CORE AREA 2CARDIOVASCULAR

Topic C

CASE STUDYMr. DD

60 years oldsmoker > 40 yearsConditions: CHF and COPDMedications:

- enalapril 10mg BD- frusemide 80mg mane- salbutamol MDI prn

Mr. DD’s Biochemistry Results

Electrolyte Result Reference Interval

Sodium 117 132-144 mmol/L

Potassium 4.5 3.3-4.7 mmol/L

Bicarbonate 26 22-32 mmol/L

Urea 2.1 2.5-6.5 mmol/L

Osmolality 260 280-300 mmol/kg

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

Clinical Manifestations of SIADH

Cardinal signs of SIADH are:Hyponatremia; serum Na+ < 120mmol/L Serum hypo-osmolality (overhydration)Normal acid-base and potassium balanceConcentrated urineLow blood urea and nitrogen levels

Clinical 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 diseases Neurological (CNS) disordersMedications e.g. psychoactive drugs, oral hypogylcaemics and substances e.g. nicotine

HypernatraemiaAcute 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.

Treatment 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.

Which 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 problems

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.

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.

Conclusion 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.

DigoxinUsed to treat heart failureNarrow therapeutic window:

Normal dose: 1-2 ng/mLToxic 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 disordersHalo vision

Hence serum levels should be monitored. This is achieved with RIA, EMIT and ELISA.

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

Advantages and disadvantages of RIA

Advantages:• Sensitivity(10-10-10-11 M)• Specificity• Determines the concentration of both

macromolecular antigens & small haptensDisadvantages:• High level of wastage and expensive• Short shelf life of radioisotope • Labour-intensive• Radiation Exposure

EMIT

Homogenous & Competitive immunoassay systemSeparation 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.

Components of the EMIT

Drug to be measured is the hapten part of the antigen Antibody binding the enzyme-hapten conjugation, inhibiting the enzyme activityBuffered substrateEnzyme covalently linked to pure drug such as glucose-6-phosphate dehydrogenase

Procedure of the EMITMix sample of serum with a solution containing antibody, enzyme-hapten complex & buffered substrate. Incubate at 37ºC for short time

• Measure rate of absorbance changed at 340nm by UV-visible spectroscopy

• Determine Δ Absorbance from reaction rate and drug concentration

• Non-linear relationship between Δ Absorbance and concentration

Procedure Cont’d

Determine standard curve and obtain concentration of the analyte from standard curve

                                                             

   Fig 3.8.1 EMIT Assay Components in

Action

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

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 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 antigen

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

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.

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.

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

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.

Competitive 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.

Comparison of EMIT & ELISA

EMITMeasures haptens (Small molecules)

DrugHormoneMetabolite

Faster than ELISA(No need to

separate free and bound enzyme labels)

•ELISAMeasures

macromolecules•Antigens•Antibodies

Greater Sensitivity

ReferencesBaylis 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, 417-420Izzedine 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 71:503-507Verbalis 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 2002 http://www.endocrinology.ed.ulca.edu/siadh.htmArthur 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 125I-radioimmunoassay at two Australian Centres. Therapeutic Drug Monitoring. 18(6): 672-677. Caplan A & Jack R. Clinical Chemistry; interpretation and techniques. 4th edition. Williams& Winkins; London 1995