Establishment Of Enzymatic Kinetic Assay Method Of Serum Argininosuccinate Lyase Activity & Its Clinical Practice

Liver disease is one of principal diseases that threaten human healthy. There has been a high occurrence of liver diseases in China. Liver cancer is the second"killer"of malignant tumor in China. The morbidity of hepatoma is increasing about a million in the world every year, and half of them are in China. There are more than three hundred million patients with type B hepatitis in the earth, and one hundred and fifty million infected persons are in China, which is approximately a half over the world. Type B hepatitis infected person is more than 9% total population in our country, quarter person of them (about thirty million) are with chronic type B hepatitis. Because a number of them are not diagnosed, they would long-term hide in health person, which influence quality of life of patient himself as well as the health other people severely. The threat of liver diseases to human being is an important and urgent problem.Hepatocyte injury is the common pathologic process in the early stage of various liver diseases,at this time, there are seldom clinical symptoms, signs and imageology changes. Therefore, the diagnosis of liver diseases in early stage is mainly dependent on laboratory investigations, in which enzymology assay is the first indicator. More than tens of serum enzyme markers have been studied for the diagnosis of liver diseases, but the majority of them are not accepted by clinician due to various reasons. Now, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are still extensively used as primary enzymatic markers of hepatic cell injury over the world. However, clinician and medical laboratory scientist have known that both enzymatic markers have unsatisfactory sensitivity, especially poor specificity in the diagnosis of liver diseases. In view of these facts, to explore a new specific and sensitive serum enzymatic marker is very necessary for definite diagnosis of liver diseases.Substantially all urea produced in human body is made in the liver. Argininosuccinate lyase (ASL, EC 4.3.2.1) is one of key enzymes of urea cycle that produces urea. ASL is primarily present in the cytoplasm of hepatocyte. It also was found in intestine and kidney in a small amount, and a trace activity in other tissues, such as heart, lung and spleen. There is no activity found in muscular tissue. The distribution characteristics in the body suggest that ASL has relative high liver specificity. There are some studies showed that ASL is a new autoantigen in autoimmune liver diseases. The antibody of ASL is a liver specific autoantibody that is not present in serum and other body fluids in patients with other autoimmune diseases outside liver. Therefore ASL may be an ideal marker of liver cell injury. Present study was divided into three parts. Firstly, human liver ASL was separated and purified, then human ASL tissue distribution characteristics were systematically studied. The establishment and methodological evaluation of an automatic analysis method for the determination of serum ASL activity were performed in the second part. Finally, the diagnostic efficiency of serum ASL activity assay for liver diseases was investigated and compared with other laboratory indices of liver cell injury.PART ONEObjective To investigate ASL distribution characteristics in main tissues or organs of human, and to separate and purify ASL in liver cell.Methods The homogenates of human main organs or tissues were prepared, then ASL activities and protein concentrations in various homogenate were measured, the specific activities (U/g protein) were calculated. ASL in liver cell was separated and purified by ion exchange chromatography. The purified product was identified with SDS-PAGE.Results Specific activity of liver cell (3.94U/g protein) was the highest in all investigated organs or tissues of human, the second was kidney that was about one tenth of liver specific activity. Brain, heart, intestine, pancreas, and lung were all with small ASL specific activity. Other tissues or organs had little specific activity. The product purified by ion exchange chromatography and dialysis showed only a single electrophoresis strap, which molecular mass was about 50 kD.Conclusions Our results demonstrate that human ASL primarily presented in liver cell. The ASL specific activity in liver cell is the highest in investigated tissues or organs of human body.PART TWOObjective To establish a continuous monitoring assay method of serum ASL activity, which could be performed with automatic biochemistry analyzer. To carry out the evaluation for established method.Methods According to the chemical reaction catalyzed by ASL and the working characteristics of automatic biochemistry analyzer, we designed a enzyme coupled reaction system with high specific. The reaction conditions were optimized by the orthogonal experiment. Following the relevant documents of International Federation of Clinical Chemistry (IFCC), the methodological evaluation of this method was completed, including precision, recovery, stability of reagent, lowest detectable limit, linear range, interference tests, and so on.Results Based on the coupled enzyme reactions, a new kinetics assay method of ASL activity was set up that could be applied on automatic biochemistry analyzer, in which NAD+-NADH was used as the indicator system. The optimal reaction conditions, such as concentration of substrate, pH, reaction time, reaction temperature, and so on, were determined. The investigations of methodological evaluation exhibited that the precision of this method was good indicated by the 4.0% (3.6%~4.7%) intraassay coefficient of variation, and 5.9% (4.0%~8.5%) interassay coefficient of variation; recovery was 100.5% (93.3%～107.2%), linear range was among the range of 0~167.7 U/L (r>0.999). The lowest detection limit was approximately 0 U/L. The interference test showed that there were no interferences while bilirubin <342μmol/L, and anticoagulants including Na2C2O4, K2EDTA, Na2EDTA,citrate sodium, heparin lithium or heparin sodium at their routine anticoagulant concentrations. However, when Hb>0.06 g/L, a positive interference with a concentration-dependent manner was found. The reagents were stable when stored at 2～8℃for up to 20 weeks.Conclusions A continuous monitoring assay for the determination of serum ASL activity is successfully established, which is suitable the performance of automatic biochemistry analyzer equipped in most clinical laboratories. This method has many advantages, such as high detection speed; widen linear range, small volume of sample consumption, higher specificity and sensitivity. Therefore the method is completely fit the application of clinical laboratories. PART THREEObjective Using established the automatic analytical method for the determination of serum ASL activity, the clinical diagnostic efficiency of serum ASL activity for diagnosis of different liver diseases was investigated.Methods Two hundred ninety one patients with various liver diseases were enrolled as the liver disease group, including 31 acute hepatopathy, 165 chronic hepatopathy (73 chronic hepatitis B patients, 39 chronic hepatitis C patients, 25 alcoholic hepatitis patients, 16 drug induced hepatitis patients and 12 autoimmunity hepatitis patients), 67 cirrhosis patients, 28 primary liver cancer patients. Controls included 247 patients exclude liver disease (non-liver disease patient group) and 32 healthy volunteers (healthy control group). Venous blood samples were drawn in the subjects and sera were separated. Serum ASL and traditionally enzymatic markers of liver disease such as ALT, AST, GGT, LDH, ALP, and also the total bilirubin (Tbil) level were determined. In the chronic liver patients, the concentrations of cystatin C (Cys C) and tissue inhibitors of metalloproteinase (TIMPs) were tested. Additionally, Liver histopathologic examinations were performed in 31 patients with hepatopathy. The diagnostic efficiency of all indices was evaluated according to the principles of Evidence-Based Laboratory Medicine (EBLM). The follow-up investigations on 4 different hepatic diseases were carried.Results Analysis of variance showed that serum ASL had no significant difference between non-liver disease group and healthy group (F=1.108,P=0.355), but other enzyme indices, including ALT and AST, and Tbil were all significantly different (P<0.05 or P<0.01). As compare to the healthy group, ASL was more than 19 times increase in liver diseases group, and there was definitely no overlap of ASL activity ranges between two groups. However, ALT and AST only rose 1.5～4 times in other liver disease except acute hepatitis patients, and their activity ranges were partly overlap among the liver disease group, non-liver disease group and healthy control group. The increased degrees of serum ASL levels in various liver diseases were hepatocellular carcinoma > acute hepatitis > liver cirrhosis > chronic hepatitis. The range of ASL levels in chronic liver diseases group had completely no overlap as comparing to the health control group or non-liver disease group, but both ALT and AST had markedly overlap. Receiver operating characteristics (ROC) curves revealed that the sensitivity and specificity ASL (at cut-off value 8 U/L) to diagnose liver diseases were 99.7% and 97.8% respectively. However, the sensitivity and specificity of ALT and AST were low (both at cut-off value 40 U/L), 97.6% and 33.3% for ALT, 83.8% and 36.5% for AST, respectively. The total diagnostic efficiency indicated by the area under the ROC curve (AUC) (95% confidence interval, CI) for ASL was 1.000 (0.999～1.000), for ALT was 0.755 (0.716～0.793), and for AST was 0.686 (0.643～0.729), respectively.The sensitivity and specificity of ASL to diagnose chronic liver diseases were 99.4% and 97.8% respectively. AUC (CI) was 1.00 (0.999～1.000). However, the sensitivity and specificity were 63.6% and 34.1% for ALT, 61.8% and 37.3% for AST, respectively. AUC (CI) of ALT was only 0.556 (0.498～0.631) and AST was 0.545 (0.488～0.602), respectively.Pearson correlation analysis of the histopathologic examination results showed that ASL concentrations (86.9±26.5) was a positive correlation (r=0.417, P=0.019) with the scores of histopathologic inflammation grading (9.83±3.36). In hepatic fibrosis patients, ASL activity was positively correlated with Cys C and TIPMs levels. Follow-up investigation found that the dynamic variation of serum ASL activity was coincident with the clinical pathologic processes of different liver diseases and the changes of infection amount of hepatitis viruses.Conclusions Serum ASL activity is a sensitive and specific marker indicating liver cell injury. The diagnostic efficiency of ASL on liver diseases is much more superior to the traditional enzyme markers such as ALT and ALT etc. Serum ASL assay is hopeful to become a routine laboratory diagnostic item of liver injury, and extensively applied in clinical practice.