Activities of several enzymes in serum and heparinized plasma from rats

The activities of lactic dehydrogenase, malic dehydrogenase, aspartate aminotransf erase, fructose-1,6-diphosphate aldolase and isocitric dehydrogenase were higher in serum from rat blood that had clotted at room temperature for 1 h than in serum separated from blood immediately. During clotting, the activities of alanine aminotransferase, sorbitol dehydrogenase, glutamic dehydrogenase, fructose-1-phosphate aldolase and ornithine carbamyl transferase did not increase. When blood was heparinized and stored at room temperature for l or 2 h, with the exception of lactate dehydrogenase the activities of these enzymes in the plasma prepared from that blood did not increase with time.Phosphate buffer was used in all but the ornithine carbamyl transferase assay. Tris-HCl or triethanolamine buffers were unsuitable because when they were used the assay mixtures became opaque when some of the heparinized samples were analyzed. In the supernatant fraction from sonicated blood, activities over 10 mU/ml were obtained for lactic dehydrogenase, malic dehydrogenase, aspartate aminotransferase, fructose-1,6- diphosphate aldolase, alanine aminotransferase and sorbitol dehydrogenase.     

Multivariate analysis of plasma enzyme profiles in severe head injury

We followed the changes in the activities of four enzymes (aldolase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase) in plasma for four days after head injury. The progression of the changes differs significantly between survivors and nonsurvivors. Stepwise discriminant analysis, involving the four enzymes, allowed us to divide 81 to 92% of head-injured patients (n = 280 selected without conscious bias) correctly into the two groups as early as 72 h after trauma. Most of the patients who were misclassified according to our biochemical criteria had received phenobarbital for sedation. Valuable prognostic information in head injury evaluation may thus be obtained by daily determination of enzymatic activities of these four enzymes.     

Enzymatic assessment of myocardial injury after infarction or cardiac surgery. Is isoenzyme analysis required?

Estimation of enzyme release in plasma requires knowledge of the fractional catabolic rate constant (FCR) for the elimination enzyme activity from plasma. However, the total plasma content of such enzymes usually consists of several isoenzymes with different values of FCR. Thus, the use of a single overall value for FCR may cause error. This problem was studied by determination of the plasma isoenzyme activities of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and alpha-hydroxybutyrate dehydrogenase in patients after cardiac surgery and after acute myocardial infarction. Values of FCR and the cumulative release of activity in plasma are estimated for separate isoenzymes and for total enzyme activity. Results are compared with the enzyme content of myocardium, skeletal muscle and blood cells. It is concluded that isoenzyme separation is not required for the quantitative use of such data. The implications for the validation of enzymatic estimation of cardiac injury are discussed. The results indicate that local inactivation of enzymes after cardiac injury must be limited.     

Serum guanase activities after myocardial infarction.

Serum guanase, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase and hydroxybutyrate dehydrogenase activities were measured in 290 blood samples from 96 consecutive patients admitted to a Coronary Care Unit. Elevated serum guanase activities (greater than 2 U/l) were found in 19 patients (20%). The magnitude and frequency of these elevations did not negate the value of guanase as a "liver function test", since all cases with raised guanase also had abnormal serum alanine aminotransferase activities. This fact, together with other information in the literature, indicated that elevated serum guanase activity following myocardial infarction was consequent upon some degree of sub-clinical hepatic necrosis. Caution must be exercised when serum asparate aminotransferase is used as an index of heart muscle necrosis unless guanase or some other "liver specific" enzyme is known to be normal, or unless creatine phosphokinase or hydroxybutyrate dehydrogenase activities are elevated.     

Enzyme changes and morphometric analysis of bile ducts in experimental bile duct obstruction.

Chronic bile duct obstruction causes a marked proliferation of bile ductules within the rat liver plus an increase in the activities of hepatic gamma-glutamyl transpeptidase, 5'-nucleotidase and alkaline phosphatase. To determine if the increase in the activities of these enzymes within the liver simply reflects the increase in bile duct mass, we subjected rats to bile duct ligation for periods up to one week and compared the activities of these enzymes within liver tissue with bile duct volume, determined by morphometric analysis. The activities of two enzymes not useful in the diagnosis of chronic cholestasis, aspartate aminotransferase (GOT) and alanine aminotransferase (GPT), were also measured. There was no correlation between the proliferation of bile ductules and the activity of any of these enzymes. Bile duct volume increased 4.9-fold within 24 h after ligation and rose steadily, reaching a value of 13 times control in one week. Alkaline phosphatase activity increased 3.6-fold within 24 h after bile duct ligation and then was relatively constant. Gamma-glutamyl transpeptidase and 5'-nucleotidase activity both fell 24 h after ligation but were slightly increased after 48 h. Enzyme activities of each were almost twice control at 120 and 168 h. Alanine aminotransferase activity fell steadily during the period of bile duct ligation, while aspartate aminotransferase was unchanged. The change in gamma-glutamyl transpeptidase and 5'-nucleotidase activity within the liver cannot be due simply to hypertrophy of bile duct epithelium.     

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.     

"Lysosomal" enzyme activities in red blood cells of normal individuals and patients with homozygous beta-thalassaemia.

Four hydrolases, beta-galactosidase, beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase were examined in red blood cells (RBC) of normal donors and patients with homozygous beta-thalassaemia. Highly sensitive fluorimetric substrates were used to determine the specific activities of these enzymes. In order to avoid contamination by lysosomal activities derived from white blood cells (WBC), the mature RBV were separated from other blood elements by cellulose chromatography. The hydrolase activities in normal RBC were detected only in their plasma membranes and were found to be considerably lower than in WBC or platelets. In thalassaemic RBC, hydrolase activities were present in both plasma membranes and in the soluble fraction. The normoblast fraction contributed most of the hydrolase activity found in these preparations, suggesting the presence of lysosomal particles in thalassaemic RBC. No differences in the enzymatic activities were found when purified membranes of mature RBC from thalassemic and normal preparations were compared. The origin and roles of these hydrolytic enzymes in normal and thalassaemic RBC membranes are not known.     

Erythrocytic Oxidative Stress Indices and Haemato-biochemical Changes in Fluorotic Cattle from Industrial Fluorotic Zone of Odisha

Effect of chronic fluorosis on haemato-biochemical parameters and oxidant-antioxidant equilibrium in cattle of industrial fluorotic zones of district Angul, Odisha, India was studied. Ten fluorotic cattle of visible clinical signs of endemic area and ten healthy cattle of non-fluorotic area were selected for the present study. Plasma fluoride level along with haemato-biochemical parameters was studied. Erythrocytic oxidative stress parameters like catalase, superoxide dismutase and lipid peroxidase were examined from 10 % red blood cell hemolysate. Statistical analysis of these two groups revealed altered haemato-biochemical parameters. There is high plasma fluoride level in the fluorotic cattle as compared to the healthy ones. The blood samples of the fluorotic cattle showed significant decrease (P ≤ 0.05) in haemoglobin, total erythrocyte count, total leucocyte count and packed cell volume as compared to healthy cattle. Differential leukocyte picture showed significant decrease (P ≤ 0.05) in neutrophil count in fluorotic cattle while increase in eosinophil, monocyte and lymphocyte percentage as compared to cattle of non fluorotic zone. Fluoride affected cattle revealed significantly higher (P ≤ 0.05) levels of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine level, whereas significant decrease (P ≤ 0.05) in plasma calcium level and increase in plasma phosphorous level was recorded in fluorotic cattle as compared to healthy ones. Marked increase (P ≤ 0.05) in lipid peroxidation whereas decrease in catalase and superoxide dismutase activities was observed in fluorotic cattle as compared to healthy ones.     

Some glycolytic enzymes in normal cerebrospinal fluid, brain tissue and blood plasma of infants.

The activities of several glycolytic enzymes (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase) as well as glycerol-1-phosphate dehydrogenase and (Mg2+)ATPase in normal cerebrospinal fluid (CSF) and blood plasma samples, from 12 healthy infants, aged 2-18 months, and in supernatants from brain tissue slices, taken during neurosurgical operations from infants of the same range of age were estimated. The values obtained confirm the high activity of the above enzymes found in animal brains, and indicate an independence of these activities in blood plasma and CSF. The origin of the activities of the investigated enzymes in CSF seems to be mainly, if not, exclusively, from brain tissue. This might be useful for detection of brain tissue damage as was earlier proven with LDH activity in CSF.     

Enzyme-activities in serum and plasma of man, dog and rat, and the variation of these enzyme activities during storage of the blood]

The following enzymes were determined in the serum and plasma of man, dog and rat: alanine aminotransferase, asparate aminotransferase, alkaline phosphatase, lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase. The enzyme assays were performed on an Eppendorf-Enzymautomat 5010 using optimised conditions at 25 degrees C. The enzyme-activities changed by variable amounts during standing of the blood. This concerned mainly lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase and aspartate aminotransferase in the serum of the rat. In human serum and in dog serum, and in the plasma of man, dog and rat this effect was only less pronounced.     

Enzyme measurements by mass: an interim review of the clinical efficacy of some mass measurements of prostatic acid phosphatase and the isoenzymes of creatine kinase

The effects of simultaneous administration of thiamine, niacin or vitamin b₂₁ with vitamin E on plasma vitamin E levels were studied in 20 adult male volunteers belonging to the low socio-economic class. The effect of vitamin E on the nutritional status of pyridoxine, riboflavin and thiamine as judged by the erythrocyte enzymes, aspartate aminotransferase, glutathione reductase and transketolase, respectively was also studied.None of the members of the B-complex vitamins studied here had any effect on plasma vitamin E levels. This was in contrast to the observation made earlier that pyridoxine and riboflavin can reduce plasma vitamin E.There was a transient reduction in both the basal and stimulated activities of erythrocyte aspartate aminotransferase, the significance of which needs further investigation.     

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。     

Mitochondrial enzymes in human serum: comparative determinations of glutamate dehydrogenase and mitochondrial aspartate aminotransferase in healthy persons and patients with chronic liver diseases

We measured the activities of two mitochondrial enzymes, the mitochondrial form of aspartate aminotransferase (EC 2.6.1.1) and glutamate dehydrogenase (EC 1.4.1.2), in the serum of apparently healthy persons (n = 84) and patients suffering from chronic liver diseases (n = 43). The distribution of activities for glutamate dehydrogenase, but not mitochondrial aspartate aminotransferase, was sex-dependent. The upper limits of the reference intervals (99th percentile) at 37 degrees C were 3.2 U/L for mitochondrial aspartate aminotransferase, 6.4 U/L for glutamate dehydrogenase (women), and 11.0 U/L for glutamate dehydrogenase (men); there was a weak correlation between the activities of both mitochondrial enzymes (r = 0.439). In patients with chronic liver diseases we found a greater increase in the activity of glutamate dehydrogenase than of mitochondrial aspartate aminotransferase and the correlation between the two mitochondrial enzymes was stronger. The diagnostic sensitivity and specificity of either mitochondrial enzyme was less than that of total aspartate aminotransferase, alanine aminotransferase (EC 2.6.1.2), or gamma-glutamyltransferase (EC 2.3.2.2).     

Two dry-reagent systems evaluated for determinations of enzyme activities

We evaluated the Kodak Ektachem DT60/DTSC and the Boehringer Mannheim Reflotron for measuring activity concentrations of six enzymes. The Ektachem CVs for low concentrations of aspartate aminotransferase and alanine aminotransferase were high. As compared with the Ektachem and a routine "wet-chemistry" system, values for aspartate aminotransferase and alanine aminotransferase were lower as measured by the Reflotron, because no pyridoxal 5'-phosphate is included in the Reflotron slides. Activity concentrations of gamma-glutamyltransferase and creatine kinase measured with the Ektachem and with the routine procedure did not agree well, possibly because the Ektachem gave too-high results for the enzyme activities. Assays of several commercial test sera indicated that test results by the dry-chemistry and routine procedures are not interconvertible. This contrasts with our previous experience, in which between-test agreement for several analytes was acceptable.     

Effects of neuroleptic phenothiazines on the activities of aminotransferases and gamma-glutamyltransferase in serum and liver

Serum alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase activities were monitored in psychiatric patients receiving normal doses of phenothiazine neuroleptics over a 30-day period. The first two enzymes showed slight initial increases and a subsequent return to normal, while the third showed a slight increase. In rats, dosage levels exceeding those used in human therapy produced much larger increases in the catalytic concentrations of all three enzymes in serum (1.4, 0.7 and 0.5 above the control value, respectively), and somewhat smaller increases in the liver homogenates of these animals.     

Protective effect of S-nitrosylated alpha(1)-protease inhibitor on hepatic ischemia-reperfusion injury

S-Nitrosylated compounds (nitrosothiols; RS-NOs) function as nitric oxide (NO) reservoirs and preserve the antioxidant activities of NO. We found remarkable cytoprotection by an S-nitrosylated protease inhibitor from human plasma, S-nitroso-alpha(1)-protease inhibitor (S-NO-alpha(1)-PI) that possesses a completely nitrosylated SH group, in hepatic ischemia-reperfusion injuries in rats. Liver ischemia was induced in rats by occluding both the portal vein and hepatic artery for 30 min and was followed by reperfusion. S-NO-alpha(1)-PI and control compounds such as native alpha(1)-PI, an NO synthase (NOS) inhibitor, and standard RS-NOs were given via the portal vein just after reperfusion was initiated. Liver injury was evaluated by measuring the extracellular release of liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). Infiltration of neutrophils and induction of apoptosis and heme oxygenase-1 (HO-1) in the liver were also examined. Maximal liver injury occurred at 3 h after reperfusion and then decreased gradually. Not only did S-NO-alpha(1)-PI treatment (0.1 micromol; 5.3 mg/rat) greatly reduce elevation of liver enzymes in plasma, as well as neutrophil accumulation and apoptotic change in liver, it also improved the impaired hepatic blood flow as assessed by laser Doppler flowmetry and potentiated the induction of HO-1 in the liver. Although native alpha(1)-PI moderately reduced liver injury, low molecular weight RS-NOs such as S-nitrosoglutathione and S-nitroso-N-acetyl penicillamine produced no obvious protective effect. An NOS inhibitor exacerbated the hepatic ischemia-reperfusion injuries. These results suggest that S-NO-alpha(1)-PI exerts a potent cytoprotective effect on ischemia-reperfusion liver injury by maintaining tissue blood flow, inducing HO-1, and suppressing neutrophil-induced liver damage and apoptosis.     

Initiation of lipid peroxidation (LPO) in blood during intraoperative mechanical autotransfusion--is hepatotoxicity of lipid peroxidation products of clinical significance?

Machine autotransfusion using cell-saver is a well-established method of saving homologous blood during extensive surgical procedures. The processing of blood may induce the initiation of lipid peroxidation (LPO) with the release of hepatotoxic products. A series of 42 patients undergoing primary (n = 20) or revision (n = 22) hip arthroplasty comprised the study group. Patients received an average of 1,260 ml of autologous blood and 2.2 units of homologous packed cells. The concentration of thiobarbituric acid reactive substances (TBARS) as LPO metabolites was measured in the patients' plasma, in the autologous packed cells as well as in the supernatants of the cell-saver-processed blood. Additionally, parameters of iron metabolism, haemoglobin levels, haematocrit as well as the activities of so-called liver enzymes aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidase and cholinesterase were determined. An initiation of LPO was detectable during the process of machine autotransfusion, but this took place mainly ex vivo. High concentrations of TBARS were detectable in the supernatants after cell-separation processing. We observed a decline in haemoglobin concentration and haematocrit during the perioperative period. Postoperatively, we found a significant iron deficiency as a consequence of the perioperative blood loss. There was not sufficient evidence of a postoperative liver disorder induced by toxic metabolites of LPO. To sum up, there is only a low contamination of the organism with LPO products during the process of machine autotransfusion. Therefore, an induction of liver damage seems to be improbable.     

Systemic short-term fibrinolysis with high-dose streptokinase in acute myocardial infarction: time course of biochemical parameters

The course of plasma catalytic activities of total creatine kinase, creatine kinase isoenzyme MB, total, cytoplasmatic and mitochondrial aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, glutamate dehydrogenase and concentrations of myoglobin, urea, acidic alpha 1-glycoprotein and creatinine were followed in 33 patients suffering from acute myocardial infarction. All patients were randomized in a double-blind, prospective study. One group (18 patients) was infused with streptokinase 1.5 X 10(6) units/90 minutes; the control group received routine continuous i.v. heparin treatment (1000 units/h). Ten hours after completion of the study protocol, treatment of both groups of patients was continued with heparin, 1000 units/h and Aspisol, 1 g/day2). Streptokinase treatment induced earlier wash-out and therefore earlier peak levels of several enzymes: total creatine kinase (11 hours), creatine kinase isoenzyme MB (6 hours), total and cytoplasmatic aspartate aminotransferase (6 hours) and lactate dehydrogenase (9 hours). Total creatine kinase peak catalytic activity and myoglobin peak concentration were higher in the group receiving thrombolytic therapy. A significantly different course of catalytic activity between both treatment groups was found for total creatine kinase and creatine kinase isoenzyme MB, total and cytosolic aspartate aminotransferase, lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase. The course of mitochondrial aspartate aminotransferase catalytic activity was different only 12 hours after the beginning of treatment. The shift of several catalytic activities to an earlier peak level in plasma may indicate reperfusion of ischaemic myocardium due to thrombolytic therapy.     

Hepatic function assessed (in rats) during chemotherapy with some anti-cancer drugs.

Using rats, we studied how best to assess hepatic damage after administering therapeutic doses of each of five anti-cancer drugs or of the hepatotoxin, carbon tetrachloride. As indexes, we compared measurement of the concentration of administered antipyrine in plasma with measurement in serum of alpha-fetoprotein or of the activities of five enzymes that reportedly best reflect hepatic damage. The biological half-life of antipyrine in the plasma was increased more than threefold on pretreating the rats with any of the five cytotoxic drugs or with carbon tetrachloride. In contrast, the concentrations of alpha-fetoprotein, alkaline phosphatase, gamma-glutamyltransferase, or glutamate dehydrogenase were not consistently increased. Of the enzymes tested in serum, aspartate aminotransferase and ornithine carbamoyltransferase best indicated hepatic impairment resulting from the treatment with anti-cancer drugs. Our results imply that determination of the pharmacokinetics of marker drugs such as antipyrine better indicates hepatic dysfunction induced by cytotoxic agents than does measurement of the enzymes liberated into serum as a result of damage to liver mitochondria.     

Catalytic enzyme activity concentration in plasma of man, sheep, dog, cat, rabbit, guinea pig, rat and mouse. Approach to a quantitative diagnostic enzymology, I. Communication

For the most convenient laboratory animals in experimental medicine such as the sheep, dog, cat, rabbit, guinea pig, rat and mouse, the catalytic concentrations of seventeen enzymes and the protein and albumin concentrations in plasma were determined. The corresponding data for man were taken from the literature. The experimental conditions were selected so as to minimize the influence on catalytic activity, variance and statistical distribution pattern of such factors as handling procedures, the choice of blood drawing technique, the choice of anaesthetic agent for distinct species and the preparation procedure of plasma from the blood specimen. Because these considerations have been largely neglected to date in experimental enzymology, normal values for most enzymes require revision and were in some instances established for the first time in certain species. Our use of assay conditions, which were identical for all animal species, satisfies current requirements in clinical chemistry with respect to standardization and optimization of enzyme catalytic activity concentration measurement, and allowed comparison of these enzymes. The catalytic activities established this way will be the basis for a later comparative study of their kinetics of adjustment in plasma.