Enzyme changes in experimental biliary obstruction

Changes in the activities of alkaline phosphatase, 5'nucleotidase and γ-glutamyl transpeptidase in serum and liver of bile-duct ligated rats were studied over a period of 8 days after operation. All three enzymes showed increased activity in the liver, but for 5'nucleotidase and γ-glutamyl transpeptidase the increases were not detectable before 48 h, whereas for alkaline phosphatase the maximum rise was at 12 h. All three activities in the serum were significantly elevated at 12 h after operation. Maximum levels were reached at 24 h for alkaline phosphatase and 5'nucleotidase but not before 48 h for γ-glutamyl transpeptidase. These results show that bile duct ligation causes an early increase in synthesis of hepatic alkaline phosphatase whereas the incresase in 5'nucleotidase and γ-glutamyl transpeptidase is delayed until the phase of bile duct proliferation. The early changes in serum activities of 5'nucleotidase and γ-glutamyl transpeptidase do not appear to be due to a rapid increase in hepatic synthesis of these enzymes.     

肝损伤酶活性联合检测在肝胆疾病诊断中的相关分析

目的探讨肝损伤酶活性联合检测在肝胆疾病诊断中的临床意义。方法对健康对照组36例和各类肝胆疾病组204例患者的丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、乳酸脱氢酶、腺苷脱氨酶、线粒体天门冬氨酸转氨酶、碱性磷酸酶、γ-谷氨酰转移酶、5′-核苷酸酶活性进行测定与分析。结果肝胆疾病组丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、乳酸脱氢酶、腺苷脱氨酶、线粒体天门冬氨酸转氨酶、碱性磷酸酶、γ-谷氨酰转移酶、5′-核苷酸酶活性均高于健康对照组（P〈0．01）。结论肝损伤酶活性联合检测有利于肝胆疾病的鉴别诊断及疗效观察。     

Activity of gamma-glutamyl transpeptidase in serum of patients receiving anticonvulsant of anticoagulant therapy.

1. Effects of chronic anticoagulant therapy in heart patients and anticonvulsant therapy in epileptics on gamma-glutamyl transpeptidase activity in serum were investigated. 2. The enzyme was elevated in 22% of 18 patients receiving anticoagulants. In these patients prothrombin time was also abnormally high. 3. 84% of 65 epileptics exhibited elevated gamma-glutamyl transpeptidase activity, 67% of which were not associated with elevated alkaline phosphatase or aspartate aminotransferase activities. In these latter cases, involvement of the liver was not apparent. 4. Possible relationships of anticonvulsant mediated enzyme induction or hepatic toxicity to elevated gamma-glutamyl transpeptidase activity in serum in epileptics is discussed.     

Biochemical changes in liver, kidney and blood associated with common bile duct ligation

Common bile duct ligation (CBDL) in rats was used to induce liver disease and secondary kidney damage. The biochemical changes in the liver, kidney and plasma were studied at 3, 6, 10 and 21 days post CBDL. The observed alterations climaxed at the 6th day following ligation. Renal, activities of aldolase (ALD), lactic dehydrogenase (LDH), isocitric dehydrogenase (ICDH), sorbitol dehydrogenase (SDH), and alkaline phosphatase (ALP), were lowered in CBDL rats. Further, microsomal Na,K-ATPase and Mg-ATPase and mitochondrial oxidative-phosphorylation were inhibited. In the liver from CBDL rats the activities of aspartate aminotransferase (AST), Mg-ATPase and ALP were elevated, while SDH, ALD, malic dehydrogenase (MDH), LDH, malic enzyme (ME) and Na,K-ATPase were lowered. Plasma enzymes, AST, ALP, MDH, LDH, ALD, acid phosphatase (ACP) and ICDH and the metabolites bile acids, bilirubin, creatinine and urea were elevated. Addition of bile acids or bilirubin at concentrations comparable to those found in the plasma of CBDL rats, to the reaction mixture of the various enzymes strongly inhibited most, particularly mitochondrial oxidative phosphorylation. High concentrations of these substances in the blood may explain the development of renal failure during liver disease and its reversibility when liver function returns to normal.     

The activity of gamma-glutamyltransferase after bile duct ligation in guinea pig.

The activity of gamma-glutamyltransferase was studied in guinea pig after bile duct ligation. In serum, an abrupt increase in activity up to 10--20 times the normal value was found 3 h after obstruction and the mean activity over the first 3 days following the operation was some 8 times the normal value. In liver, however, a small decline in activity could be demonstrated. The administration of cycloheximide did not influence the acute increase in serum activity. Bile duct ligation caused marked increases in serum bile acid levels which initially paralleled the serum gamma-glutamyltransferase activity. It is suggested that the increased serum activity may arise from the solubilization by bile acids of liver membrane-bound enzyme.     

Biochemical changes in liver and blood during liver fattening in rats

Excessive fat accumulation in the liver is a common metabolic disorder seen in humans and animals. Fatty liver was induced in the rat by feeding the animals with a sucrose rich diet containing 1% orotic acid for 2-3 weeks. In the sera from fatty liver rats there were significant changes in the level of alanine aminotransferase (+ 68.7%), malic dehydrogenase (+ 77.8%), gamma-glutamyl transpeptidase (- 53.4%) and total lipids (+ 26.6%). There were small to no changes in the levels of aspartate aminotransferase, glucose-6-phosphate dehydrogenase, lactic dehydrogenase, aldolase, malic enzyme, 6-phosphogluconic acid dehydrogenase, alkaline phosphatase and albumin. In fatty liver, significant differences were seen in the levels of glucose 6-phosphate dehydrogenase (+ 235%), malic enzyme (+ 170%), gamma-glutamyl transpeptidase (+ 113%), 6-phosphogluconate dehydrogenase (+ 63%), aspartate aminotransferase (+ 35.6%), malic dehydrogenase (+ 38%), lactic dehydrogenase (+ 37%), and alanine aminotransferase (- 23%). Comparison of the non-fatty part with the fatty part of the fatty liver showed larger changes in the non-fatty part of the liver, suggesting that during the fattening process, there is an induction of enzymes in the liver reaching a peak prior to lipid accumulation, declining thereafter during liver fattening. The increase in NADPH-generating lipogenic enzymes suggests that accumulated fat in the liver is at least partially from de-novo increased synthesis in the liver.     

Changes in activation of aspartate aminotransferase by pyridoxal 5'-phosphate after experimental liver damage in rabbits

The aspartate aminotransferase activity with and without pyridoxal 5'-phosphate supplementation was examined in mitochondrial and cytoplasmic preparations from fresh human heart and liver samples. The apoenzyme was fully saturated in all cases. Liver cell damage was produced by ischaemia and carbon tetrachloride poisoning in two groups of rabbits. The activity of aspartate aminotransferase with and without pyridoxal 5'-phosphate was measured in the plasma and in cytoplasmic and mitochondrial preparations from both groups. After carbon tetrachloride poisoning the enzyme activity in the plasma increased within 2 h but was not enhanced by pyridoxal 5'-phosphate. Following ischaemia, plasma enzyme activity only increased between 4 and 8 h and was progressively stimulated by pyridoxal 5'-phosphate. Up to 15 h after carbon tetrachloride poisoning the liver cytoplasmic and mitochondrial apo-enzyme remained fully saturated with co-enzyme. In contrast, a pronounced loss of co-enzyme occurred in both fractions of the ischaemic group. These results suggest that the type of injury and not necessarily the organ affected could determine the degree of activation of aspartate aminotransferase by pyridoxal 5'-phosphate observed in human myocardial infarction and liver disease.     

gamma-Glutamyl transpeptidase activity in liver disease: serum elevation is independent of hepatic GGTP activity

gamma-Glutamyl transpeptidase activity was measured in liver and serum from 110 patients undergoing diagnostic liver biopsy, including patients with alcoholic liver disease, fatty liver not due to alcohol, primary biliary cirrhosis, persistent hepatic disease, chronic active hepatitis and normal livers. Serum gamma-glutamyl transpeptidase was markedly elevated in patients with alcoholic liver disease and primary biliary cirrhosis while mean hepatic gamma-glutamyl transpeptidase activity was significantly increased only in the alcoholic liver disease group. There was considerable overlap of individual enzyme values among the different disease groups. There was no inhibitors or activators of liver gamma-glutamyl transpeptidase in any of these disorders. The increased liver activity was not related to the degree of hepatic fibrosis or cirrhosis. There was no correlation between hepatic and serum gamma-glutamyl transpeptidase activity. Hepatic and serum gamma activities were equally increased in individuals with alcoholic liver disease whether or not they were drinking at the time of the study. The data suggest that increased hepatic gamma-glutamyl transpeptidase activity is neither specific for alcoholic liver disease nor essential for serum GGTP to be elevated.     

Induction of rat liver alkaline phosphatase: the mechanism of the serum elevation in bile duct obstruction

Bile duct ligation in the rat leads to a rapid increase in hepatic and serum alkaline phosphatase activity. Within 12 hr after bile duct ligation, hepatic alkaline phosphatase has increased 7-fold and serum alkaline phosphatase activity 2(1/2)-fold. The elevation in the serum activity is completely due to an increase in an isozyme that appears to originate in the liver. This serum isozyme and liver phosphatase, both partially purified by DEAE-cellulose column chromatography, have identical Michaelis constants, pH optima, and rates of heat denaturation. These isozymes migrate identically when subjected to electrophoresis on polyacrylamide gel, and their migration rates are equally slowed after neuraminidase digestion. The data suggest that the rise in hepatic alkaline phosphatase activity is dependent on de novo protein synthesis. Cycloheximide, in a dose that inhibited incorporation of leucine-(14)C into protein by 68%, inhibited the rise in liver phosphatase by 98% and that in serum by 80%. The rise in liver phosphatase activity could not be accounted for by simple retention of alkaline phosphatase that would normally appear in bile. The rise in liver activity after bile duct ligation was 240 times greater than the amount of phosphatase that normally appears in bile over a similar period of time. Cycloheximide had no effect on the bile duct ligation-induced changes in the serum and liver glutamic pyruvic transaminase.     

High levels of secretory IgA and free secretory component in the serum of rats with bile duct obstruction

In the rat , ligation of the bile duct induces a rapid and progressive elevation of the IgA levels in serum. The increase is about 4-fold at 1 h, 15-fold at 1 day, and 30-fold at 1 wk after ligation. The additional IgA is of the secretory type. Free secretory component also appears in serum after bile duct obstruction; it does not continue to increase and occasionally disappears from serum after prolonged ligation. The increase in serum IgA levels is selective. These changes are totally reversible if the bile duct is reopened at 1 day after ligature. These findings confirm the role of the rat liver in the transfer of circulating IgA into the bile.     

Serum alkaline phosphodiesterase I in experimental biliary obstruction in the rat

Alkaline phosphodiesterase I was present in rat liver at approx. 100-fold greater activity than alkaline phosphatase, and in rat bile at approx. 25-fold greater activity. Rat serum alkaline phosphodiesterase I was increased 6-fold whilst serum alkaline phosphatase was increased only 2-fold 96 h after bile duct ligation. In contrast to alkaline phosphatase, hepatic alkaline phosphodiesterase I was not affected by bile duct ligation, suggesting its raised serum activity was due to bile regurgitation rather than overspill of the enzyme from liver into blood. Gel filtration showed that 8 and 96 h after bile duct ligation the serum contained a high molecular weight form of alkaline phosphodiesterase I. It is suggested that alkaline phosphodiesterase I offers a potentially useful indicator of biliary obstruction in the rat.     

Adaptive hepatic changes in mild stenosis of the common bile duct in the rat

Adaptive hepatic changes were investigated in rats with mild stenosis of the common bile duct and in sham-operated controls. The studies were performed 24 h and 7–12 days postoperatively. A continuous intravenous infusion of taurocholic acid at stepwise-increasing rates was performed to explore the responses to bile acid effects. During the infusion, bile flow and the outputs of bile acids, phospholipids, cholesterol, alkaline phosphatase and gamma glutamyl transpeptidase were studied. At the end of the infusion, hepatic morphometric measurements were performed. In other experimental sets, biliary excretions of horseradish peroxidase, a marker of microtubule-dependent vesicular transport in the hepatocyte, and sulphobromophthalein, a well-known organic anion model, were studied. In other rats, bile acid pool size and composition were determined by depletion of bile. The results in rats with mild stenosis maintained for 24 h showed a greater susceptibility to the toxicity of taurocholic acid, as revealed by the abrupt decrement in bile flow at high rates of infusion, and increased outputs of phospholipids and canalicular enzymes. Conversely, rats with mild stenosis maintained for 7–12 days showed decreased bile acid maximum secretory rate and biliary outputs of phospholipids and canalicular enzymes, as well as hepatocyte hypertrophy. These findings may explain the limited hepatic and systemic repercussion of experimental mild stenosis of the common bile duct and help us to understand the early stages of constriction of the common bile duct in man.     

Serum and liver glycylproline dipeptidyl aminopeptidase activity in rats with experimental hepatic cancer

Changes of glycylproline dipeptidyl aminopeptidase (GPDA) and gamma-glutamyl transpeptidase (gamma-GTP) activities were compared in the serum and liver tissue of rats with hepatic cancer induced by 3'-methyl DAB. Serum glycylproline dipeptidyl aminopeptidase activity in rats with the azo dye-induced hepatic cancer was significantly higher than that in healthy rats, but the increase was not so extensive compared with that of gamma-glutamyl transpeptidase. The specific activity of glycylproline dipeptidyl aminopeptidase was decrease in the microsomal fraction and increased in the supernatant fraction of hepatic cancer tissue, whereas that of gamma-glutamyl transpeptidase was increased in both microsomal and supernatant fractions. These results suggest that the mechanisms, whereby serum activities of these two enzymes were increased in rats with hepatic cancer, were different from each other.     

Allopurinol dose is important for attenuation of liver dysfunction after normothermic ischemia: Correlation between bile flow and liver enzymes in circulation

We have investigated the effect of two doses of allopurinol (ALL) (100 and 50 mg/kg) administered i.v. on liver function after 1 h of normothermic ischemia. ALL given in a concentration of 100 mg/kg significantly improved bile output after 1 and 24 h of reperfusion. Hepatocyte injury reflected by alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) in plasma was also significantly reduced at 24 h, but not at 1 h of reperfusion compared with controls. ALL administered at a concentration of 50 mg/kg had some protective effect. Significant correlation between circulating liver enzymes and bile output at 24 h after reperfusion indicates an important pathophysiologic link between hepatocyte function and injury in this time window.     

Time course of serum protein changes after strenuous exercise of the forearm flexors.

The time course of changes in serum proteins and other blood constituents after eccentric exercise of the forearm flexors by six nonweight-trained female subjects (age, 19.7 +/- 1.9 years) was investigated. Eccentric muscle actions are those in which the muscle lengthens as it exerts force, as when a person lowers a weight. Serum levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, myoglobin, as well as urea nitrogen, uric acid, creatinine, calcium, and phosphorus were examined before and for 6 days after exercise. Creatine kinase increased dramatically (peak value ranged from 6740 to 24,200 U/L) and aspartate aminotransferase, lactate dehydrogenase, alanine aminotransferase, and myoglobin followed the same time course as creatine kinase, but their peak values were lower. These proteins did not increase significantly until 48 hours after exercise and reached peak values 3 to 5 days after exercise. Alkaline phosphatase, gamma-glutamyl transpeptidase, uric acid, urea nitrogen, creatinine, calcium, and phosphorus showed no change. There is either a delay in muscle protein release by damaged muscle fibers, or the proteins are unable to leave the interstitial area for the 24 to 48 hour period after exercise. Because of the long delay, care should be taken when blood protein levels are interpreted in persons who have exercised strenuously (even if only for a short period of intense effort) several days before any diagnostic tests are performed.     

Dehydroepiandrosterone prevents oxidative injury in obstructive jaundice in rats

We investigated the effect of dehydroepiandrosterone (DHEA) on oxidative injury in obstructive jaundice using three groups of rats: sham-operated group; common bile duct (CBD) group--the CBD was ligated; and DHEA group--DHEA administration followed CBD ligation. Liver function tests were performed using blood samples, and malondialdehyde concentration (MDA), superoxide dismutase activities (SOD), glutathione peroxidase (GPx), and total glutathione (tGSH) concentrations were measured in liver tissue. Serum alkaline phosphatase, gamma-glutamyltransferase and alanine aminotransferase activity were significantly elevated in the CBD group compared with the other groups. Serum aspartate aminotransferase and total bilirubin were highest in the CBD group; the MDA concentration was higher in the CBD group than the sham group. There were no significant differences in GPx activity among the groups. SOD activity and tGSH concentration were significantly lower in the CBD group than the other groups. DHEA may protect hepatic tissue against oxidative injury in obstructive jaundice by decreasing MDA concentration and increasing SOD activity and tGSH concentration.     

Critical role of plasminogen activator inhibitor-1 in cholestatic liver injury and fibrosis

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein known to correlate with hepatic fibrosis. However, whether or not PAI-1 plays a causal role in this disease process had not been directly tested. Therefore, wild-type or PAI-1 knockout (PAI-1(-/-)) mice underwent bile duct ligation. Mice were sacrificed either 3 or 14 days after surgery for assessment of early (i.e., inflammation) and late (i.e., fibrosis) changes caused by bile duct ligation. Liver injury was determined by histopathology and plasma enzymes. Accumulation of extracellular matrix was evaluated by Sirius red staining and by measuring hydroxyproline content. Hepatic expression of PAI-1 was increased approximately 9-fold by bile duct ligation in wild-type mice. Furthermore, early liver injury and inflammation due to bile duct ligation was significantly blunted in PAI-1(-/-) mice in comparison with wild-type mice. Although PAI-1(-/-) mice were significantly protected against the accumulation of extracellular matrix caused by bile duct ligation, increases in expression of indices of stellate cell activation and collagen synthesis caused by bile duct ligation were not attenuated. Protection did, however, correlate with an elevation in hepatic activities of plasminogen activator and matrix metalloprotease activities. In contrast, the increase in tissue inhibitor of metalloproteases-1 protein, a major inhibitor of matrix metalloproteases, caused by bile duct ligation was not altered in PAI-1(-/-) mice compared with the wild-type strain. The increase in hepatic activity of urokinase-type plasminogen activator was also accompanied by more activation of the hepatocyte growth factor receptor c-Met. Taken together, these data suggest that PAI-1 plays a causal role in mediating fibrosis during cholestasis.     

Elevated level of serum Mn-superoxide dismutase in patients with primary biliary cirrhosis: possible involvement of free radicals in the pathogenesis in primary biliary cirrhosis.

Serum Mn-superoxide dismutase (Mn-SOD) was determined in patients with various liver diseases including 31 patients with primary biliary cirrhosis (PBC), 46 with hepatocellular carcinoma (HCC), 17 with liver cirrhosis (LC), 23 with chronic hepatitis (CH) and 12 patients with obstructive jaundice with an enzyme-linked immunosorbent assay using a specific monoclonal antibody. The serum level in patients with PBC (407 +/- 35 ng/ml, mean +/- SEM; n = 31) was significantly increased (p less than 0.01) compared with those of other liver diseases. Mn-SOD level did not correlate with total bilirubin level, gamma-glutamyl transpeptidase activity, alkaline phosphatase activity, alanine aminotransferase activity, IgM, or with ceruloplasmin level in the sera of the patients. When the patients with PBC were histologically subdivided into four groups according to Scheuer's classification (Scheuer PJ. Primary biliary cirrhosis. In: Scheuer PJ, ed. Liver biopsy interpretation. 3rd ed. London: Bailliere Tindall, 1980:47-56), a high level of serum Mn-SOD was noticed in the early stage as well as in the advanced stage of the disease. Immunoblot analysis confirmed the reactivity and specificity of the monoclonal antibody to the enzyme protein in the patients' sera. Immunostaining of a liver biopsy specimen from the patients with PBC revealed increased expression of the enzyme protein in damaged epithelial cells of interlobular bile ducts, bile ductules, and degenerated hepatocytes. These data suggested that free radicals including superoxide anion are possibly involved in the pathogenesis of the disease and Mn-SOD may play some role in a protection against the superoxide anion.     

Some laboratory correlates of drinking habits.

The effect of drinking habits on the frequency distributions of eight biochemical or haematological test results was studied in 7915 patients attending a multiphasic health testing centre. Increasing incidences of abnormal results with increasing alcohol intake, at levels of alcohol intake habitual for a large proportion of the population, were found for plasma gamma-glutamyl transpeptidase, triglycerides and uric acid, and for erythrocyte mean corpuscular volume. Of four frequently used liver function tests, aspartate aminotransferase, alkaline phosphatase, bilirubin, and albumin, only aspartate aminotransferase was strongly affected by drinking habits. These findings have relevance for the detection of individuals whose drinking habits are harmful to them, and for the interpretation of 'profile' results.