1. Clinical significance & Methods
Enzymes
Clinical significance & Methods

2. Measurements of enzymes
in serum
within a tissue
enzymes as markers of disease,
Injury to tissue releases cellular substances that can be used as plasma markers of tissue damage.
enzyme release is highly specific for cell death in some settings.

3. Some enzymes are predominantly found in the specialized tissue
while others, more widely distributed, have tissue specific isoenzymes or isoforms
The timing of the enzyme's diagnostic window
early indicators
late indicators
Several enzymes have diagnostic utility

4. Overlap occurs for some enzymes
may be used for investigating disease in several organs.

5. Enzymes of clinical interest MUSCLE ENZYMES
More commonly measured
CK, LD
CK; adenosine triphosphate: creatine N-phosphotransferase

7. Inhibitors
excess Mg2+
Many metal ions, such as Mn2+ , Ca2+ , Zn2+ ,Cu2+
sulfhydryl-binding reagents
Iodoacetate

8. The distribution of these isoenzymes in the various tissues
CK is a dimer (B and M)
the products of loci on chromosomes 14 and 19, respectively.
BB (or CK-1), MB (or CK-2), and MM (or CK-3).
numbered on the basis of their electrophoretic mobility, with the most anodal form receiving the lowest number.
The distribution of these isoenzymes in the various tissues

10. Serum CK activity is subject to a number of physiological variations.
Sex, age, muscle mass, physical activity, and race all interact to affect serum activities

11. Clinical Significance
Serum CK activity is greatly elevated in all types of muscular dystrophy.
may be increased long before the disease is clinically apparent.
Serum CK activity characteristically falls as patients get older and as the mass of functioning muscle diminishes with the progression of the disease.

12. Long time Physical inactivity reduce serum CK
Skeletal muscle that is diseased or damaged (fetal reversion)
Renal failure , increase CK
Serum CK activity demonstrates an inverse relationship with thyroid activity.
Following a myocardial infarction
MB isoenzyme

14. The assay of CK activity
Coupled enzyme methods
NADP+ to NADPH, monitored spectrophotometrically.

15. Methods for the Separation & Quantification of CK Isoenzymes
CK activity in serum is relatively unstable and is rapidly lost during storage.
Average stabilities are less than 8 hours at room temperature, 48 hours at 4°C, and 1 month at -20°C.
Methods for the Separation & Quantification of CK Isoenzymes
Electrophoresis & various immunological methods.

16. Enzymes of clinical interest
Widely used enzymes
Aspartate aminotransferases (AST)
Alanine aminotransferases (ALT)
ALP

17. The aminotransferases
catalyze the interconversion of amino acids to 2-oxo-acids
L-aspartate:2-oxoglutarate aminotransferase;AST
L-alanine:2-oxoglutarate aminotransferase; ALT

20. Pyridoxal-5'-phosphate ↔ pyridoxamine-5' –phosphate
Both the coenzyme-deficient apoenzymes and the holoenzymes may be present in serum.
All factors affecting the rate of reaction must be optimized and controlled

21. Transaminases are widely distributed throughout the body.

22. Clinical Significance
Liver disease is the most important cause of increased transaminase activity in serum.
In most types of liver disease, ALT activity is higher than that of AST;
exceptions
alcoholic hepatitis, hepatic cirrhosis, and liver neoplasia.
elevated even before the clinical signs and symptoms of disease

23. ALT is the more liver-specific enzyme.
Peak values of transaminase activity occur between the 7th and 12th days
Medications
nonsteroidal antiinflammatory drugs, antibiotics.
ALT is the more liver-specific enzyme.
elevations of ALT activity persist longer than do those of AST activity.

24. After AMI, increased AST activity appears in serum
AST activity also is increased in some types of muscle diseases
Also serum CK
Mitochondrial AST (m-AST) activity
extensive liver cell degeneration and necrosis.
the ratio between m-AST and total AST activities
typical of alcoholic hepatitis

25. The increased AST activity might reflect decreased clearance
Macro-AST

26. Measurement of Transaminase Activity
coupling the transaminase reactions to specific dehydrogenase reactions.
Pyruvate formed in the ALT reaction is reduced to lactate by LD.

27. The change in absorbance per minute (ΔAlmin) is proportional to the micromoles of NADH oxidized and in turn to micromoles of substrate transformed per minute.
AST activity in serum is stable for up to 48 hours at 4°C.
ALT stability is better maintained at -70°C.

28. Methods for the Separation & Quantification of AST Isoenzymes
Reference Intervals
AST; 31 U/L for women and 35 U/L for men
Methods for the Separation & Quantification of AST Isoenzymes
electrophoresis, selective inhibition, and immunoassays.
anionic (cytoplasmic AST) and a cationic band (m-AST)

29. m-AST (healthy individuals )
About 5% to I0% of the activity of total AST in serum
3.0 U/L.

30. ALKALINE PHOSPHATASE ALP
Orthophosphoric monoester phospho hydrolase
alkaline optimum
hydrolysis of a large variety of naturally occurring and synthetic substrates
ALP activity is present in most organs of the body
especially associated with membranes and cell surfaces
In the mucosa of the small intestine and proximal convoluted tubules of the kidney, in bone (osteoblasts), liver, & placenta

31. ALP exists in multiple forms
Metabolic function
Lipid transport in the intestine
Calcification process in bone
ALP exists in multiple forms

33. activators of the enzyme, Inhibitors
divalent ions, Zn2+
Inhibitors
Phosphate, cyanide ions,…
The ALP activity in the sera of healthy adults
Liver
Skeleton

34. Clinical Significance
Common causes of elevation
Liver
Hepatobiliary disease
bone
Bone disease associated with increased osteoblastic activity
Carcinoplacental isoenzymes
derepression of the placental ALP gene
non placental isoenzymes
Modified forms of ALP

35. Determination of Alkaline Phosphatase Activity
The rate of formation of 4-NP at 405 nm is monitored

36. Determination of Alkaline Phosphatase Activity
Serum or heparinized plasma, free of hemolysis, should be used.
Freshly collected serum or up to 4 hours RT*
Frozen specimens; ALP activity increases
kept at room temperature for 18 to 24 before assay
*Room temperature

37. Reference Intervals
ALP activities in serum vary with age.

38. Assays for ALP isoenzymes are needed when:
Methods for the Separation and Quantification of Alkaline Phosphatase Isoenzymes
Assays for ALP isoenzymes are needed when:
the source of an elevated ALP in serum is not obvious
to monitor the disease activity and the effect of appropriate therapies
Electrophoretic mobility
Stability to denaturation by heat or chemicals
Response to the presence of selected Inhibitors

39. Improvement of electrophoretic separation
anodal mobility,
The liver, most rapidly
Bone ALP, slightly lower
Intestinal ALP
Placental isoenzyme
Discrete band overlying the diffuse bone fraction
Improvement of electrophoretic separation
treated briefly with neuraminidase
Electrophoresis in the presence of wheat germ lectin

40. Placental ALP is heat stable Other evidences Immunological methods
65°C for 30 min
Other evidences
eg ;measurement of GGT
Immunological methods
Intestinal or placental ALPs.

41. Acid Phosphatase (ACP)
Include all phosphatases
that hydrolyze phosphate esters with an optimum pH of less than 7.0.
Produced by
Primarily, prostate gland,
also found in
erythrocytes, platelets, leukocytes, bone marrow, bone, liver, spleen, kidney, and intestine.

42. Acid Phosphatase (ACP)
ACP is present in
Lysosomes,
Extra lysosomal
The lysosomal and prostatic enzymes
strongly inhibited by D-tartrate ions,
the erythrocyte and bone isoenzymes
are not inhibited.

43. Acid Phosphatase (ACP)
Normal serum ACP
The majority of the normally low ACP activity of (unhemolyzed) serum is of a
Tartrate-resistant type (TR-ACP)
Probably originates mainly in osteoclasts.
Increased
Physiologically in growing children
Pathologically in conditions of increased osteolysis and bone remodeling.
High concentrations of TR-ACP in serum
Reflect increased osteoclastic activity, whether appropriate as in normal bone growth, or damaging

44. The only nonbone condition
Gaucher's disease
of spleen, a lysosomal storage disorder,
elevated activities of TR-ACP are found in serum,
abnormal macrophages in spleen and other tissues, overexpress ACP

45. Acid Phosphatase (ACP)
ACP-determining genes
At least four have been identified and mapped.
ACPs are labile
more than 30% of the ACP activity may be lost in 3 hours at room temperature.
Acidification of the serum
specimen to a pH below 6.5 aids in stabilizing the enzyme activity.

46. Acid Phosphatase Prostatic acid phosphatase (PAP),
an optimum pH of 5 to 6,
very labile at a pH of greater than 7.0
very labile at temperature greater than 37°C.
Distinguished from other acid phosphatases by
Using tartrate,
Strongly inhibits the prostatic form.
Select substrates that are more specific for PAP
thymolphthalein monophosphate and β-naphthol phosphate.
Hydrolyzed by PAP much more quickly

47. Acid Phosphatase The clinical use of PAP
as a screening tool for prostate cancer.
to help stage prostate cancer,
to correlate with the prognosis of the disease,
to monitor therapy.
Elevated serum PAP may be seen in
malignant conditions,
osteogenic sarcoma, multiple myeloma, and bone metastases of other cancers.
in some benign conditions,
Osteoporosis, benign prostatic hyperplasia and hyperparathyroidism.

48. Acid Phosphatase The clinical use of PAP
has been replaced by PSA
PAP is not as sensitive as PSA for screening or for detection of early cancer.
restricted to confirmation of metastatic prostate cancer and staging of prostate cancer.
Currently the method of choice for PAP
Measurement of enzymatic activity.

49. Acid Phosphatase Measurement of ACP Principle: Methods
Continuous-monitoring methods of ACP activity
Immunological methods
Principle:
Thymolphthalein monophosphate is hydrolyzed by prostatic ACP at pH 5.4 and 37°C.
The reaction stopped after 30 min by addition of NaOH-Na2CO3 solution
This develops the alkaline color of the liberated Thymolphthalein
Measured at 595 nm

50. Acid Phosphatase Specimens:
Serum should be immediately separated from erythrocytes and stabilized by the addition of acetic acid to lower the pH to 5.4
Under these conditions, ACP activity is maintained at room temperature for several hours,
for up to a week if the serum is refrigerated, and for 4 months if stored at -20°C.

51. Acid Phosphatase Specimens:
Hemolyzed serum specimens are contaminated with considerable amounts of the erythrocyte tartrate-resistant isoenzyme and should be rejected.
Chylous sera should be avoided
Interference with measurement due to turbidity

52. Acid Phosphatase