Clinical Pathology Enzymology

OUTLINE

REFERENCES:

PPT

• RECORDING

Note: Intro to Enzymology was not included in the trans. You can study the ppt or the book for that. Kahit wag na kasi wala naman sa unicorn. ;)

*R. cont.loves enzymemada。

I. DIAGNOSTIC ENZYMOLOGY

• Why are enzyme measurements useful?
• What information do they give?
•  Examples of enzymes
• Principles of enzymatic assays
• Enzymatic assays vs immunoassay of enzymes

• Membrane and cell integrity are also sensitive to “normal” metabolism

• Some enzyme release reflect normal cell proliferation  / turnover
•  => RELEASE/ LEAKAGE depends on number of cells present & Number of cells damaged
• Amount of enzyme may depend on induction of cellular synthesis - e.g. Toxins (EtOH) in liver*inducers

II. MEASUREMENT OF SERUM ENZYMES

• Normally intracellular

• They are not elevated except in organ or cell damage by disease, physical trauma or whatever conditions which cause it to rupture. The reason why you have a normal level is because of normal turnover rate. Cells are not permanent especially the normal epithelial cell lining that has a certain life span. When they die, enzymes are secreted in the serum.

• When in blood → indicates cell or membrane damage, organelle damage, or cell death
• Quantitatively measure  cell/tissue damage
• Fairly non-invasive test → easily repeated for disease monitoring

INFORMATION FROM SERUM ENZYME MEASUREMENTS

• Presence or absence of disease
• Organs involved
• Etiology of disease: differential diagnosis
• Extent of disease – stage (mild, moderate or severe)
• Disease time course or monitoring
• Recovery vs worsening
• Prognosis
• Result in units μmol product per min
• Carefully specified conditions e.g. substrate concentration not rate limiting
• International units per liter (IU/L or U/L)
•  Standardization of conditions and reagents
• Comparable results world-wide
• Sample used: serum or plasma
• Seldom measured on tissue biopsies

ENZYME DEFICIENCY

• A variety of metabolic diseases are now known to be caused by deficiencies or malfunctions of enzymes.
• Albinism, for example, is often caused by the absence of tyrosinase, an enzyme essential for the production of cellular pigments*rt pku. (REMEMBER).

• It is not the melanin that is absent. It is tyrosinase, which leads to decrease in melanin.

• The hereditary lack of phenylalanine hydroxylase results in the disease phenylketonuria (PKU) which, if untreated, leads to severe mental retardation in children.

ENZYME UNITS

• Result is in unit/mmol product per minute
• Kingarmstrong
• Somogy
• Reitman Frankel (RFU)
• Spectrophotometric
• Katal
• International Unit (IU/L)

ENZYMES ESTIMATED FROM:

• Whole blood, serum, plasma – MOST COMMON SAMPLE USED
• Red blood cells
• Urine
• Sweat, CSF, Saliva, Semen, Amniotic fluid, Tears
• Tissues: Brain, Heart, Liver, Kidney, Muscle – LESS COMMONLY USED (REMEMBER)

• No role of enzyme determination in histopath samples*more fluid enzyme

ENZYMES MEASURED IN ROUTINE CLINICAL EXAMINATIONS

• Aspartate Amino Transferase [AST] – Heart/Liver
• Alanine Amino Transferase [ALT] – Liver/Heart
• Alkaline Phosphatase [ALP] – Bone/Intestine/Biliary lining epithelium
• γ-Glutamyl Transferase (γ GT) – Liver
• Creatine Kinase [CK] – Muscle
• Lactate Dehydrogenase [LDH] – Heart, Liver, Muscle, RBC
• Some enzymes have many isoforms like CK. Each isoform represents predominantly a specific organ. Eg. CKMB, CKMM, CKBB
• Isoenzymes

• α - Amylase – Pancreas
• Acid Phosphatase – Prostate (tissue specific isoform)*pataste
• Bone disease - alkaline phosphatase
• Muscle disease/myocardial infarction - Creatine kinase
• Pancreatic disease/carcinoma and acute pancreatitis - α-amylase
• Malignant disease - NO specific markers

• Many enzymes may change

• It depends on the tissue involved.

• Prostate Diseases (esp Ca)

• Acid phosphatase
• Prostate Specific Antigen (PSA)

PROVED DIAGNOSCURACY (Need to be memorized)

• Measure several enzymes
• Calculate ratios
• Separate isoenzymes electrophoretically or biochemically (inhibitors)

• Example: Lactate dehydrogenase

• To remember:

• Rationale/nature of enzymes as diagnostic markers
• Enzymatic determinations vs.  measurements by  immunoassays

• Determination of enzymes are still very important in  clinical chemistry but are not necessarily best determined by enzymatic assays
• Immunoassays vs Enzyme activity measurement

IV. IMMUNOASSAY

• More sensitive than enzyme activity measurement
• Uses antibodies and antigens to measure the activity, unlike in enzymatic determination which uses catalytic reactions to measure the activity.

• more the activity = more enzyme

• Biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine using the reaction of an antibody or antibodies to its antigen.
• The assay takes advantage of the specific binding of an antibody to its antigen.
• Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the antigen (if there is antigen available, a very high proportion of it must bind to the antibody).
• Both the presence of antigen or antibodies can be measured. For instance, when seeking to detect the presence of an infection, the concentration of antibody specific to that particular pathogen is measured. For measuring hormones such as insulin , the insulin acts as the antigen.*antiinsulin
• Immunoassay can be made much more sensitive than enzymatic assays.*enzymada have to be burned pa, assay is easy
• Many enzymatic assays are still well established, well performing, inexpensive and easily available on commercial analysers
• Both assay types (immuno and enzymatic) are being used today.

ISOENZYME: ISOZYME*iso same quality

• May appear in multiple forms, with slightly different chemical or other characteristics.
• May be produced in different organs, although each performs essentially the same function.
• The various forms are distinguishable in analysis of blood samples, which aids in the diagnosis of disease.
• Isoenzymes that catalyze the same physiologic reaction may also appear in different forms in different animal species.
• Isoenzymes or Isozymes are physically distinct form of same enzyme having same specificity, but are present in different tissues of same organism, in different cell compartment.
• Useful for diagnosing diseases of different organs.
• Homomultimer: All the units are same.
• Heteromultimer: Sub units are different. These are produced by different genes. *Multi,count, units, measured
• Fractionation and measurement of the three distinct CPK isoenzymes have replaced the use of total CK (or CPK) levels to accurately localize the site of increased tissue destruction.

• CK-BB is most often found in brain tissue.         *B,brain
• CK-MM and CK-MB are found primarily in skeletal and heart muscle.*MM,mr muscle; mb ll heart

• Subunits of CK-MB and CK-MM, called isoforms or isoenzymes, can be assayed to increase the test's sensitivity.

V. MAIN CARDIAC ENZYMES