Isoenzymes are multiple forms of enzymes that arise from genetically determined differences in primary structure. Isoforms arise from post-translational modifications. Lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), and acid phosphatase (ACP) are clinically important enzymes that exist as isoenzymes. LDH isoenzymes can indicate tissue damage like myocardial infarction. CK isoenzymes are measured to detect heart attacks. ALP isoenzymes are elevated in bone and liver diseases. Isoenzyme patterns are determined through properties like electrophoretic mobility, heat stability, and inhibitor response.

1. Isoenzymes : Diagnostic
Methods and Importance
Dr. RAJEEV RANJAN
Resident, Dept. of Lab. Medicine
AIIMS, New Delhi
17th July, 2019
1

2. Overview
• What are Isoenzymes
• Difference between Isoenzymes & Isoforms
• Clinically Important Isoenzymes :
oLactate dehydrogenase (LDH)
oCreatine kinase (CK)
oAlkaline phosphatase (ALP)
oAcid Phosphatase (ACP)
oAmylase
oLipase
2

3. The term "multiple forms of the enzyme . . ." should be used as a broad term
covering all proteins catalyzing the same reaction and occurring naturally in a single
species.
The term "isoenzyme" or "isozyme" should apply only to those multiple forms of
enzymes arising from genetically determined differences in primary structure and not
to those derived by modification of the same primary sequence .
Isoforms are highly related gene products which differ due to post translation
modification but perform essentially the same biological function.
Isoenzymes & Isoforms
IUPAC-IUB Commission on Biochemical Nomenclature (CBN)
Nomenclature of Multiple Forms of Enzymes, Recommendations 1976

4. Difference between Isoenzymes and Isoforms
Isoenzymes Isoforms
 Catalyze the same reaction
 Have different genetic loci
 Different from each other
o Location-wise,
o Electrophoretically,
o Immunologically,
o Physically, and
o Biochemically.
 Catalyze the same reaction
 Have same genetic loci
 Have different post translational modifications
 May be different or same

5. Properties used to identify Isoenzymes
S.No Property Example
1. Electrophoretic mobility Isoenzymes of Lactate dehydrogenase have different electrophoretic mobility
2. Heat stability Alkaline phosphatase isoenzymes are either heat labile or stable
3. Inhibitor An inhibitor can inhibit only one isoenzyme of an enzyme eg. Acid phosphatase
4. Km Glucokinase and hexokinase
5. Cofactors Mitochondrial isocitrate dehydrogenase requires NAD+ , cytosolic form requires NADP+
6. Tissue localisation LDH 1 is present in heart, LDH 5 in muscle
7. Antibodies For creatine kinase, each isoenzyme can be bound only by a specific antibody
5

6. Lactate Dehydrogenase
Lactate dehydrogenase (LDH) (EC 1.1.1.27) is an enzyme present in a wide variety
of organisms
Molecular weight- 134 kDa & it is a tetramer
o M (A) - muscle – chromosome 11(basic)
o H (B) - heart – chromosome 12(acidic)
Lactate dehydrogenase, reversibly converts lactate to pyruvate, in different tissues.
LDH consists of 5 isoenzymes – LDH1, LDH2, LDH3, LDH4 & LDH5
These isoenzymes are separated by cellulose acetate electrophoresis at pH 8.6
Normal values:
o Serum -100 -200 U/L
o CSF - 7 -30 U/L
o Urine - 40 -100 U/L
6

7. Enzymes Classification
Class Designation Function
EC 1 Oxidoreductase Catalyze oxidation/reduction reactions
EC 2 Transferases Transfer a functional group
(e.g : a methyl or phosphate group)
EC 3 Hydrolases Catalyze the hydrolysis of various bonds
EC 4 Lyases Cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases Catalyze isomerization changes within a single molecule
EC 6 Ligases Join two molecules covalent bonds
7

8. For Example : Lactate dehydrogenase
8

9. Isoenzyme of LDH
9

10. Electrophoretic Separation of LDH Isoenzymes
10

11. LDH Isoforms
No. of
Isoenzyme
Subunit
make up of
isoenzyme
Electrophoretic
mobility at
pH8.6
Activity at
60° for 30
minutes
Tissue origin Elevated In Percentage in
Human Serum
(Mean)
LDH 1
Heat Resistant
( H4) Fastest Not
destroyed
Myocardium,
RBC, Kidney
Myocardial Infarction
(MI)
30%
(LDH1 - 20-34%)
LDH 2
Heat Resistant
(H3M1) Faster Not
destroyed
Myocardium,
RBC, Kidney
Kidney disease,
Megaloblastic anemia
35%
(LDH2 - 28-41%)
LDH 3 (H2M2) Fast Partly
destroyed
Brain Leukemia, Malignancy 20%
(LDH3 – 15-25%)
LDH 4
Heat Labile
(H1M3) Slow Destroyed Lung, Spleen Pulmonary Infarction 10%
(LDH4 – 8-16%)
LDH 5
Heat Labile
Inhibited by Urea
(M4) Slowest Destroyed Skeletal
muscle, Liver
Skeletal Muscle and
Liver diseases
5%
(LDH5 – 6-15%)
11

12. LDH isoforms
 An example of two duplicated genes becoming specialized to different tissues.
 Isozymes are also differentially expressed in different developmental stages.
o Before birth the heart is more anaerobic compared with adulthood.
o Indeed, before birth the main isozyme in the heart is the M4, and with time it switches to HM3 (at
birth), to H2M2 and HM3 at 1 year after birth, and to H3M and H4 after 2 years.
 Normally LDH - 2(H3M1) level in blood is greater than LDH -1, but this pattern is
reversed in myocardial infarction, this is called ‘ flipped pattern ’.
 Atypical forms of LDH
o6th isoenzyme LDH – X
o7th isoenzyme LDH – 6
12

13. Clinical Significance of LDH Isoenzymes
Condition Types of LDH isoenzymes increased
Normal serum LDH2 (H3M) is predominant isoenzyme & LDH5 is rarely seen
Myocardial infarction LDH1(H4) > LDH2
Megaloblastic anemia (50 times upper limit of LDH1 & LDH2)
Muscular dystrophy LDH5 (M4) is increased.
Toxic Hepatitis with Jaundice (10 times more LDH5)
Renal disease - Tubular necrosis or pyelonephritis LDH3
Pulmonary embolism LDH3 (massive destruction pf platelets)
Neoplastic diseases Total LDH is increased
Breast cancer, malignancies of CNS, prostatic carcinoma LDH5 is increased
Leukaemias LDH2 & LDH3 levels are increased
Malignant tumors of Testis & Ovaries LDH2, LDH3 & LDH 5 levels are increased
13

14. Clinical Significance of LDH isoenzymes in CSF
 Bacterial meningitis – LDH4 and LDH5
 Viral meningitis - LDH1
 Metastatic tumors - LDH5
14

15. Reaction catalyzed by LDH
15
Reaction is reversible & uses NAD+ as a coenzyme

16. Methods of Estimation of LDH
 IFCC Liquid method/ or optional DGKC method.
 Electrophoretic separation is the only procedure commercially
available to demonstrate LDH isoenzymes.
16

17. Creatine Kinase (CK)
• It catalyses creatine to creatine phosphate
• EC 2.7.3.2
• Adenosine triphosphate : creatine N-phosphotransferase
• Dimeric enzyme (82 kDa)
• Normal serum value:
o 4 - 60 IU/L
• Enzyme unstable in serum
• CK consists of 3 isoenzymes
• Activity lost due to sulfhydryl group oxidation at active site
• Dimer (each of 41000 Da) subunits :
o B (brain) – chromosome 14
o M (muscle) – chromosome 19
17

18. Creatine Kinase
• It is an important enzyme in energy metabolism.
• Immediate source of ATP in contracting muscle.
• 3 isoenzymes are separated by electrophoresis.
• All 3 isoenzymes in cytosol
18
Isoenzyme Tissue of Origin Elevated in Mean Percentage
in Blood
Electrophoretic
Mobility
CK-1 CK-BB Brain, Prostate, GI tract, Lung, Bladder,
Uterus, Placenta
CNS
diseases
0% Maximum
(Fast moving)
CK-2 CK-MB Myocardium/ Heart Acute MI 0-3% Intermediate
CK-3 CK-MM Skeletal Muscle, Myocardium 97-100% Least
(Slow moving)

19. Concentrations of Tissue CK Activity
Tissue Isoenzymes, (%)
Relative CK Activity CK-BB CK-MB CK-MM
Skeletal muscle (Type I, Slow twitch, or Red fibres) 50,000 < 1 3 97
Skeletal muscle (Type II, Flow twitch, or White fibres) 50,000 < 1 1 99
Heart 10,000 < 1 22 78
Brain 5.000 100 0 0
Smooth muscle 5,000 100 0 0
Gastrointestinal tract 5,000 96 1 3
Urinary Bladder 4,000 92 6 2
19
Table : Approx. Concentrations of Tissue Creatine Kinase Activity (Expressed as Multiples of CK Activity Concentrations in Serum) and Cytoplasmic Isoenzyme Composition

20. Electrophoretic Separation of CK Isoenzymes
20

21. Reaction catalyzed by CK
21

22. Clinical Significance of CK
• CK & Heart attack :
oCK2 isoenzymes is very small, (2% of total CK activity) & undetectable in plasma.
oIn myocardial infarction (MI), CK2 levels are increased within 4 hrs, then falls rapidly.
oTotal CK level is elevated upto 20-folds in MI.
• CK 1 elevated :
oVery Low Birth Weight (VLBW) Newborns
oBrain damage in neonates
oNeurological injury
• CSF :
o>200 U/L – Death
o100 – 200 U/L – Survive with Neurological deficits
o<100 U/L – Good chance of recovery
22

23. Elevated CK
• Elevated CK 1 :
oAdenocarcinomas of GI tract
oCarcinoma lung
oCa prostate, bladder, testes, kidneys, breast, ovaries, uterus, CNS, Leukemia, Lymphoma
and sarcoma
• Elevated CK 2 :
oMyocardial infarction
oHead injuries
oSubarachnoid haemorrhage
oExercise
• Elevated CK 3 :
oMuscular dystrophies (DMD- 10000 IU/L)
oMyopathies
oHypothyroidism (5 fold more than normal value, CK 2 is also elevated)
23

24. Clinically Significant Enzymes in MI
24

25. Atypical forms of CK
• Fourth form - CK-Mt (chromosome 15)
oSevere illness
oMalignant tumors
• Macro-CK
oType 1- CK BB complexed with IgG
oType 2- Oligomeric CK-Mt
25

26. Methods of Estimation of CK
 IFCC Liquid method @ 370 C
 CK catalyzes the conversion of CrP to Cr with concomitant phosphorylation of ADP to ATP.
 The ATP produced is measured by Hexokinase (HK)/glucose-6-phosphate dehydrogenase
(G6PD) coupled reactions that ultimately convert NADP+ to NADPH (reduced form of NADP)
 Monitored Spectrophotometrically at 340 nm
 The assay is optimized by adding :
o N-acetylcysteine to activate CK
o EDTA to bind Ca2+ and increase the stability of the rkn mixture
o Adenosine pentaphosphate (Ap5A) in addition to AMP to inhibit adenylate kinase (AK)
26

27. Alkaline Phosphatase (ALP)
• EC 3.1.3.1
• Catalyzes alkaline hydrolysis of phosphate monoesters
• Optimum activity occurs at pH 9-10
• Membrane bound glycoproteins
• Zinc containing metalloenzyme
• Requires Mg++ for activity
• Various isoenzymes and isoforms.
27

28. Reaction catalyzed by ALP
28

29. Biological Functions of ALP
• Bone : calcification and mineralization process
• Intestine : lipid transport
29

30. Encoded by at least four different genes-
1. Placental
2. Germ cell
3. Intestinal
4. Tissue non-specific
Post translational modifications
a. Liver
b. Kidney
c. Bone
ISOENZYMES
ISOFORMS
30
Isoenzymes and Isoforms of ALP

31. Different isoenzymes of ALP
31
Fig : Main Physiological and Pathological expression of genes encoding Human Alkaline Phosphatase
(Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics)

32. Source of ALP in Plasma and Urine
 In Plasma:
oLiver
oBone
oIntestine (very little)
oKidney (negligible)
oPlacenta (during pregnancy)
 In Urine:
oRenal tissue (not from Plasma)
32

33. Clinical Significance of ALP:
 Normal serum levels:
o Children – 180 – 1200 U/l
o Adults – Male – 100 – 270 U/l
Female – 100 – 240 U/l
 Increased levels:
Physiological
o Growing children - 1.5 - 3 times
o Pregnancy - 2 - 3 times
Pathological
o Liver diseases
o Cholestasis
o Extra hepatic obstruction - 10-12 times
o Intra hepatic obstruction - up to 3 times
o Parenchymal liver disease - moderate rise (<3 times normal)
33

34.  Bone Disease
o Paget’s disease
o (10-25 times)
o Osteogenic sarcoma
o Rickets
o Osteomalacia
34

35. Clinical Significance
• Hepatobiliary disease
• Hepatic carcinoma
• Hepatic metastases
• Paget’s disease (10 – 25 times)
• Bone cancer
• Healing of bone fracture
• Osteomalacia and rickets
• Hyperparathyroidism
• Ca of Ovary, Uterus - Regan isoenzyme
• Metastatic Ca of pleural surfaces – Nagao isoenzyme
35

36. Isoenzyme & Isoform Separation
1. Electrophoretic mobility :
36

37. 2. Heat stability: Placental form is most resistant.
o Placenta > liver > bone
3. Stability for denaturing agents:
oResistance to Urea : Placenta >liver>bone
4. Response to inhibitors:
o L-Phenylalanine — specially inhibit intestinal type and have little effect on
bone and liver enzyme
5. Immunological techniques:
o Monoclonal antibodies for tissue specific ALP
37

38. Methods for Estimation of ALP
 Optimized Determination of ALP in body fluids (DGKC Method)
 DGKC – (Deutsche Gesellschaft Fur Klinische Cheime)
 Principle :
38
p-Nitro-phenylphosphate + H2O AP p-Nitrophenolate + Phosphate

39. Acid Phosphatase (ACP)
• EC 3.1.3.2
• Include all phosphatases with optimal activity below a pH -7.0
(optimum pH for activity 5-6)
• Major sources:
oProstate (richest)
oLysosomes
oRBCs, WBCs, platelets,
oLiver, spleen
oBone (osteoclasts)
39

40. Inhibition by dextro rotatory tartrate ions
⁺ ⁻
Lysosomal Bone
Prostatic Erythrocytes
Leucocytes
40
Tartrate resistant acid phosphatase
(TRAP)
 4 ACP determining genes identified :
o Erythrocyte ACP gene – Ch-2
o TR-ACP (Osteoclasts & Other tissue macrophages – Alveolar & Kupffer cells, Type 5 ACP) – Ch-19
o TR-ACP 5a (Tartrate-inhibited Lysosomal ACPs) – Ch-11
o TR-ACP 5b (Tartrate-inhibited Prostatic ACPs) – Ch-13

41. Clinical Significance of ACP
 Normal Values: 1.8-8.8 IU
 Elevated ACP seen in-
o Carcinoma prostate
 Increased osteoclast activity
o Paget’s disease
o Hyperparathyroidism
o Osteoclastoma
 Gaucher’s Disease (lysosomal storage disorder)
 Hairy cell leukemia
41

42. Method of Estimation of Acid Phosphatase
Immunoassays for serum TR-ACP have been developed that preferentially detect isoform 5b.
One Method uses a monoclonal antibody to bind serum TR-ACP in a solid phase format.
o After the capture of, osteoclastic enzyme (Type 5B) is specifically determined by measuring its activity at
optimal pH 6.1.
Another assay uses 2 monoclonal antibodies generated against purified bone TR-ACP 5b.
o One of the antibodies captures active intact isoform, while the 2nd eliminates interference of inactive 5b
fragments in serum.
After the immunoreaction, activity of bound TR-ACP 5b is measured.
42

43. Amylase
• EC 3.2.1.1
• Molecular weight - 54 -62 kDa
• 1,4 α-D Glucan glucanohydrolase
• Acts only at α 1-4 glycosidic bonds
• Calcium metalloenzyme, with calcium essential for functional
integrity and are activated by anions such as Chloride & Bromide
• From salivary gland and pancreas
• Enzymes are products of 2 closely linked loci on chromosome 1
• Optimum pH - 6.9 to 7.0

44. Hyperamylasaemia
Pancreatic disease
o Acute Pancreatitis
o Chronic ductal obstruction
o Pancreatic trauma
o Pancreatic carcinoma
Non – Pancreatic abdominal conditions
o Biliary tract disease
o Perforated peptic ulcer
o Intestinal obstruction
o Peritonitis
o Ruptured ectopic pregnancy
Salivary gland disorders
o Mumps
o Parotitis
o Calculi
Tumor Hyperamylasaemia
o Lung carcinoma
o Oesophageal carcinoma
o Breast carcinoma
o Ovarian carcinoma
Renal insufficiency

45. Clinical Significance of Amylase Estimation
• Used as a screening test for acute pancreatitis
• Highly sensitive marker but not very specific
• Rise in serum level is rapid but transient
• Diagnosis is confirmed by serum lipase

46. Sources of Amylase
• Two isoenzymes:
o P type (Pancreatic)
oS type (Non-Pancreatic)
• Sources of amylase:
o Major sources : Pancreas, Salivary gland
oMinor sources: Small Intestine, Testes, Ovaries, Fallopian Tubes, Striated Muscle,
Lungs, Adipose tissue
• Only plasma enzyme normally present in urine (Mol wt. - 54 to 62 kDa)

47. Methods of Estimation of Amylase
 Enzymatic colorimetric test according to IFCC method.
 Test with 4,6-Ethylidene-(G7)-1,4-nitrophenyl-(G1)- 𝜶,D-maltoheptaoside
as substrate.
 Expected Value :
o Serum/plasma – 28 – 100 U/l
o 𝜶-Amylase/Creatinine quotient - ≤ 310 U/g
o Spontaneous Urine - ≤ 460 U/l
47

48. Reference IFCC Method

49. Lipase
EC 3.1.1.3
Molecular weight - 48 kDa
LPS gene resides on chromosome - 10
Hydrolyses glycerol esters of long chain fatty acids
Origin - Pancreas, Intestinal and Gastric mucosa
Serum Level – 0-160 U/L
Serum from Acute Pancreatitis patient have 3 enzymes : (Lott JA et al ,1991)
o L1, L2 are pancreatic isoenzymes of Lipase (EC 3.1.1.3)
o L3 is probably pancreatic carboxyl ester Lipase (EC 3.1.1.13)
Methods of Estimation – Enzymatic colorimetric test
49