concentrations but inhibited above 2 mM. Activations were greater at low temperature (5 degrees C) and decreased with increasing temperature. The extent of activation was largely unaffected by the concentration of either substrate. Examination of the organic peroxidase activity of the enzyme revealed inhibition by 2,4,5-T and 2,4-D rather than activation.

Purification and characterization of hepatic glutathione S-transferases of rhesus monkeys. A family of enzymes similar to the human hepatic glutathione S-transferases.

Thirteen forms of glutathione S-transferase were purified from the livers of female rhesus monkeys (Macaque mulatta). Most (74.7%) of the activity in the hepatic cytosol adhered well to the GSH affinity column and could be eluted only with the addition of GSH to the eluting buffer. The predominant isoenzymes (n = 5) in this 'high-affinity' fraction had alkaline pI values (greater than 9.0) and contained a subunit with an Mr value of 24,000. All of these isoenzymes had high organic peroxidase activity and, on the basis of amino acid analysis, substrate specificities and affinity for non-substrate ligands, appear to belong to the family of glutathione S-transferases that have been termed alpha [Mannervik, Alin, Guthenberg, Jensson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206]. Also within the high-affinity fraction was an isoenzyme with an acidic (5.8) pI value. This acidic isoenzyme was composed of a unique subunit (Mr 23,000). The N-terminal sequence (ten residues) of this acidic enzyme was identical with that of a human form that is referred to as pi. The predominant form of enzyme in the 'low-affinity' (eluted from the GSH affinity column with an increase in buffer pH) fraction was a homodimer of a 26,000-Mr subunit. It had an alkaline pI (greater than 9.0) but it lacked organic peroxidase activity. The N-terminal sequence (ten residues) of this enzyme was identical with that of a human enzyme referred to as mu. The substrate specificities and affinity for non-substrate ligands of this monkey enzyme also were similar to those of the human enzyme. In conclusion, the liver cytosol of rhesus monkeys contains a number of glutathione S-transferase isoenzymes that are very similar to the human hepatic enzymes.

Differential activation and inhibition of different forms of rat liver glutathione S-transferase by the herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T).

The predominant forms of the dimeric enzyme glutathione S-transferase were purified from rat liver. Forms YbY'b and YbYb (also known as forms C and A, respectively) could be almost completely inhibited by 2,4-dichlorophenoxyacetate (2,4-D). Half-maximal inhibition was obtained at 0.5 mM 2,4-D. Inhibition was seen even at extrapolated infinite concentrations of both substrates for YbYb but not YbY'b. These same forms could also be inhibited 70 to 80% by 2,4,5-trichlorophenoxyacetate (2,4,5-T) with half maximal inhibition occurring at 0.2 mM. Glutathione S-transferase form YaYa was maximally inhibited by 72 and 30%, respectively, by 2,4-D and 2,4,5-T. The 30% inhibition of YaYa caused by 2,4,5-T was shown to reduce the nearly complete inhibition caused by a previously characterized inhibitor, namely bile acids. This suggests competition for a common binding site on the enzyme. In contrast to the above results, it was found that form YcYc (also termed AA) was activated 2.7-fold by 2,4,5-T and 1.4-fold by 2,4-D. This activation could be blocked by chenodeoxycholate which, by itself, did not affect the activity of the enzyme. The effects of 2,4,5-T and 2,4-D on the heterodimer YaYc (also termed form B) were intermediate between their effects on YaYa and YcYc, suggesting that each subunit contributes its unique property to the heterodimer. The microsomal membrane-bound form of glutathione S-transferase was also examined and found to be inhibited by both 2,4-D and 2,4,5-T. However, unlike the inhibitions of soluble forms, 2,4,5-T caused more extensive inhibition than 2,4-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.

Inhibition of human cationic glutathione S-transferase by nonsubstrate ligands.

Inhibition of a major hepatic form of human cationic glutathione S-transferase by bilirubin, biliverdin, indocyanine green and chenodeoxycholic acid was investigated as a function of pH (range = 6.5 to 9.1). Changes in pH had little effect on the extent of inhibition by indocyanine green. However, inhibition by bilirubin, biliverdin and chenodeoxycholic acid was found to be pH-dependent, with markedly less inhibition at the high values of pH. The reduced inhibition at the high values of pH could not be ascribed to a failure of the enzyme to bind the nonsubstrate ligand. Instead, the complete inhibition observed at pH 6.5 became partial (hyperbolic) inhibition at pH 9.1. This behavior can be ascribed to the binding of the nonsubstrate ligands at a site other than the active site, i.e., at high values of pH there is formation of an enzyme-substrate-inhibitor complex which still retains considerable catalytic activity. At physiologic values of pH (7.0), the human transferase was completely inhibited by saturating concentrations of the tested nonsubstrate ligands. This is in contrast to our previous studies performed with the rat transferases where, although inhibition also was affected by buffer pH, some forms of the enzyme retained significant catalytic activity at pH 7.0 despite high concentrations of nonsubstrate ligands. We conclude that the ability of the human cationic glutathione S-transferases to serve as enzymes of detoxification in the presence of high intracellular concentrations of nonsubstrate ligands may be significantly reduced, and this may render the cholestatic liver unusually susceptible to injury by toxic electrophiles.

Hepatic hydrothorax.

Ascites is a common manifestation of portal hypertension in patients with cirrhosis. Approximately 5% of patients with cirrhosis may develop a pleural effusion. This is usually right sided. In the absence of cardiac or lung disease, the presence of a pleural effusion in a cirrhotic patient is known as hepatic hydrothorax. Small volumes of fluid within the pleura may be associated with significant respiratory symptoms which require the clinician to rapidly remove the fluid. The development of hepatic hydrothorax is secondary to passage of ascites from the abdomen to the pleural space via defects in the diaphragm. Once the diagnosis of hepatic hydrothorax is established with certainty, medical therapy with salt restriction and diuretics is initiated. When these measures are ineffective the patient has refractory hepatic hydrothorax. Based on current studies, transjugular intrahepatic portal systemic shunts appear to be the most effective form of treatment for these patients.