4Syndrome 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 urineADH is Anti-Diuretic Hormone which is responsible for increasing the kidney’s response to water reabsorption – hence less diuresis.Osmolality is an indicator of cell hydration. Low values indicate over hydration. Hypo-osmolality may result from CHF and diuretic use.
5Clinical Manifestations of SIADH Cardinal signs of SIADH are:Hyponatremia; serum Na+ < 120mmol/LSerum hypo-osmolality (overhydration)Normal acid-base and potassium balanceConcentrated urineLow blood urea and nitrogen levels60% SIADH present with dilutional hyponatraemia.
6Clinical Manifestations of SIADH (cont’d) 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. tumoursPulmonary lesions and other lung diseasesNeurological (CNS) disordersMedications e.g. psychoactive drugs, oral hypogylcaemics and substances e.g. nicotineSIADH may be caused by: Neoplasia – eg cancers. Neurological- eg meningitis and brain tumours, Lung diseases- eg asthma, pneumonia, TB and Drugs – eg anticancers, antidepressants, diuretics, nicotine and ectasy
7Hypernatraemia 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 hyponatraemiaSerum Na+ < 125 mmol/LPatients 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.Sodium is responsible for regulating water transfer between cells to maintain extracellular fluid.Low osmolality suggests the cause of hypoNa is caused by fluid overload. (Low osmolality indicates overhydration)
8Treatment of SIADHAims 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) 600–1200mg 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.Frusemide acts to inhibit the reabsorption of sodium and chloride ions at the loop of Henle, which results in fluid excretion.Democycline is a tetracycline antibiotic, which is not used as an antibacterial, but antagonises the effect of ADH within the kidneys to promote diuresis in patients with SIADH.
9Which of Mr. DD’s 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 problemsACE inhibitors block the conversion of angiotensin I to II in the PNS not CNS.
10How 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.Explanation why smokers have increased risk: higher occurrence of airway obstruction, airway mucus cell histological changes leading to a change in mucous consistency and prevalence of a productive cough.
11How 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.
12Conclusion of SIADHPotential risk of Mr DD’s 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 sodiumCOPD and nicotine may contribute to SIADH by increasing the release of ADH.
13Digoxin Used to treat heart failure Narrow therapeutic window: Normal dose: 1-2 ng/mLToxic dose: >2 ng/mLSigns of toxicity:Early clinical warning signs include anorexia, nausea, vomiting, malaise, listlessness, fatigue and generalized weakness/dizziness, insomniaCardiac rhythm disordersHalo visionHence serum levels should be monitored. This is achieved with RIA, EMIT and ELISA.Digoxin may also be used to treat cardiac arrhythmias.
14Radioimmunoassay (RIA) Involves incubation of:Limited amount of specific antibody with a fixed amount of radio-labelled antigenSerum unlabeled antigenLabelled and unlabelled antigens compete for the binding site on the antibody
15Advantages and disadvantages of RIA Sensitivity( M)SpecificityDetermines the concentration of both macromolecular antigens & small haptensDisadvantages:High level of wastage and expensiveShort shelf life of radioisotopeLabour-intensiveRadiation Exposure
16EMIT Homogenous & Competitive immunoassay system Separation using specifically of antibody-antigen binding and quantification using enzyme reactionEMIT is reliant on enzyme activity. Enzymatic activity is severely reduced when it becomes bound to antibody, thus making the separation from hapten unnecessary.
17Components of the EMITDrug to be measured is the hapten part of the antigenAntibody binding the enzyme-hapten conjugation, inhibiting the enzyme activityBuffered substrateEnzyme covalently linked to pure drug such as glucose-6-phosphate dehydrogenase
18Procedure of the EMITMix sample of serum with a solution containing antibody, enzyme-hapten complex & buffered substrate. Incubate at 37ºC for short timeMeasure rate of absorbance changed at 340nm by UV-visible spectroscopyDetermine Δ Absorbance from reaction rate and drug concentrationNon-linear relationship between Δ Absorbance and concentration
19Fig 3.8.1 EMIT Assay Components in Action Procedure Cont’dDetermine standard curve and obtain concentration of the analyte from standard curveFig EMIT Assay Components in Action
20Advantages and disadvantages of EMIT Enzyme stabilityAutomated spectrophotometerInexpensive ($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 M)Lower limit of quantification than RIA methodAdvantages – stable enzyme used rather than an isotope in RIA, Use of automated spectrophotometer allows results to be obtained easily and simplifies the process, inexpensive ($25-65 per test) this makes one of the most common tests used currently.
21Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is a widely used method for measuring the concentration of molecules (e.g. hormone and drug) in serum or urineIn 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 antigenThere are two types of ELISA techniques: Sandwich and competitive.Tubes are filled with antigen solution to be assayed. Any antigen present bind to immobilised antibody molecules
22ELISA- Sandwich technique Antigen-specific antibody (monoclonal) is attached to a solid phase surface e.g. inner surface of test tubeTubes are filled with antigen solution to be assayed. Any antigen present bind to antibody molecules
23Sandwich 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.The antibody part of the conjugate binds to any antigen molecules that were bound previously creat
24Sandwich 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.
25ELISA- 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
26Competitive 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.If there is a lot of antigen, the colour change will be more intense, where as lower concentration of antigen will have a lower intensity in colour change.
27Competitive ELISA- Quantification Colour of the solution is inversely proportional to amount of antigenThe 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.
28Comparison of EMIT & ELISA Measures haptens (Small molecules)DrugHormoneMetaboliteFaster than ELISA(No need to separate free and bound enzyme labels)ELISAMeasures macromoleculesAntigensAntibodiesGreater Sensitivity
29ReferencesBaylis PH, The International Journal of Biochemistry and Cell Biology. 35 (2003) p1495 – 1499Choi 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,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:Verbalis JG, Best practice and Research clinical endocrinology and metabolism. Vol 17, No.4, p471 – 503, 2003The Merck manual 17th Edition Centennial edition 1997Australian Pharmaceutical Formulary and Handbook 18th Edition 2002Arthur 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):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 125I-radioimmunoassay at two Australian Centres. Therapeutic Drug Monitoring. 18(6):Caplan A & Jack R. Clinical Chemistry; interpretation and techniques. 4th edition. Williams& Winkins; London 1995