1. CORE AREA 2 CARDIOVASCULAR Topic C

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

3. Mr. DD’s Biochemistry Results

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
ADH 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.

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
60% SIADH present with dilutional hyponatraemia.

6. 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. tumours
Pulmonary lesions and other lung diseases
Neurological (CNS) disorders
Medications e.g. psychoactive drugs, oral hypogylcaemics and substances e.g. nicotine
SIADH 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

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.
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)

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) 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.

9. 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
ACE inhibitors block the conversion of angiotensin I to II in the PNS not CNS.

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.
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.

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.

12. Conclusion of SIADH
Potential 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 sodium
COPD and nicotine may contribute to SIADH by increasing the release of ADH.

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.
Digoxin may also be used to treat cardiac arrhythmias.

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

15. Advantages and disadvantages of RIA
Sensitivity( 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

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.

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

18. Procedure of the EMIT
Mix 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

19. Fig 3.8.1 EMIT Assay Components in Action
Procedure Cont’d
Determine standard curve and obtain concentration of the analyte from standard curve
                                                               
Fig EMIT Assay Components in Action

20. Advantages and disadvantages of EMIT
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 M)
Lower limit of quantification than RIA method
Advantages – 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.

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
There 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

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

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.
The antibody part of the conjugate binds to any antigen molecules that were bound previously creat

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.

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

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.
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.

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.

28. Comparison of EMIT & ELISA
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

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,
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Verbalis JG, Best practice and Research clinical endocrinology and metabolism. Vol 17, No.4, p471 – 503, 2003
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Australian Pharmaceutical Formulary and Handbook 18th Edition 2002
Arthur S. (1985). Role of serum digoxin assay in patient management. The American Journal of Cardiology. 5(suppl 5): 106A-110A.
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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):
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