core area 2 cardiovascular topic c. case study mr. dd 60 years old smoker > 40 years conditions: chf...

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CORE AREA 2 CARDIOVASCULAR Topic C Slide 2 CASE STUDY Mr. DD 60 years old smoker > 40 years Conditions: CHF and COPD Medications: - enalapril 10mg BD - frusemide 80mg mane - salbutamol MDI prn Slide 3 Mr. DDs Biochemistry Results Slide 4 Syndrome of Inappropriate Anti-Diuretic Hormone (SIADH) Is characterised by the sustained and inappropriate release of ADH from the posterior pituitary gland. Continued release of ADH despite fluid intake, interferes with the osmoregulation of thirst. Ingested fluid is retained and the extracellular fluid expands and cells become hypo- osmolar. Patients excrete small amounts of concentrated urine Slide 5 Clinical Manifestations of SIADH Cardinal signs of SIADH are: Hyponatremia; serum Na + < 120mmol/L Serum hypo-osmolality (overhydration) Normal acid-base and potassium balance Concentrated urine Low blood urea and nitrogen levels Slide 6 Clinical Manifestations of SIADH (contd) Anorexia, nausea, vomiting, abdominal cramps, muscle weakness and fatigue. CNS effects: abnormal mental status, seizures, hallucinations, headaches and confusion. Common causes of SIADH include: Malignancies e.g. tumours Pulmonary lesions and other lung diseases Neurological (CNS) disorders Medications e.g. psychoactive drugs, oral hypogylcaemics and substances e.g. nicotine Slide 7 Hypernatraemia Acute hyponatraemia Serum Na + < 115 mmol/L in 48 hours. Cerebral oedema results in symptoms of headache, nausea, restlessness and drowsiness. Should be corrected quickly to 130 mmol/L to prevent permanent brain damage. Chronic hyponatraemia Serum Na + < 125 mmol/L Patients can present with mild symptoms or be asymptomatic (50% of patients). No brain oedema. Rate of correction 0.5 mmol/hr till Na + reaches 130 mmol/L. Slide 8 Treatment of SIADH Aims to decrease fluid retention in order to treat dilutional hyponatraemia. This is achieved with Frusemide, a loop diuretic. Other treatments aimed to treat hyponatraemia include fluid intake restriction to 0.5 1L daily, or Demeclocycline (tetracycline AB) 6001200mg daily if fluid restriction is insufficient. However, demeclocycline is inappropriate for Mr. DD, as it may cause irreversible nephrotoxicity in patients with oedema forming disorders e.g. CHF. Slide 9 Which of Mr. DDs medications are associated with hyponatraemia & SIADH? Enalapril (ACE-Inhibitor) is associated with hyponatraemia, but is not documented to cause SIADH. ACE-I blocks conversion of Angiotensin I to II Increased circulation of angiotensin II, may stimulate thirst and the release of ADH and hyponatremia. Frusemide (loop diuretic)- used for treatment of SIADH, but can cause hyponatraemia. It works with high efficacy at the loop of Henle to block Na + and Cl - reabsorption. Salbutamol- unlikely to cause these problems Slide 10 How does COPD predispose to hyponatremia or SIADH? COPD is a condition which is characterised by chronic bronchitis and progressive airway obstruction. There are many drugs and disease states which may cause SIADH. COPD and nicotine are examples of these, however the direct MOA is unknown. Slide 11 How does CHF predispose to SIADH and hyponatraemia. CHF (Congestive Heart Failure) is a condition where there is an accumulation of fluid within the body caused by the heart pumping inefficiently. An accumulation of body fluid results in dilution of solutes such as sodium i.e. dilutional hyponatremia results. Diuretics are used to treat CHF which depletes the body of solutes and therefore contributes to hyponatraemia. Slide 12 Conclusion of SIADH Potential risk of Mr DDs developing SIADH presenting as low osmolality and hyponatraemia is high: Pathology results; low sodium and especially low urea are indicative of SIADH. Use of frusemide, which interferes with the reabsorption of sodium, and enalapril, which causes hyponatremia. Fluid retention caused by CHF causing dilution of sodium COPD and nicotine may contribute to SIADH by increasing the release of ADH. Slide 13 Digoxin Used to treat heart failure Narrow therapeutic window: Normal dose: 1-2 ng/mL Toxic dose: >2 ng/mL Signs of toxicity: Early clinical warning signs include anorexia, nausea, vomiting, malaise, listlessness, fatigue and generalized weakness/dizziness, insomnia Cardiac rhythm disorders Halo vision Hence serum levels should be monitored. This is achieved with RIA, EMIT and ELISA. Slide 14 Radioimmunoassay (RIA) Involves incubation of: Limited amount of specific antibody with a fixed amount of radio-labelled antigen Serum unlabeled antigen Labelled and unlabelled antigens compete for the binding site on the antibody Slide 15 Advantages and disadvantages of RIA Advantages: Sensitivity(10 -10 -10 -11 M) Specificity Determines the concentration of both macromolecular antigens & small haptens Disadvantages: High level of wastage and expensive Short shelf life of radioisotope Labour-intensive Radiation Exposure Slide 16 EMIT Homogenous & Competitive immunoassay system Separation using specifically of antibody- antigen binding and quantification using enzyme reaction EMIT is reliant on enzyme activity. Enzymatic activity is severely reduced when it becomes bound to antibody, thus making the separation from hapten unnecessary. Slide 17 Components of the EMIT Drug to be measured is the hapten part of the antigen Antibody binding the enzyme-hapten conjugation, inhibiting the enzyme activity Buffered substrate Enzyme covalently linked to pure drug such as glucose-6-phosphate dehydrogenase Slide 18 Procedure of the EMIT Mix sample of serum with a solution containing antibody, enzyme-hapten complex & buffered substrate. Incubate at 37C for short time Measure rate of absorbance changed at 340nm by UV-visible spectroscopy Determine Absorbance from reaction rate and drug concentration Non-linear relationship between Absorbance and concentration Slide 19 Procedure Contd Determine standard curve and obtain concentration of the analyte from standard curve Fig 3.8.1 EMIT Assay Components in Action Slide 20 Advantages and disadvantages of EMIT Advantages: Enzyme stability Automated spectrophotometer Inexpensive ($25-$65 per test) EMIT 2000 lower cross sensitivity of digoxin compared to RIA and ELISA. Disadvantages: Mainly for small molecule detection eg. Steroid hormones or thyroxine (T4) Less sensitive compared to ELISA or RIA (sensitivity range 10 -6 -10 -8 M) Lower limit of quantification than RIA method Slide 21 Enzyme-Linked Immunosorbent Assay (ELISA ) ELISA is a widely used method for measuring the concentration of molecules (e.g. hormone and drug) in serum or urine In this case study the molecule is digoxin, and it is detected using antibodies that have been made against it, i.e. for which digoxin is the antigen Slide 22 ELISA- Sandwich technique Antigen-specific antibody (monoclonal) is attached to a solid phase surface e.g. inner surface of test tube Tubes are filled with antigen solution to be assayed. Any antigen present bind to antibody molecules Slide 23 Sandwich technique (cont) An enzyme-labeled antibody specific to the antigen (conjugate) is added. After washing away any unbound conjugate, the substrate solution is added, which in presence of the enzyme, changes colour. Slide 24 Sandwich ELISA-Quantification The concentration of the coloured product formed is measured in a spectrophotometer. The intensity of the colour is proportional to the concentration of bound antigen. Slide 25 ELISA- Competitive technique Specific antibody is attached to a solid- phase surface. Test specimen, which may or may not contain the antigen, and an enzyme-labeled antigen specific to the test antigen (conjugate) are added together Slide 26 Competitive technique (cont) Chromogenic substrate is added, in which presence of the enzyme, changes colour. Colour change intensity is proportional to the amount of antigen present. Slide 27 Competitive ELISA- Quantification Colour of the solution is inversely proportional to amount of antigen The test solution of unknown antigen is compared with standard solutions of known concentrations of antigen to competitively inhibit the indicator antibody binding. Increased antigen decreases the amount of bound antibody. An inhibition curve, is a function of antigen concentration, can be derived using the results from the standard solutions. Slide 28 Comparison of EMIT & ELISA EMIT Measures haptens (Small molecules) Drug Hormone Metabolite Faster than ELISA (No need to separate free and bound enzyme labels) ELISA Measures macromolecules Antigens Antibodies Greater Sensitivity Slide 29 References Baylis PH, The International Journal of Biochemistry and Cell Biology. 35 (2003) p1495 1499 Choi M.H., Kim M.K., Cho H.C., Kim M.S., Lee E.A, Paeng I.R, Cha G.S Enzyme Linked Competitive Binding Assays for Digoxin Bulletin of the Korean Chemical Society 2001, 22, 417-420 Izzedine H, Fardet L, Launay-Vacher V, Dorent R, Peticlerc T, Deray G. (2002). ACE-I induced syndrome of SIADH: Case report and review of literature. Clinical Pharmacology & Therapeutics 71:503-507 Verbalis JG, Best practice and Research clinical endocrinology and metabolism. Vol 17, No.4, p471 503, 2003 The Merck manual 17 th Edition Centennial edition 1997 Australian Pharmaceutical Formulary and Handbook 18 th Edition 2002 Arthur S. (1985). Role of serum digoxin assay in patient management. The American Journal of Cardiology. 5(suppl 5): 106A-110A. Radembino N., Poirier J., Jaillon P. (1999). Improved sensitivity of digoxin assay by modification of the EMIT 2000 method. Therapeutic Drug Monitoring. 21(2): 256-258. Saccoia N.C., Hackett L.P., Morris R.G., Ilett K.F. (1996). Enzyme-multiplied immunoassay (EMIT 2000) digoxin assay compared with fluorescence polarization immunoassay and amerlex 125 I-radioimmunoassay at two Australian Centres. Therapeutic Drug Monitoring. 18(6): 672-677. Caplan A & Jack R. Clinical Chemistry; interpretation and techniques. 4 th edition. Williams& Winkins; London 1995