Isoenzymes Dr. Vidya.D Asst. Professor, College of Pharmacy, PHL – 213 Biochemistry - I Isoenzymes Dr. Vidya.D Asst. Professor, College of Pharmacy, Prince Sattam Bin Abdul Aziz University, Kingdom of Saudi Arabia

Isoenzymes Isoenzymes or isozymes are multiple forms of same enzyme that catalyze the same chemical reaction Different chemical and physical properties: Electrophoretic mobility Kinetic properties Amino acid sequence Amino acid composition

Lactate Dehydrogenase Lactate dehydrogenase (LDH) is an enzyme present in a wide variety of organisms EC 1 = oxidoreductase. EC 1.1 = acting on the CH-OH group of the donor. EC 1.1.1 = With NAD or NADP as acceptor. EC 1.1.1.27 = L-lactate dehydrogenase. Molecular weight- 32 kD & it is tetramer M (A) -muscle –chromosome 11(basic) H (B) -heart – chromosome 12(acidic)

Lactate dehydrogenase, reversibly converts lactate to pyruvate, in different tissues. LDH consists of 5 iso-enzymes – LDH1,LDH2,LDH3,LDH4 & LDH5 These isoenzymes are separated by cellulose acetate electrophoresis at pH 8.6 Normal values: Serum -100 -200 U/L CSF - 7 -30 U/L Urine - 40 -100 U/L

LDH reaction

LDH isoforms

LDH isoforms Isoenzyme name Composition Electrophoretic migration Present in Elevated in LDH 1 Heat resistant ( H4) Fastest moving Myocardium, RBC, kidney myocardial infarction LDH2 (H3M1) Kidney disease, megaloblastic anemia LDH3 (H2M2) brain Leukemia, malignancy LDH4 Heat labile (H1M3) Lung, spleen Pulmonary infarction LDH5 Inhibited by urea (M4) Slowest moving Skeletal muscle, Liver Skeletal muscle and liver diseases

Clinical significance of LDH In normal serum, LDH2 (H3M) predominant isoenzyme & LDH5 is rarely seen. In myocardial infarction, LDH1(H4) levels are greater than LDH2. Megaloblastic anemia (50 times upper limit of LDH 1 and LDH 2) Muscular dystrophy, LDH5 (M4) is increased. Toxic hepatitis with jaundice (10 times more LDH5)

Renal disease- tubular necrosis or pyelonephritis, pulmonary embolism LDH 3 (massive destruction of platelets) Total LDH is increased in neoplastic diseases. LDH5 is increased in breast cancer, malignancies of CNS, prostatic carcinoma. In leukaemias, LDH2 & LDH3 levels are increased. In malignant tumors of testis & ovaries, LDH2, LDH3 & LDH 5 levels are increased.

In CSF: Bacterial meningitis – LDH4 and LDH5 Viral meningitis - LDH1 Metastatic tumors - LDH5 Neonatal cases of intracranial haemorrhage associated with seizures and hydrocephalus

Creatinine phosphokinase (CPK) It catalyses creatine to creatine phosphate. Normal serum value: 15-100 U/L for males & 10-80 U/L for females. CPK consists of 3 isoenzymes. Each isoenzyme of CK is a dimer; Molecular weight of 40 kD. The subunits are called B for brain (chromosome -14) & M for muscle (chromosome -19)

Creatine Phosphokinase (CPK) It is an important enzyme in energy metabolism. Immediate source of ATP in contracting muscle.

Three Iso-enzymes are separated by electrophoresis. CPK-1 (also called CPK-BB) is found mostly in the brain & lungs. CPK-2 (also called CPK-MB) is found mostly in the heart. CPK-3 (also called CPK-MM) is found mostly in skeletal muscle.

Creatine phosphokinase isoenzymes SUB-UNIT TISSUE % IN SERUM CK1 Fast moving BB Brain 1 CK2 2% of total MB Heart 5 CK3 Slow moving MM Skeletal muscle 80

Clinical significance of CK CPK & heart attack: CPK2 isoenzymes is very small, (2% of total CPK activity) & undetectable in plasma. In myocardial infarction (MI), CPK2 levels are increased within 4 hrs, then falls rapidly. Total CPK level is elevated upto 20-folds in MI.

CPK & Muscle diseases CPK level is elevated in muscular dystrophy (500- 1500U/L) CPK level is highly elevated in crush injury, fracture & acute cerebrovascular accidents. Estimation of total CPK is employed in muscular dystrophies & CPK-MB isoenzyme is estimated in myocardial infarction.

Atypical forms of CK Two atypical isoforms. Macro-CK (CK-macro) Formation: Formed by aggregation CK-MB with IgG, sometimes IgA. Also formed by complexing CK-MM with lipoproteins. Electrophoretically migrates between CK-MB & CK-MM. Occurs frequently in women above 50 years.

CK-Mi (Mitochondrial CK-Isoenzyme) Formation: It is present bound to the exterior surface of inner mitochondrial membrane of muscle, liver & brain. It exist in dimeric form & oligomeric aggregates & molecular weight 35,000 Electrophoretically, migrates towards cathode & is behind CK-MM band. It is not present in normal serum.

Clinical significance It is present in serum when there is extensive tissue damage causing breakdown of mitochondrial & cell wall. Its presence in serum indicates cellular damage, seen in malignancies.

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