Aspartate Aminotransferase Assays Research Articles

Activities of aspartate and alanine aminotransferases in the MollicutesA. laidlawii MG,M. pneumoniae FH, andM. salivarium VV

A radiochemical method was developed for the assay of aspartate aminotransferase and alanine aminotransferase activities in Mollicutes. Using [1-C14]α-ketoglutarate as the amino group acceptor in transamination, we found that the fermentative speciesAcholeplasma laidlawii MG of the family of Acholeplasmataceae, the fermentativeMycoplasma pneumonia FH of the family of Mycoplasmataceae, and the nonfermentativeMycoplasma salivarium VV, also of the family of Mycoplasmataceae, all had aspartate aminotransferase and alanine aminotransferase activities. The radioactive product was identified as [1-C14]l-glutamic acid.Mycoplasma pneumoniae andM. salivarium had very low activity of alanine aminotransferase. Both aminotransferases had a partial requirement for pyridoxal 5′-phosphate and were strongly inhibited by 0.1 mM aminooxyacetate.

Simultaneous Assay of Aspartate Aminotransferase and Alanine Aminotransferase by Flow Injection Method Using Immobilized Enzyme Reactors

A simultaneous assay of two enzymes by a flow injection analytical (FIA) method using enzyme-immobilized open-tubular reactors in parallel is described. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were studied. This FIA system has an additional channel for evaluation and correction for the error resulting from pyruvate coexisting in sample sera. The results obtained by the FIA method corresponded well with those obtained by a conventional UV method. The recognition of the signal pattern from three channels of the FIA system will make possible the primary screening of diseases.

Evaluation of commercially formulated aspartate aminotransferase and alanine aminotransferase activity determinations by the scandinavian committee on enzymes and IFCC methods as modified for use with automated enzyme analysers

The performance characteristics of the Scandinavian Committee on Enzymes (SCE) methods for the assay of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using six automated enzyme analysers. The reagent formulation did not include pyridoxal phosphate (PLP). An optimal operating mode was defined for each instrument and precision was assessed in greater detail on four instruments. A points rating system was devised to place the instruments in the following order of proficiency: IL Multistat III, LKB-8600, Gilford 3500, ABA 100. In contrast to AST, the ALT activity of patient samples was unstable at -20 degrees C over periods as short as seven days. The performance characteristics of the IFCC methods for assay of AST and ALT activities were determined by using three automated enzyme analyzers in order to assess the effect of PLP upon precision and activity of four quality control sera, and to compare the SCE and IFCC methods. Precision of AST assays did not alter on omission of PLP from the IFCC formulation, while that of ALT assays showed slight deterioration. The decrease in activity on omitting PLP was variable with each instrument. A points-rating system was devised to place the methods in the following order of precision: AST: IFCC (-PLP) 118, IFCC 109, SCE 61; ALT: IFCC 125, IFCC (-PLP) 97, SCE 66. The IFCC methods offer better precision, and the overall change on omitting PLP is minimal.

The Diagnostic accuracy of three recommended methods for serum aspartate aminotransferase assays in patients suspected of myocardial infarction and hepatobiliary diseases

Serum aspartate aminotransferase (AST) activity was measured by the methods recommended by the Scandinavian Committee on Enzymes (SCE) and by the International Federation of Clinical Chemistry (IFCC) with pyridoxal phosphate (PLP) and without (-PLP) in one laboratory at 37 degrees C with the Abbott ABA-100 and in another at 30 degrees C with the IL Multistat III. Reference ranges were determined on 195 healthy hospital staff. Sera from 102 patients with suspected hepatobiliary disease (HBD) and 104 with suspected myocardial infarction (MI) were assayed at both laboratories by all three methods. Based on the above reference ranges, all assays with each method at both hospitals were abnormal in 59 of 67 cases with HBD and 53 of 55 with MI. In aggregate, all three methods yielded comparable rates of misclassification (20-23). The SCE method gave highest false negatives (18) and lowest false positives (5); the IFCC method gave lowest false negatives (1) and highest false positives (20); intermediate values of 8 false positives and 12 false negatives were given by the IFCC (-PLP) method. Using receiver operating characteristic (ROC) curves, the SCE method was clearly superior at 30 degrees C, and the IFCC (-PLP) method was marginally superior at 37 degrees C. However, when the decision threshold corresponded with a 2.5% false positive rate in the non-HBD, non-MI patients, the SCE method gave the lowest false negatives at both temperatures and, on the basis of the present data, must be considered to be the method of choice for AST activity determinations.

17] Glutamate-aspartate transaminase from microorganisms

Publisher Summary Various methods have been developed for the assay of aspartate aminotransferase. Most of these depend on the determination of oxalacetate or L-glutamate. Out of these, the Karmen method is used most widely, particularly in the field of clinical analysis, in which oxalacetate is determined with malate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. A new method introduced recently, determines 2-oxoglutarate produced in the reverse reaction is with 2-hydroxyglutarate dehydrogenase. A method for the determination of aspartate aminotransferase activity after electrophoretic separation of the isoenzymes has also been developed. The purification procedure consists of several steps: preparation of cell-free extract, ammonium sulfate fractionation, first Diethylaminoethyl (DEAE)-cellulose column chromatography, heat treatment, Sephacryl S-200 chromatography, second DEAE-cellulose chromatography, and hydroxyapatite chromatography.

Effects on analgesic and antirheumatic drugs on the assay of serum enzymes.

Interference by some commonly used analgesic and antirheumatic drugs in the spectrophotometric and colorimetric assays of serum enzymes was examined. None of the investigated methods was significantly influenced by caffeine, phenacetin, ibuprofen or indomethacin. Acetylsalicylic acid affected the continuous assays of creatine kinase and lactate dehydrogenase (with pyruvate as substrate), and the colorimetric assay of alanine aminotransferase. Aminophenazone interfered with the colorimetric method for determination of aspartate aminotransferase and alanine aminotransferase, while phenobarbital interfered only with the continuous method for lactate dehydrogenase (with L-lactate as substrate). Ketoprofen interfered with the colorimetric assays of lactate dehydrogenase and aspartate aminotransferase, while diclofenac affected the continuous assay of aspartate aminotransferase. None of the tested drugs interfered with the continuous methods for the determination of alkaline phosphatase and alpha-hydroxybutyrate dehydrogenase.

Preincubation of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the SMAC: comparison with revised DuPont aca method and recommended IFCC method.

The method for continuous-flow assay of aspartate aminotransferase with the Technicon SMAC was modified to include preincubation of the serum enzyme with pyridoxal 5'-phosphate, to be consistent with the recommendations of IFCC and the Standards Committee of AACC. Preliminary estimates of the imprecision of the modified method on SMAC gave day-to-day standard deviations of 5.3 U/L at mean of 48 U/L (n = 66) and 6.2 U/L at 155 U/L (n = 61). Added bilirubin, sodium pyruvate, ascorbic acid, and endogenous lipids did not interfere. Comparison of results for 50 samples by this method with those by the manual IFCC method gave y = 1.1113x - 0.3 U/L, Sy/x = 4.4 U/L, and r = 0.997. Similar data are presented for the revised AST method for the DuPont aca discrete analyzer. Clinical data show that AST activities increase by as much as 200% when the serum is preincubated with pyridoxal 5'-phosphate.

Improved serum-initiated aspartate aminotransferase assay by inhibition of lactate dehydrogenase with oxamate.

We describe a serum-inititated aspartate aminotransferase (EC 2.6.1.1) assay that obviates the need for added lactate dehydrogenase (EC 1.1.1.27) in the reagent system. Interference from the oxidation of NADH by endogeneous lactate dehydrogenase is eliminated by adding sodium oxamate, a competitive inhibitor of the enzyme. The advantages of oxamate inhibition over lactate dehydrogenase addition are a shorter preincubation period, an increase in the linear range from less than 600 to more than 1400 U/L, no interference from above-normal concentrations of ketoacids, and elimination of bias between serum-initiated and the standard oxoglutarate-initiated assays for aspartate aminotransferase.

Improved serum-initiated aspartate aminotransferase assay by inhibition of lactate dehydrogenase with oxamate.

Abstract We describe a serum-inititated aspartate aminotransferase (EC 2.6.1.1) assay that obviates the need for added lactate dehydrogenase (EC 1.1.1.27) in the reagent system. Interference from the oxidation of NADH by endogeneous lactate dehydrogenase is eliminated by adding sodium oxamate, a competitive inhibitor of the enzyme. The advantages of oxamate inhibition over lactate dehydrogenase addition are a shorter preincubation period, an increase in the linear range from less than 600 to more than 1400 U/L, no interference from above-normal concentrations of ketoacids, and elimination of bias between serum-initiated and the standard oxoglutarate-initiated assays for aspartate aminotransferase.

Improved serum initiated aspartate aminotransferase assay by inhibition of lactate dehydrogenase with oxamate

We describe a serum-inititated aspartate aminotransferase (EC 2.6.1.1) assay that obviates the need for added lactate dehydrogenase (EC 1.1.1.27) in the reagent system. Interference from the oxidation of NADH by endogeneous lactate dehydrogenase is eliminated by adding sodium oxamate, a competitive inhibitor of the enzyme. The advantages of oxamate inhibition over lactate dehydrogenase addition are a shorter preincubation period, an increase in the linear range from less than 600 to more than 1400 U/L, no interference from above-normal concentrations of ketoacids, and elimination of bias between serum-initiated and the standard oxoglutarate-initiated assays for aspartate aminotransferase.

Measurement of aspartate aminotransferase activity: effects of oxamate.

Oxamate, a potent inhibitor of lactate dehydrogenase, is shown also to inhibit aspartate aminotransferase activity, both in human serum and in purified isoenzymes of human origin. The inhibition was competitive with respect to 2-oxoglutarate for both isoenzymes. The apparent Ki was 29 mmol/L for the cytoplasmic enzyme and 17 mmol/L for the mitochondrial enzyme. Noncompetitive inhibition was found between oxamate and aspartate. At saturating concentrations of substrate (2-oxoglutarate greater than or equal to 15 mmol/L, L-aspartate greater than or equal 150 mmol/L) oxamate inhibited the mitochondrial enzyme but had less effect on the cytoplasmic isoenzyme. Oxamate at 40 mmol/L inhibited the enzyme in serum by 11 and 9% in assays containing 2-oxoglutarate at 6.7 and 15 mmol/L, respectively. This concentration of oxamate inhibited enzyme activity in serum by 5% more than did the same concentration of Cl- (itself an inhibitor). Oxamate (less than or equal to 30 mmol/L) had no measurable effect on the stability or activity of porcine malate dehydrogenase. Until the effects of its inhibitory properties are considered, addition of oxamate to suppress lactate dehydrogenase-mediated side reactions in the assay of aspartate aminotransferase cannot be recommended.

Determination of initial enzyme reaction rates from spectrophotometric recordings: A rapid method

The initial enzyme reaction rate on a recording spectrophotometer can be expressed as an angle. The tangent of this angle multiplied by a factor derived from the chart speed, chart width, absorbance range, and sample dilution will give the enzyme activity of the sample in mU/ml. The enzyme activity can be tabulated for all angles between 0° and 90° at 0.5° intervals and enzyme activity can be directly read from the table after measuring the value of the angle with a protractor. This technique has been applied to serum cholinesterase assays on the SP 800 recording spectrophotometer and to serum aspartate aminotransferase assays on the LKB 8600 reaction rate analyzer. The method has been shown to be simple, rapid, and more precise than the usual methods of reading these recordings. The sensitivity of the method is known (unlike the usual methods) and the relative error can be calculated. The accuracy of the method is similar to the other manual methods. This technique is quite general and it can be applied to all initial enzyme reaction rate determinations.