Ischaemic Heart Disease Group D

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Ischaemic Heart DiseaseGroup D
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Scenario

• Mr Pex (55 yrs) has taken these medications for 2
  years
• Perhexiline 100mg bd- anti-angina
• Gliclazide 80mg bd- hypoglycaemic agent
• Fluoxetine 20mg d- antidepressant
• It is suspected that Mr. Pex has perhexiline
  toxicity because he has signs and symptoms of
  hepatotoxicity and peripheral neuropathy
• How it should be managed
• Place of Monitoring levels of Perhexiline
• Explanation of any medications that could be
  responsible for the abnormalities in the clinical
  chemistry profile
• Whether the abnormalities are likely to be
  ongoing
• Recommended management for this patient with
  respect to his medication regimen

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Clinical chemistry profile
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Perhexiline

• indicated for angina
• plasma/blood perhexiline concentrations must be
  maintained within the range 0.15-0.6mg/L (0.5-
  2umol/L)
• Perhexiline is metabolised via CYP450 2D6
  (patient genetic variability- fast, intermediate,
  slow metabolisers) resulting in variable
  clearance.
• Genetic Polymorphism approximately 10 of the
  population are slow metabolisers of perhexiline
  and are at a higher risk of toxicity, thus
  requiring decreased doses
• Perhexiline and its metabolites undergo
  extensive hepatic metabolism and are excreted in
  bile and urine (ratio 12 respectively)
• It has a low therapeutic index so therapeutic
  drug monitoring is essential.
• Therapeutic Drug Monitoring factors
  Individualised dosage Target range
  Concentration related effects (therapeutic and
  adverse) Narrow Therapeutic Index Desired
  therapeutic effect is difficult to monitor.

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Perhexiline pharmacokinetics

• gt80 of perhexiline maleate is absorbed from the
  GIT after oral dosing
• Large volume of distribution, that is probably
  related to the tissue binding of perhexiline and
  its metabolites
• It crosses the BBB (lipophilic)
• Highly protein bound (gt90). Some binding to
  erythrocytes
• Saturable rate of hepatic metabolism (genetic
  polymorphism of CYP2D6)
• Several metabolites with unknown pharmacological
  activity
• Racemic mixture- 2 Enantiomers

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Perhexiline toxicity

• long term- usually occurs gt 3 months of continued
  therapy
• Directly related to perhexiline blood
  concentrations
• Hepatotoxicity- elevation in serum liver enzymes
  (AST aspartate aminotransferase, ALT alanine
  aminotransferase, alkaline phosphatase, LDH
  lactate dehydrogenase). Monitoring is essential
  at least every month.
• In Mr Pex AST, ALT Alkaline Phosphatase, LDH
  elevated. These liver enzymes show that the liver
  is damaged. They increase with liver dysfunction
• Aminotransferases- ALT, AST are sensitive
  indicators of hepatic inflammation and necrosis.
  ALT is more specific as it is mainly found in the
  liver.
• Alkaline Phosphatase primarily made in liver,
  but also in bones. Sensitive marker of damage.
• LDH LDH4, LDH5 appear mainly in the liver and in
  skeletal muscles
• Total Bilirubin (conjugated unconjugated) low

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Hepatotoxicity

• Hepatitis and Cirrhosis have been reported.
  (Higher AST vs. ALT may be indicative of
  cirrhosis but chronic alcohol consumption must be
  excluded)
• Though, more specifically the non-alcoholic
  fatty liver disease Non-alcoholic Steatohepatitis
  (NASH) is associated with perhexiline maleate
• NASH is usually asymptomatic and may progress to
  cirrhosis within 7 years (inflammation and
  necrosis)
• Abnormal liver function test results are present
• Markers Serum aminotransferase activities
  usually lt4 fold above the upper limit of normal
  moderately elevated serum ALT and AST
• Insulin resistance may occur and may be unrelated
  to the presence/absence of obesity

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Non-alcoholic steatohepatitis

• No biological test to positively identify it
• The following diagnostic approach can be used by
  health care professionals, to show hepatotoxicity

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Peripheral neuropathy

• Occurs when nerves connecting spinal cord and
  brain to other parts of the body become damaged
• 3 different types mono- (damage to a single
  nerve) multiple mono- (two or more nerves)
  poly-neuropathy (many nerves throughout the body)
• Most common causes drugs eg. perhexiline,
  diabetes
• Patient signs and Symptoms
• (begin in hands or feet may spread throughout
  the limbs)
• Tingling, prickling, numbness
• Sharp pain
• Decreased or lack of sensation
• Muscle weakness
• Lack of muscle control
• Burning or freezing sensations
• Extreme sensitivity to touch
• Loss of balance or coordination

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Tests for signs and symptoms

• Signs of muscle cramping/twitching and muscle
  weakness
• Skin sensitivity to temperature changes, touch,
  vibration, pinpricks
• Observation for clinical signs of hepatic
  involvement eg. Weakness, loss of appetite,
  weight loss
• Nerve function tests eg. EMG (Electromyography-
  measures muscle electrical activity)
• Analysis of serum enzymes (AST, ALT, alkaline
  phosphatase, LDH) and bilirubin- abnormalities or
  persistent elevations
• Blood Glucose measurements- persistent/marked
  hypoglycaemia
• Weight- excessive weight loss (gt10 initial
  weight)
• Perhexiline plasma levels- therapeutic target
  range

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Gliclazide

• Symptoms of hypoglycaemia
• Comfusion
• Agitation
• Tachycardia
• Tremor
• Hypothermia
• May progress to coma/convulsions

• Risk Factors
• Advanced age
• Poor nutrition
• Alcohol
• Renal disease
• Hepatic disease
• Concurrent medications

Rarely associated with increases in ALP, AST and
bilirubin Hypersensitivity can occur with
accumulation
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Fluoxetine (1)

• Metabolised by 2D6, as is perhexiline
• Clinically significant drug interactions occur
  when
• Therapeutic index of substrates is narrow
  (applies to perhexiline)
• Isozyme involved in competition is the primary
  metabolic pathway (applies to perhexiline and
  fluoxetine both pathways are saturable at
  therapeutic concentrations)
• Concentration of inhibiting substrate reaches
  sufficient levels in vivo (demonstrated for both)
  

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Fluoxetine (2)

• Liver enzyme elevations may occur (reported in
  about 0.5 of patients receiving SSRIs)
• gt58,000 reports of hepatic ADRs with SSRIs
  other medications
• 493 suspected to be due to fluoxetine
• 12 acute hepatitis (6 were on concurrent
  medications)
• 5 asymptomatic increase in serum transaminases
• 80 with paroxetine
• 65 caused by sertraline
• 54 attributed to fluvoxamine

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Management of Mr. Pex

• Perhexiline?
• Discontinue
• Reassess angina (severity, frequency of attacks)
  and initiate alternative treatment
• Fluoxetine?
• Reassess the need for this treatment
• Phenotyping may be of some value if PM status is
  suspected
• Alternative agent could be used (different class
  of antidepressant, or try fluvoxamine/sertraline
  which are less potent CYP2D6 inhibitors and less
  frequently associated with hepatic ADRs
• Gliclazide?
• Assess glycaemic control (ongoing)
• Determine hepatic and renal function
• Change of agent may be necessary

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Bilirubin

• Outline the chemical basis for the
  spectrophotometric analysis of bilirubin
• Look at the various techniques used to obtain
  values for total and direct bilirubin
• Examine the problems and variations of different
  approaches used to examine patient bilirubin
  profiles

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Bilirubin

• bile pigment formed from the breakdown of
  Haemoglobin
• found in serum as unconjugated, conjugated and
  delta
• Unconjugated bilirubin is insoluble in aqueous
  solution, bound to serum albumin and represents
  bilirubin prior to hepatic processing
• Conjugated bilirubin is formed subsequent to
  hepatic processing and is yielded by unconjugated
  bilirubins esterification of its two proprionic
  side groups with glucoronic acid.
• Delta bilirubin represents bilirubin which is
  covalently bound to albumin
• Since the liver is involved in the enzymatic
  modification of bilirubin, the serum plasma
  concentration is used as a test for hepatic
  function
• Elevation in unconjugated bilirubin can occur as
  a result of excessive bilirubin production eg.
  haemolysis, or the inability to conjugate or take
  up bilirubin from the circulation
• Elevations in conjugated bilirubin are commonly
  seen in hepatocellular or biliary dysfunction

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Analysis of bilirubin in serum

• The most widely used methods for the measurement
  of serum bilirubin are based on the diazo reaction

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Analysis of bilirubin in serum

• Jendrassik Grof Assay
• Uses caffeine-benzoate which acts as an
  accelerator
• Caffeine benzoate displaces unconjugated
  bilirubin from albumin perhaps making it more
  water soluble by disruption of the internal
  hydrogen bonds ? making bilirubin more readily
  available for reaction with the diazo reagent
• Performed at alkaline pH
• Azobilirubins produced in these reactions are
  measured spectrophotometry at 600nm
• Evelyn Malloy Assay
• Uses methanol to dissociate albumin
• Performed at acidic pH
• Absorbances are measured at 560nm producing red
  or purple colour

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Direct bilirubin

• Conjugated Bilirubin Diazotized sulfanilic
  acid ? Azobilirubin B (Isomers 1 2)
• Measures the majority of conjugated and delta
  bilirubin and a variable but small percentage of
  unconjugated bilirubin
• To prevent measurement of unconjugated bilirubin,
  the serum should be diluted with HCl first

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Total bilirubin
DIRECT BILIRUBIN Conjugated Bilirubin
Diazotized sulfanilic acid ? Azobilirubin B
(Isomers 1 2)   INDIRECT BILIRUBIN Total
Bilirubin Direct Bilirubin Indirect Bilirubin
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Other methods used to obtain bilirubin fractions

• High Performance Liquid Chromatography
• Measures four bilirubin fractions in serum
• Unconjugated bilirubin
• Delta bilirubin
• Bilirubin monocongugate
• Bilirubin diconjugate

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Other methods (cont.)

• Direct Spectrophotometric Method
• Measures conjugated and unconjugated bilirubin
  and calculates delta bilirubin as the difference
  between the sum of these and total bilirubin
• Based on the absorbance of unconjugated bilirubin
  at 454nm and Hb at 540nm
• Enzymatic Methods
• BOX
• Bilirubin ½ 0² ? Biliverdin H2O
• Based on enzyme bilirubin oxidase
• Oxidation rates depend on the pH of the reaction
  mixture
• Maximum
  oxidation
• Conjugated pH 4.5
  10
• Unconjugated pH 6
• Delta pH 4

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Problems with techniques

• Despite the advances of these methods, in the
  clinical laboratory they still have limitations.
  Some of these are related to bilirubin's
  instability and insolubility in water, but there
  are also problems of assay interference, lack of
  pure conjugated bilirubin standards, and
  interpretation of bilirubin fractions depending
  on the method on use

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Diazo method

• Limitations include
• The assumption that direct and indirect
  bilirubins represent conjugated and unconjugated
  bilirubins, respectively although in several
  assays this may be incorrect.
• The lack of adequate standards for calibration of
  the direct bilirubin assay. Direct bilirubin
  assays have used unconjugated bilirubin for assay
  calibration, not a particularly stable compound
  and is not ideal because assays should be
  calibrated with the analyte that they are
  designed to measure. Many manufacturers now use
  synthetic forms of bilirubin in their calibration
  and control material, and it is hoped that use of
  these synthetic variants will help improve the
  accuracy of the direct bilirubin assay
• Direct bilirubin assay is dependent on reaction
  conditions, especially pH and often
  underestimates conjugated bilirubin , this leads
  to inaccuracies in indirect bilirubin

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Direct spectrophotometry

• The assay is only suitable for serum neaonates
  (usually less than 2 - 3 weeks of age) because
  other pigments, notably carotene, start to appear
  as infants get older and cause interference at
  454nm.
• Studies have shown that such spectrophotmeteric
  methods are not only rapid, easy to carry out,
  requiring small samples but also are less
  influenced by factors like hemoglobin
  concentration and hemolysis

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HPLC

• Originally limited by inadequate fraction
  quantitation and the large sample size
  requirement
• Modifications allowed measurement of all four
  fractions using small sample size. More recently
  developed procedures do not precipitate albumin
  and lose delta bilirubin
• Delta bilirubin represents bilirubin covalently
  bound to plasma proteins, predominately albumin.
  This binding unlike that of unconjugated
  bilirubin, is resistant to physical, chemical,
  and enzymatic treatments
• HPLC is considered the gold standard as it
  measures all four fractions, however, it is
  extremely expensive, elaborate and time consuming
  for routine clinical use. It is also labor
  intensive and requires specialized equipment and
  therefore not a method suited for a laboratory
  required to perform bilirubin 7 days per week,
  often on a 24-hour basis. It does however remain
  a method to which the more commonly used
  procedures can be compared

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Specimen requirements

• Because both conjugated and unconjugated forms of
  bilirubin are photo-oxidised on exposure to UV
  light, it is recommended that sample should be
  protected from light. Bilirubin is unstable and
  light sensitive and therefore the assay should be
  carried out within 2 hours of sample collection.
  If a longer delay is unavoidable, refrigerate the
  sample. Bilrubin is stable in the refrigerator
  (40C) for 3 days. Samples can be frozen at -700C,
  to keep bilirubin stable for 3 months

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Measurement in urine

• Because conjugated but not unconjugated bilirubin
  is excreted in urine, uniary examination may be
  used as a simple screen to determine whether high
  levels of bilirubin is due to prehepatic causes
  or to hepatic or post hepatic disorders.
• The urine specimen to be investigated should be
  fresh. If delays are anticipated, the urine
  container should be protected from light and
  refrigerated.
• Urine bilirubin measurements are often made using
  qualatative methods such as dipsticks impregnated
  with diazo reagent, which reacts with bilirubin
  to produce a colour change.
• The conjugated forms of bilirubin can be isolated
  from bile however, are not suitable for mass
  isolation and use in calibrators and control
  material and therefore unstable

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References

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