Biomarkers in the Diagnosis of Disease

• a biomarker is a biological molecule whose concentration changes in response to a specific disease (pathological disease process in tissue) and may be produced as a corrective response by tissues that are injured or stressed
• Changes in [plasma] are directly related to the disease as biomarkers leak into plasma and can be used to monitor the onset/prognosis of diseases, and predict outcomes in response to treatments
• biomarkers are ideally only present in a diseased state as well as being stable and easy to assay
• Increases of a biomarker in plasma should be relative to tissue damage, and can usually indicate a disease process (can have low background levels)
• some examples of biomarkers include:

  • molecules of intermediary metabolism,
  • cell signalling molecules,
  • hormones,
  • enzymes,
  • structural proteins
• biomarkers are used to screen for a disease before clinical symptoms occur - they are used in conjunction with scans
• there are three stages to cellular leakage:

  • normal = background levels of cellular leakage and degradation giving background plasma levels
  • reversible damage = increases in the amounts of leakage from the damaged cell - enzymes, small cytosolic molecules, changes in the production of messenger or signalling molecules
  • cell death = disintegration of cell structure with loss of molecules as before PLUS cellular structural proteins like troponins in MI
• reversible damage to cell structure occurs often in liver cells as that is the only organ able to self-repair cells
• cells which undergo cell death and disintegration of cell structure are unable to recover - cardiac troponins after experiencing myocardial infarction undergo this (anoxia)
• Enzymes that occur in plasma in the physiological situation can be divided into 2 groups:

  • those with clearly defined action e.g. renin, complement, and coagulation factors.
  • Those with no known function in blood and are only present because they are released from cells as a result of cell turnover. These are usually at a very low level
• Plasma levels are a result of the balance between release from the tissue and removal, Small molecules e.g. amylase are filtered by the glomeruli, but most enzymes are probably removed by reticuloendothelial cells, this varies from a few hours to a few days.
• In pathological situations, biomarker enzyme levels may rise due to:

  • cell damage,
  • increased cell turnover,
  • cell proliferation
  • increased enzyme synthesis,
  • obstruction to secretion,
  • decreased clearance from the plasma.
• elevated enzyme levels due to pathological increased cell damage can be due to ischaemia or toxic substances
• elevated enzyme levels due to pathologically increased cell turnover can be due to normal active growth ALP which is high in the first year and at puberty or tissue repair after fractures
• elevated enzyme levels due to pathological cell proliferation can be due to malignant disease
• elevated enzyme levels due to pathological increased enzyme synthesis can be due to stimulation by disease or drugs such as alcohol in liver cells or cancer
• elevated enzyme levels due to pathologically increased obstruction to secretion can be due to duct blockage to enzymes which are normally present in exocrine secretions and may be regurgitated into the blood
• When selecting useful plasma enzymes for diagnostic measurements i.e. determining when damage has occurred, and if so to what extent. It is important to consider the sensitivity of the test - this largely depends on the tissue to plasma ratio of enzyme activity, which for most enzymes of clinical value lies between 1,000 in 1 and 10,000 in 1
• The activity of plasma enzymes is measured and needs standardised assay conditions for reference ranges, e.g. temp, pH etc.
• plasma enzymes are given in international units /L.
• IU= “the amount of enzyme which under given assay conditions will catalyse the conversion of 1 mmol of substrate per minute”
• In the disease processes, plasma enzymes are usually described in the values as X upper level of reference limit (X URL)
• plasma enzyme concentrations are sometimes measured using immunoassays
• enzyme measurement can lack specificity for particular cells/tissues as most enzymes can occur in many different tissues - making it difficult to determine which tissues have been affected.
• tests can be overcome by the use of enzyme combinations and/or isoenzymes
• isoenzymes are slightly different forms of an enzyme which have similar catalytic activities, often differing in some other measurable property e.g. sensitivity, heat stability or sensitivity to inhibitors
• clinically important plasma enzymes include:

  • lactate dehydrogenase (LD, LDH)
  • creatine kinase (CK)
  • aspartate aminotransferase (AST)
  • alanine aminotransferase (ALT)
  • alkaline phosphate (ALP)
  • gamma-glutamyl transferase (GGT)
• lactate dehydrogenase exists as a tetramer with combinations of 2 monomers giving rise to 5 isoenzymes, each having differing electrophoresis mobilities.
• LHD 1 is found mainly in the cardiac muscle and RBCs
• LDH2 is foud mainly in cardiac muscle, RBCs and kidney
• LDH 3 is found mainly in the pancreas
• LDH 4 is found mainly in the lung
• LDH 5 is found mainly in the liver
• total LDH levels are non-specific indicators of disease, LDH 5 is the major form found in the liver and so can be used to monitor liver function
• LDH 1 and 2 are the major cardiac muscle forms however they are no longer used in the diagnosis of myocardial infarctions due to a lack of specificity
• Cardiac muscle and RBCs contain LDH 1(H4) as their predominant isoenzyme which shows a greater catalytic activity with a-hydroxybutyrate as a substrate (rather than lactate) - giving rise to the alternative name of a-hydroxybutyrate dehydrogenase (HBD) or cardiac-specific LDH
• artefactual-raised LDH can be due to haemolysis or delayed separation of plasma
• markedly raised LDH (>5x URL) can be caused by circulatory failure, myocardial infarction, and some haematological disorders such as megaloblastic anaemia, acute leukaemias and lymphomas - usually at 20x URL
• markedly raised LDH (>5x URL) can be seen in smaller increases in disorders of erythropoiesis such as thalassaemias, myelofibrosis, haemolytic anaemias, renal infarction, and renal transplant rejection
• moderately raised LDH levels can be caused by viral hepatitis, malignancy, skeletal muscle disease, pulmonary embolism, and infectious mononucleosis