Immunochemical quantitation of isoenzymes of aspartate aminotransferase and lactate dehydrogenase

The applicability of immunochemical techniques to the determination of aspartate aminotransferase (AspAT, EC 2.6.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) isoenzymes in human serum are reviewed. In the case of AspAT, the human enzymes of mitochondrial (m-AspAT) and cytosolic (s-AspAT) origin were purified to homogeneity from liver and erythrocytes respectively and used to prepare isoenzyme-specific anti-sera in rabbits. Immunoprecipitation and immunoinhibition assays using partially purified antibodies or monovalent Fab fragments were found to provide better accuracy and precision than column chromatographic, electrophoretic, or differential kinetic techniques. A variety of immunochemical techniques were examined for the determination of enzyme protein including radioimmunoassay, turbidimetric procedures, and an assay using the indium slide technique. In the last, purified isoenzyme was absorbed as a monolayer to the surface of an indium metal film upon glass. The enzyme retains immunological reactivity, allowing the specific binding of antibody at the surface. The minimum detectable concentration by this technique is greater than 50 micrograms/L of enzyme protein; results suggest that normal and patient sera contain considerably more immunologically reactive s- and m-AspAT than catalytically active enzyme.     

Reversible loss of lactate dehydrogenase isoenzymes and lactate dehydrogenase activity in patient's serum

An abnormal lactate dehydrogenase (LD; EC 1.1.1.27) electrophoretogram (only one band, at the application site) and a low LD activity (7 U/L) was seen for a patient's serum during storage at 22 and 4 degrees C. Both reverted to normal when the serum was incubated at 37 degrees C.     

Effect of reaction initiator on human lactate dehydrogenase assay.

Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.     

Radioisotopic assay of aspartate and alanine aminotransferase.

The activities of aspartate and alanine aminotransferases in biological samples were assessed through a novel and sensitive procedure, based on the conversion of [U-14C]2-ketoglutarate to L-[U-14C]glutamate. In human plasma, the generation of L-[U-14C]glutamate was proportional to the volume of plasma (20-60 microL) and to the length of incubation (30-90 min). The reaction velocity was related to the temperature with a Q10 close to 1.7 for aspartate aminotransferase and 2.0 for alanine aminotransferase. At 37 degrees C, the 95% confidence interval in healthy subjects ranged from 5.1-18.8 U/mL (mean value 11.9 U/L) for aspartate aminotransferase and from zero to 20.1 U/L (mean value 9.9 U/L) for alanine aminotransferase. The intra-assay coefficient of variation did not exceed 2.5%. The present method was also applied to homogenates prepared from rat pancreatic islets, liver, heart, parotid glands, and erythrocytes, using no more than 40 micrograms wet weight of tissue per sample, and could thus be used in small biological samples, such as those obtained by needle biopsy.     

High-pressure liquid-chromatographic separation of lactate dehydrogenase isoenzymes.

The isoenzymes of lactate dehydrogenase (EC 1.1.1.27) were separated with excellent resolution in less than 1 h by high-pressure anion-exchange chromatography. These isoenzymes were determined in some tissue extracts and high-activity serum samples by this technique.     

Use of purified lyophilized human lactate dehydrogenase isoenzymes in a study of the measurement of lactate dehydrogenase activity

We examined the stability of human lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes 1, 2, and 3--purified to specific activities of about 200 kU/g--when lyophilized in a buffered stabilized matrix of bovine albumin. Each isoenzyme was prepared at two activity concentrations and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. LD-1 activity decayed with zero-order kinetics, LD-2 and LD-3 with first-order kinetics. The extrapolated half-lives of these preparations at -20 degrees C varied between 80 and 530 years. Stability of reconstituted samples stored at 4 degrees C was excellent for LD-1 but poor for LD-2 and LD-3. We suggest that preparations of human LD-1 be further investigated as a possible reference material.     

Studies on alkaline phosphatase isoenzymes. Relation to gamma-glutamyltransferase and lactate dehydrogenase isoenzymes.

Gamma-Glutamyltransferase (GT) and isoenzymes of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) have been studied in 282 cases with increased S-ALP and in 18 chronic alcoholics with normal routine liver tests. There was a high degree of correlation between S-GT and the bile (alpha 1) and liver (alpha 2) fractions of S-ALP. Fractionation of alkaline phosphatases sometimes yielded clinical information, which could not be obtained by determinations of S-ALP and S-GT only. The presence of alpha 1-ALP and increased S-GT appeared to be more sensitive indicators of ethanol-induced liver involvement than other liver tests, including LDH-5/LDH-4 ratios.     

Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay

The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.     

A transient variant of serum lactate dehydrogenase isoenzymes.

An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.     

Automated separation and measurement of lactate dehydrogenase isoenzymes.

We describe a totally automated, computer-controlled system for separating and measuring the activity of lactate dehydrogenase (EC 1.1.1.27) isoenzymes. These isoenzymes in tissue extracts were separated on disposable DEAE-cellulose mini-columns. Resolution was complete except that LD-1 was not resolved from LD-2.     

Improved alanine aminotransferase assay using the microtiter plate

New procedures and new devices have been developed to improve the alanine aminotransferase (ALT) assay by using microtiter plates with 96 wells. Sera for ALT determination and reagents are distributed with a computer-controlled multiple syringe for simultaneous filling of the 96 wells. The volumes simultaneously distributed by this new multiple syringe show very little variation. The coefficient of variation (CV) is 0.6 percent (n = 96). Repeated filling of the wells with this syringe results in good reproducibility of distributed volumes (CV = 0.5%, n = 10). The temperature of sera, reagents, and mixtures, even during photometry, is actively regulated (down or up) to 25 degrees C in special chambers. Thus, the temperature varies minimally +/- 0.05 degrees C. ALT values in the low normal range as well as the high pathologic range show little variation (CV less than 2.3%) in aliquots of the same sample simultaneously or subsequently examined by the new procedure and with new devices. There is good correlation between the improved method using microtiter plates and the conventional methods (r greater than 0.9900, ALT values from 1-154 U/L). False-low and false-high values are excluded by computerized evaluation of many single determinations during the kinetic reactions in each sample. The new method is precise as well as simple, saves time, and may become important for the determination of other serum constituents on the basis of kinetics.