We have described a case of Lactate dehydrogenase (LDH) linked to IgG (kappa) immunoglobulin which LDH isoenzyme pattern shows as if six bands are present. Of these bands, one additional is located at slow gamma region and such an anomalous appearance has not previously reported. The serum contains monoclonal immunoglobulin G (kappa) of which electrophoretic mobility is at slow gamma region (same as for abnormal LDH).From reconstitution experiments, we concluded that all five LDH isoenzymes could bind to IgG prepared from the patient and those mobilities of the linked LDHs changed to cathodic side; LDH1, tailed to cathodic side; LDH2, moved to cathodic side; LDH3, located at slow LDH4; LDH4 located at fast LDH5; LDH5, located it slow gamma region; respectively. The affinity constant of each LDH isoenzyme was estimated and the values obtained from LDH1 to 5 were 0.026, 0.079, 0.166, 3.715, and 0.218. (Keg×109 liters/mol) was respectively. LDH4 evoked the highest affinity, it suggests that the site of antigen recognition of LDH linked to IgG was not associated with the structure of individual LDH-H and LDH-M subunits.
The combination of both the electrophoresis and the immunofixation and of the thin layer gel filtration and the immunofixation followed by enzyme activity staining on antigen-antibody complexes were applied to detect and identify the enzyme-linked immunoglobulins which were observed as macromolecular enzyme complexes in human sera. For any given antigen-antibody system, the optimum concentration of antigen in the serum to be analysed must be given experimentally. Moreover, removing of unreacted proteins must be carefully performed with the solution which will protect the activity of the enzyme to be detected.In the cases of LDH- and amylase-linked immunoglobulins the detection of minimum enzyme activities on Cellogel membrane were limited by staining sensitivity. Thus, two to ten times concentration of commercially available antibodies was required to give the optimum concentration of antigen-antibody complexes. In the cases of alkaline-phosphatase-linked immunoglobulins, the detection of the enzyme activity was easily amplified by the elongation of the incubation period. Thus, the optimum concentration of antigen was given without concentration of commercial antibodies.In the studies of macromolecular enzyme complexes in human sera, the immunoprecipitation technique and enzymo-immunoelectrophoresis were conventionally carried out to identify the enzyme-linked immunoglobulins. Some cases, however, still ramain ambiguous because of unskilled and unstable techniques used for analysing these macromolecular enzyme complexes. Thus, these immunofixation techniques followed by enzyme activity staining (enzyme immunofixation), described in this paper can facilitate the detection and identification of such macromolecular complexes as enzyme-linked immunoglobulins.
The usefulness of monitoring bivariates was studied and discussed. Analysis of the correlation between bivariates demonstrated that combinations of bivariates can be divided into two groups. The first group was the combination in which a close correlation between two variates could be observed in any pathophysiologic state. The monitoring of the ratio of this first group was effective for the detection of analytical mistakes and the detection of symptom poor hereditary disorders, such as pharmacogenetical disorders. The second group was one in which a high correlation between two variates was obtained in some specific pathologic stage, and the monitoring the ratio of this second group gave high predictive values for the screening of that pathologic stage. It was demonstrated that the use of bivariate monitoring was not only very simple but also provided an effective laboratory data analysis system for the laboratory routine.
Lactate Dehydrogenase(LDH) M subunit deficiency was first discovered by urinary discoloration and discrepancy in laboratory data. The response to ischemic forearm work is characteristic(absence of an increased venous lactate concentration after ischemic work and a marked increase in venous pyruvate are found). The increase of pyruvate concentration is specific to LDH-M subunit deficiency. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase(GAPDH) step, possibly due to the impaired reoxidation of NADH produced by GAPDH activity. Then the excessive NADH is reoxidized by alpha-glycerophosphate and glycerol. Therefore, ATP production is significantly impaired and muscle tissue is damaged. Molecular analysis revealed a detection of 20 base-pairs in exon 6 in LDH-M subunit deficiency. This mutation results in a frame-shift translation and premature termination.
