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Saturday, November 14, 2009

ENZYME LINKED IMMUNOASSAY AND ITS MEDICAL RELEVANCE

Enzyme linked immunoassay commonly known as ELISA allows for rapid screening and quantification of the presence of an antigen in a sample. The principle guiding ELISA is as follows; a non-specific protein is used to wash a polystyrene plate inorder to block other proteins introduced in subsequent steps during the assay. The polystyrene is then treated with a solution which has the primary antibody. This is usually an antibody against the protein of interest. The unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody.




These secondary antibodies have been linked to an enzyme that catalyses a reaction that forms a coloured product. The unbound secondary antibody is washed away, and the substrate of the antibody-linked enzyme is added.



Product formation (which is seen as colour intensity) is proportional to the concentration of the protein of interest in the sample.



Enzyme linked immunoassay is useful in the clinical laboratory for the diagnosis of Hepatitis B surface antigen, Human Immunodeficiency Virus and Herpes Simplex virus. Hepatitis is a liver disease that is associated with elevated levels of both direct (conjugated) and indirect plasma bilirubin. It is accompanied by jaundice. Human Immunodeficiency Virus results in Acquired Immune Defeciency Syndrome (AIDS).It results from opportunistic infections and ultimately results in death. Virologists and Immunologists are carrying out detailed research in the discovery of HIV vaccine, a cure to this global pandemic.

PROTEINS-ESSENTIAL BUILDING BLOCKS OF LIFE

Proteins are macromolecules found in all living things. Proteins are naturally occurring polypeptides composed of monomers called amino acids. Proteins show great diversity such as enzymes, keratin, melanin and so on. The common feature associated with proteins irrespective of their structure is specificity. For instance, haemoglobin and myoglobin specifically binds molecular oxygen, enzymes bind to specific foreign bodies during immunological responses.




Proteins can either be simple or conjugated. Simple proteins are composed of amino acids while conjugated proteins contain other compounds apart from amino acids. Examples of simple proteins are glycine, histidine, phenylalanine, tyrosine and so on while conjugated proteins include glycoproteins and proteoglycans, lipoproteins. Glycoproteins and proteoglycans have carbohydrates as their prosthetic groups while lipoproteins have lipids as their prosthetic groups.




Proteins are known to fulfill diverse biochemical roles. It is also pertinent to note that functions of proteins are understood with respect to how the structure of such proteins allow the specific binding of particular molecules. The following are functions performed by proteins viz; enzymatic activity, transport and storage, mechanical functions, movement, protection and information processing.




Enzymes are known as biological catalysts and proteins.They are capable of enhancing rates of reactions by factors of about 1012. Haemoglobin and myoglobin are transport proteins. They are able to bind specifically to molecular oxygen. Haemoglobin makes oxygen to be readily available to tissues. Ferritin,is a protein that stores iron in various tissues.



Proteins perform structural functions. For instance, collagen provides tensile strength in skin, teeth and bone. Cell membranes and cell organelles are also partly composed of proteins. Movement via the use of muscles is enhanced by the interaction between protein filaments actin and myosin with myosin facilitating the enzymatic process that converts chemical energy of ATP into mechanical energy.




Antibodies and immunoglobulins are proteins found in mammals and they confer immunity by eliciting immunological responses. They also interact with complement to phagoticize and lyse foreign cells leading to their destruction. Signal transduction can be produced from hormones and light intensity detected by specific protein receptors which are capable of transfering the signal to the target cells via the process of biosignalling.For instance, in the biochemistry of vision, rhodopsin,a visual protein located in the retinal photoreceptors called rods and cones play an invaluable role in vision.



The role of proteins as building blocks in living organisms cannot be overemphasized. Hence, the need to consume proteinous foods should be encouraged.

CANCER AND ITS TREATMENT

Cancer is a diseased growth in the body that often causes death. Cancer cells are referred to as oncogenes. Oncology is defined as the study of an abnormal mass of tissue or an abnormal cell population with a capacity for progressive growth. Persistence of growth is an important feature of cancerous or tumour cell that distinguishes them from other neoplastic cells. The neoplasm or new growth can either be benign (not dangerous) or malignant (harmful or dangerous).




Malignant tumours are divided into two groups viz; carcinomas and sarcomas. Carcinomas are composed of epithelial cells while sarcomas consist of connective tissues. Malignant cells prefer anaerobic glycolysis and produce more lactic acid than normal cells. Examples of cancers include lung cancer, breast cancer, cervical cancer and prostate cancer.




Two important concepts that are necessary for the treatment of cancers include growth fraction and cell cycle.Growth fraction has the following information; proportion of rapidly dividing neoplastic cells that effectively respond to chemotherapy, percentage of actively dividing cells in the cell cycle, an index of tumour enlargement. The cell cycle have various phases such as the synthesis phase (S-phase),mitosis phase (M-phase),gap phase represented by G1 and G2 respectively(here RNA synthesis ,protein synthesis and production of initiator proteins occur.),G0 represents the period when the cell go into a state of rest.



Cancer can be treated through the combination of three methods viz; surgery, radiotherapy and chemotherapy. Surgery involves the removal of malignant tumours from the affected part. For instance,lumpectomy involves the removal of malignant tumours from the breast. Radiotherapy employs the use of controlled radiation to treat malignant tumours. Effective chemotherapeutic approach to the treatment of cancer is based on the proper understanding of growth fraction and cell cycle concept. Growth fraction is an index of tumour enlargement and a higher growth fraction yields better results with chemotherapy.




Chemotherapeutic or anti-tumour drugs have the ability of affecting the various phases in the cell cycle. Antimetabolites are S-phase specific and examples include 5-fluorouracil,6-mecarptopurine,metotextrate and arabinosylcytosine. Vinca alkaloids are M-phase specific; two compounds are used which are vincaleucoblastine(VBL) and vincristine (VCR). Non-cell specific chemotherapeutic agents include alkylating agents such as nitrogen mustard, triethylenemelamine, and triethylene phosphorimide and antibiotics such as Actinomycin D, Adriamycin and so on.




It is also pertinent to note that anti-tumour drugs are associated with toxicity. Their therapeutic dose should be strictly obeyed. Examples of such effects include bone marrow depression, depletion of blood cells, loss of hair and so on.




The use Positron Emission Tomography (PET) scan is invaluable in the detection of cancerous growth in organs of the body.Conclusively,early detection of cancerous growth would ultimately enhance effective treatment of carcinomas and sarcomas occurring in humans.

AUTOIMMUNE DISEASES AND THEIR MEDICAL RELEVANCE

Autoimmune diseases results when tolerance to self antigens sometimes breaks down causing T or β cells or both to react against their own tissues. The response they produce contribute directly to the pathogenesis of the disease. Antibodies produced against tissue components may mediate cell or tissue destruction either alone or in conjunction with complement.



Myasthenia gravis is an autoimmune disease. Affected humans make antibodies against the acetycholine receptors on their own skeletal muscle cells. The antibodies intefer with the normal functioning of the receptors so that such patients become weak and can die since they cannot breathe.



Autoantibody to thyroglobulin leads to a disease called Hashiomoto’s thyroiditis. Systemic Lupus erythematosus is another autoimmune disease resulting from autoantibodies of nuclei, DNA,cytoplasmic soluble antigens.


ImmunoglobulinG (IgG) and immunoglobulin M (IgM) autoantibodies are associated with rheumatoid arthritis.Beta cells of the Islets of Langerhans in the pancreas are associated with Insulin Dependent Diabetes Mellitus(IDDM). Nephritis, Good pasteire’s syndrome have been linked to basement membrane autoantibodies. Parietal cells, intrinsic factor autoantibodies are associated with the disease pernicious anaemia.



Active chronic hepatitis results from smooth muscles microsomes autoantibodies.
Erythrocytes autoantibodies result in haemolytic anaemia. Primary biliary cirrhosis results from mitochrondria autoantibodies.



Chronic infection results from complement C3 autoantibodies.Complement component C3b enhances the ability of phagocytic cells such as microphages and neutrophils through their C3b receptors to bind,ingest and destroy the micro-organisms to which their C3b is bound.

IMMUNOGLOBULINS AND THEIR RELEVANCE TO THE IMMUNE SYSTEM

Immunoglobulins are antibodies. These antibodies are glycoproteins.The antibodies which make up the immunoglobulins are found in heavy chains represented by the symbols γ,α,µ,and so on.Examples of immunoglobulins include immunoglobulin G (IgG),immunoglobulin A (IgA),immunoglobulin D(IgD) and immunoglobulin E (IgE).These immunoglobulins play vital roles in the immune systems of human and they are usually associated with diseases and immune responses.



Immunoglobulin G(IgG) represented by the symbol γ is a monomeric protein with molecular weight of 146,000 and sedimentation co-effiecient 7s.It is the most abundant of the immunoglobulins contributing between 70-75% of total plasma immunoglobulins.It is able to cross the placenta and activate the complement system.It provides antibody activity in tissues,contributes to immunity against many infectious agent that are blood-borne e.g. bacteria,viruses,parasites and some fungi.
Immunoglobulin M(IgM) represented by the symbol µ and accounts for about 10% of the total serum immunoglobulin.It is a pentameric protein with a molecular weight of 970,000.IgM are highly efficient against particulate antigens such as bacteria,they bind complement with high degree of efficiency.It is of greatest importance in primary immune response to interact with a foreign antigen that is introduced into a host.IgM is associated with rheumatoid arthritis.




Immunoglobulin A(IgA) represented by the symbol α and consist of between 15-20% of the total serum immunoglobulins in humans.IgA is the predominant immunoglobulin in sero-mucous secretions such as saliva,tracheobronchial secretion,colostrums milk and urinogenital secretions.It has a molecular weight of 385,000(i.e the secretory IgA existing mainly in the dimeric form).The secretory IgA is protected from proteolysis by the secretory component.




Immunoglobulin D(IgD) constitute less than 1% of the total plasma immunoglobulin but it is known to be present in large quantities on the membranes of circulatory B-lymphocyte.It plays a role in antigen triggered lymphocytes differentiation.(I.e neutrophils, eosinophils, monocytes, and basophils).



Immunoglobulin E(IgE) is present in trace amount in serum.It is found on the surface membrane of basophils and mast cells in all humans.It may play a role in active immunity to helminthic parasites but it is more commonly associated with immediate hyper-sensitivity diseases such as asthma,hay fever and eosinophilia.

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PARKINSON’S DISEASE AND ITS TREATMENT

This is a disorder associated to dopamine oversecretion. Dopamie, a neurotransmitter
is involved in signal transduction in dopaminergic neurons. Basic symptoms include tremor, bradykinesia, reluctance to movement, and disturbed gait.




The cause of Parkinson’s disease is idiopathic.However,some clues were offered with the discovery of a selective dopamine toxicity produced by the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).MPTP was synthesized by illicit drug dealers in an attempt to produce MPPP, a synthetic heroin.When injected,MPTP produces neurological symptoms remarkably similar to Parkinson’s disease. The toxic effects of MPTP can be blocked by pargyline, an inhibitor of MAO-B which is normally distributed in serotonin containing neurons. Serotonin terminals are found in the substantia nigra.




The dopaminergic neurons of the substantia nigra project to the striatum,and their degeneration in Parkinson’s disease is associated with a reduction in the striated dopamine content. Parkinson’s disease has been linked to genetic disposition, viral infection, drugs such as resorpine deplete dopamine stores and doesnot inhibit the vessicularisation of dopamine stores. In the prevalence of Parkinson’s disease,1% of people of age 50years and above are affected. Beyond 25years,13% of such cells are lost per decade. β-N-methylaminoalanine,a plant product can also contribute to the incidence of Parkinson’s disease.




Treatment of Parkinson’s disease can be achieved via the use of the following; anticholinergic drugs, dopamine precursors, and dopamine receptor agonists such as bromocryptine. Parkinson’s disease has been treated successfully with the dopamine precursor 3,4-dihydroxyphenylalanine(L-DOPA),which unlike dopamine, is able to cross the blood-brain barrier. Inorder to prevent the rapid decarboxylation of L-DOPA in the bloodstream, decarboxylase inhibitors such as carbidopa are often given to increase the circulating levels of L-DOPA and decrease the side effects. Tolcapone is a selective inhibitor of catechol-o-methyltransferase is useful in the treatment of Parkinson’s disease. Catechol-o-methyltransferase converts L-DOPA to an inactive form, which contributes to the limited effects of L-DOPA treatment.

DIABETES MELLITUS AND TREATMENT

Diabetes mellitus is a devastating metabolic disease caused by insufficient insulin synthesis, increased insulin disruption or ineffective insulin action. The pancreas is the organ where the Islets of Langerhans is located. The beta cells found in the islets of Langerhans aid the production of insulin. Diabetes mellitus is classified into two groups viz; Insulin Dependent Diabetes Mellitus (IDDM).and Non-insulin Dependent Diabetes Mellitus (NIDDM).



Insulin Dependent Diabetes Mellitus (IDDM) is also referred to as Type 1 diabetes or Juvenile onset diabetes. It affects people below 30years or age. Inadequate amount of insulin is secreted because the beta cells of the pancreas have been destroyed. Three factors have been implicated in this destructive process of the beta cells viz; genetics, viral infections and autoimmunity (i.e the body’s immune system destroys insulin.)


Non-insulin Dependent Diabetes Mellitus (NIDDM) is also called Type2 or maturity (adult) onset diabetes.It affects people above 35years of age. This type of diabetes is caused by the insensitivity of target tissues it insulin.It occurs mostly in obese people.



Symptoms of diabetes mellitus include hyperglycemia (high blood glucose level) caused by inadequate cellular uptake of glucose, glucosuria (i.e glucose in urine).osmotic dieresis,dehydration may affect patients despite consumption of large volumes of fluids in very severe cases, polydipsia (excessive thirst) and polyphagia(excessive hunger).



When there is insufficient insulin to regulate glucose, three principal target tissues and organs (i.e. liver , adipose tissue ,and muscles) don’t absorb nutrients adequately. In severe cases of diabetes, despite increased appetite and food consumption, the failure to utilize glucose effectively results in body weight reduction.



The following methods can be used to treat both types of diabetes mellitus;diet(should include high content dietary fibres),through the use of insulin pump,beta cell replacement therapy ,pharmacotherapy(i.e sulphonyiureas ,biguanides),
alternative medicine(plant extracts).Research has shown that extracts from Loranthus bengwensis (African mistletoe) and Indian almond and several others possess hypoglycemic properties.



Diabetes mellitus is a devastating metabolic disease and should be given adequate medical attention so that it can be effectively managed and treated.

DIAGNOSTIC ENZYMES AND THEIR CLINICAL IMPORTANCE

Enzymes are substances which catalyse metabolic and biochemical reactions. They are also known as biological catalysts. They enable metabolic and biochemical reactions to occur by lowering the activation energy of such reactions. The following are examples of diagnostic enzymes viz,lactatedehydrogenase(LDH),
creatinekinase(CPK),alkaline phosphatase,aspartate aminotransferace(AST),
alanine aminotransferace(ALT),acid phosphatase,glucose-6-phosphate dehydrogenase
and so on.



It is pertinent to note that the rationale for assaying enzyme activities is based on the premise that changes in activities reflect changes that have occurred in aspecific tissue or organ. Lactate dehydrogenase is an important enzyme involved in glucose metabolism.It is a tetramer(has 2 different subunits).It has five isoenzymes which are designated LDH1(HHHH),LDH2(HHHM),LDH3(HHMM),LDH4(HMMM) and LDH5(MMMM) respectively.Isoenzymes catalyse similar reaction but migrate differently when subjected to electrophoresis.Hence,lactate dehydrogenase activity is found in cardiac muscles and red blood cells( i.e LDH1,LDH2 and LDH4),skeletal muscle(LDH5),Liver (LDH5),kidney and brain (LDH3).Hence,increased LDH activities can be used to detect myocardial infarction,leukaemia,liver cirrhosis,tumours of the central nervous system.



Creatinekinase(CPK) exists as a dimeric enzyme with three isoenzymes CPK1,CPK2, and CPK3.The subunits are designated BB,MB, and MM respectively.Total creatinekinase activity shows an increase following myocardial infarction.It is pertinent to note that it is only when CPK2(i.e MB subunit) contribute more than 6% of the total creatinekinase activity that creatinekinase is implicated in myocardial infarction.
Alkaline phosphatase is an enzyme which transport phosphate groups across cell membranes of organs.It is found in the osteoblasts(bone cells),bile canali ( in the liver),small intestinal epithelium,proximal tubule(kidney) and placenta.Increase in alkaline phosphatase occurs mainly in bone diseases such as Paget disease,Osteogenic sarcoma,liver diseases and bilary tract.



Aspartate aminotransferase (AST) is an enzyme involved in transamination reactions. Transamination reactions produce keto and amino acids simultaneously in tissues where they occur.The assay of serum AST is particularly useful in the diagnosis of myocardial infarction.Elevated AST levels is indicative of damage to the myocardium. Although AST activity is still used in the diagnosis of myocardial infarction,it has been replaced to some extent by the assay of total creatinekinase activity(especially the isoenzyme CPK2).



Serum aminotransferase activities are sensitive indicators of parenchymal liver damage.Both alanine and aspartate aminotransferases have been implicated in infective hepatitis,infectious mononucleosis and hepatocellular damage.Exposure to toxic substances such as carbontetrachloride poisoning ,therapeutic substances such as chlormphenicol, cephalosporins and paracetamol overdose have also resulted in elevated AST and ALT activities.



The serum barely detectable glucose-6-phosphate dehydrogenase activity .An elevated activity is indicative of myocardial infarction .Red blood cells contain 120 – 240 units per 1012 cells. Defeciency of this enzyme can result in hemolytic anaemia through the use of antimalarials such as primaquine, aromatic compounds ,it has also been associated to Favism (resulting from Fava beans consumption).



Acid phosphatase is found in the prostate, red blood cells, small amounts are found in bone, liver, spleen and pancreas. The assay of serum acid phosphatase is mostly used to detect prostatic carcinoma and to monitor the treatment.