CORE AREA 2 CARDIOVASCULAR Topic C

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CORE AREA 2CARDIOVASCULAR Topic C
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CASE STUDY

• Mr. DD
• 60 years old
• smoker gt 40 years
• Conditions CHF and COPD
• Medications
• - enalapril 10mg BD
• - frusemide 80mg mane
• - salbutamol MDI prn

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Mr. DDs Biochemistry Results
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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

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Clinical Manifestations of SIADH

• Cardinal signs of SIADH are
• Hyponatremia serum Na lt 120mmol/L
• Serum hypo-osmolality (overhydration)
• Normal acid-base and potassium balance
• Concentrated urine
• Low blood urea and nitrogen levels

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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

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Hypernatraemia

• Acute hyponatraemia
• Serum Na lt 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 lt 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.

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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.

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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

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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.

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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.

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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.

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Digoxin

• Used to treat heart failure
• Narrow therapeutic window
• Normal dose 1-2 ng/mL
• Toxic dose gt2 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.

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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

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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

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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.

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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

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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 ? Absorbance from reaction rate and
  drug concentration
• Non-linear relationship between ? Absorbance and
  concentration

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Procedure Contd

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

                                                
                Fig 3.8.1 EMIT Assay Components
in Action
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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

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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

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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

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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.

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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.

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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

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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.

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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.

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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

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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
  71503-507
• Verbalis JG, Best practice and Research clinical
  endocrinology and metabolism. Vol 17, No.4, p471
  503, 2003
• The Merck manual 17th Edition Centennial edition
  1997
• Australian Pharmaceutical Formulary and Handbook
  18th Edition 2002
• http//www.endocrinology.ed.ulca.edu/siadh.htm
• 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
  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