CREATINE KINASE ISOENZYMES IN SERUM. A. IN VITRO STUDIES WITH RAT CK-1 AND HUMAN SERUM. B. APPARENT MITOCHONDRIAL CREATINE KINASE IN THE SERUM OF A PATIENT WITH METASTATIC CANCER TO THE LIVER

摘要

The clinical usefulness of the measurement of cellular enzyme activity in blood or other extracellular fluids depends, in part, on the understanding of the interactions between the enzyme and the extracellular fluid. Creatine kinase (CK; E.C. 2.7.3.2) is a cellular enzyme associated with energy utilization and storage in contractible or excitable tissues. The soluble isoenzymes of CK, CK-2 and CK-3, have been studied extensively and are useful in the diagnosis and prognosis of diseases of the heart and skeletal muscle. Creatine kinase-1, mitochondrial CK (CKm) and abnormal forms of CK, as observed on electrophoretic separation of CK isoenzymes, are rare or inconsistent phenomena in the human population.;Part A. Creatine kinase-1 is present in high activities in brain and nervous tissue but is not consistently observed in the blood of patients with extensive brain damage. The high lability of CK-1 activity to heat, pH, and oxidizing reagents and the short half-life of CK-1 activity in blood when compared to CK-2 and CK-3 are data which requires a more thorough understanding if blood CK-1 is to be of clinical significance.;I have studied the interaction of CK-1, obtained from rat brain, and human serum and have defined some of the variables which must be controlled to maximize the recognition of CK-1 in human serum. The pH must be maintained between 7.0 and 7.5 to retain maximal activity and to retard the formation of CK-1', an electrophoretically identified form of CK-1 which migrates like CK-2. Cation chelators such as EDTA are detrimental to CK-1 activity during storage but prevented or reversed the formation of CK-1' from CK-1. Also, the presence of thiol reagents in the serum were detrimental to CK-1 activity during storage and prevented the formation of CK-1' from CK-1. The addition of Zn('2+) to the serum caused an increased formation of CK-1' from CK-1. Serum fractionation on Sephadex G-25 demonstrated that the serum factor(s) responsible for the formation of CK-1' from CK-1 were eluted in the protein fractions of the serum, as did Zn and Cu.;These studies demonstrated the need to determine the optimal handling and storage conditions for serum samples which may contain CK-1. The formation of CK-1' must also be considered as a possible in vivo phenomena and assay methods for CK-1 must be evaluated to determine the assay specificity to this form of the enzyme.;Part B. Creatine kinase migrating as two bands cathodal to the origin and to CK-3 on electrophoresis was observed in the serum of a patient with metastatic cancer to the liver. The more cathodal isoenzyme (CKm-2) was of high relative molecular mass, was precipitated by ammonium sulfate at 30% of saturation, and was not retarded by Sephadex G-100. Treatment with urea to a concentration of 6 mol/L caused CKm-2 to elute with proteins of a lower molecular mass on a G-100 column and shifted the electrophoretic migration to a position just cathodal to the origin (CKm-1). Antibodies to CK-1 and CK-3 did not inhibit the activity of either CKm-1 or CKm-2. Similarities between these cathodal bands of CK activity and mitochondrial CK suggest the mitochondrial origin of these isoenzymes. Different commercial reagent systems differed in the estimation of CK activity when CKm-1 and CKm-2 were present in the sample.