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Liver testing + disease, Watch lecture, AST:ALT ratio helpful - if >1…

- - - - Absorbed nutrients
- - - - Synthesis + storage of glucose units e.g. glycogen
        
        During periods of carbohydrate availability
      - Glycogenolysis
        
        During fasting
      - Gluconeogenesis
        
        Mainly occurs in liver
  - - - Lipoproteins
      - Phospholipids
      - Cholesterol
      - Endogenous triglycerides
    - - Products excreted in bile
  - - - Plasma proteins
      - Special carrier proteins
      - Coagulation factors
      - Immunoglobulins are NOT produced in liver
  - - - Vitamins A, D, E, K, B9 + B12
      - Copper
      - Fats
  - - - Drugs
      - Toxins
      - Abnormal presence e.g. high levels, unnatural substance
    - - Glucuronidation
      - Sulfation
      - Improves excretion
  - - - Haem oxygenase + bilirubin reductase
      - Bilirubin complexed with albumin + transported to liver
        
        Hepatocytes take up complex
        
        Conjugation of bilirubin with glucuronides + secretion in bile
        
        Conjugation increases solubility
        
        Bacterial breakdown to urobilinogens (excreted in faeces)
        
        20% urobilinogens reabsorbed + re-excreted in bile + urine
    - - Bilirubin-derived pigments responsible for colour
        
        Urine - Urobilin
        
        Faeces - Stercobilin
      - Pale stool indicates problems with bilirubin metabolism
    - - Yellow pigment - jaundice
      - Antioxidant property
        
        Minute amount (nanomolar) has protective function
        
        Damage caused by highly reactive reactive free radicals
        
        Inverse relationship between serum bilirubin serum conc. (within reference range) + future risk of coronary artery disease (CAD)
- - - - Direct
        
        Conjugated
        
        All water soluble
      - Indirect
        
        Unconjugated
        
        Total - direct
      - Total
        
        Sum of conjugated + unconjugated
        
        Solubilised by accelerator (caffeine-sodium benzoate)
      - Azidopyroles in serum
        
        1.Formation
        
        Diazonium salt reacts with conjugated bilirubin
        
        Caffeine-sodium benzoate + diazonium salt reacts with all bilirubin
        
        Add ascorbic acid
        
        Stop reaction
        
        Add alkaline tartrate
        
        Blue-coloured form of aziopyroles
        
        Measure absorbance at \(\lambda\) = 600nm
      - Urine
        
        Same principle as serum
        
        Different diazonium salt
        
        Must be done on fresh urine
        
        Only conjugated enter urine (unconjugated insoluble in water)
        
        Test detects ~3\(\mu\)mol/L
        
        False positives in patients on large doses of chlorpromazine
      - Bilirubin metabolites
        
        Urine strip tests
      - Detects general impairment to metabolism
      - Biliary tract involvement in pathology
    - - Total protein of biological fluids (serum, urine, CSF)
        
        Biuret test
        
        Under alkaline conditions cupric ions (Cu\(^{2+}\)) react with proteins \(\geq\)2 peptide bonds
        
        Reagent contains Na\(^+\)/K\(^+\)-tartrate to form complex with Cu\(^{2+}\) ions (maintain solubility) + iodide (antioxidant)
        
        Absorbance of Cu\(^{2+}\)-protein complex at \(\lambda\)=540nm directly proportional to conc. of protein in sample
        
        Conditions
        
        Serum preferred over plasma
        
        Fasting specimen desirable - decrease risk of lipaemia
        
        High emulsified fat conc. in blood
        
        Haemolysis should be avoided
        
        Frozen + thawed specimens thoroughly mixed before assay
      - Albumin
        
        Properties of albumin
        
        Globular protein Mr=66.3kDa
        
        Normally most abundant protein in plasma (\(\frac{1}{2}\) protein mass) - 35-50g/L
        
        Abnormally high = dehydration
        
        Typically lower in hospital in-patients
        
        Synthesised primarily by liver
        
        Functions
        
        Maintenance of colloidal osmotic pressure (COP)in extravascular + vascular spaces (continuous equilibrium between them)
        
        Transport of compounds e.g. free fatty acids, metallic ions, drugs, hormones + bilirubin
        
        Measurement
        
        Automated dye-binding methods
        
        Bromocresol green (BCG)
        
        Bromocresol purple (BCP)
        
        Absorbance of albumin-BCG complex measured at \(\lambda\)=628nm
        
        Absorbance proportional to [albumin]
        
        Serum used over plasma - overestimation from fibrinogen + heparin
        
        Erroneous if overall serum protein pattern is abnormal
        
        Fetal albumin (\(\alpha\)-fetoprotein/AFP)
        
        Normally present in fetal serum
        
        Small levels in maternal serum + urine
        
        Clears fast
        
        Abnormal levels indicate birth defects
        
        Ab test detection
        
        Undetectable past infancy
        
        Elevation in males + non-pregnant females
        
        Indication of tumours
        
        Hepatocellular carcinoma
        
        Metastases affecting liver
    - - Most coagulation factors produced in liver
      - Measured in plasma from blood sample with citrate anticoagulant (binds Ca\(^{2+}\))
      - Coagulation started by adding excess Ca\(^{2+}\) + thromboplastin (factor III)
      - Factor III degrades in sample
      - Coagulation time measured optically
      - Detects general impairment to metabolism - lack of albumin + coagulation factors
  - - - ALT - alanine
        
        ALT converts L-alanine to pyruvate
        
        Pyruvate can be detected
        
        Incubate sample with 2,4-dinitrophenylhydrazine (20 mins)
        
        Stop reaction with strong base
        
        Monitor decrease in absorbance at \(\lambda\)=340nm
        
        Pyruvate converted to pyruvate hydrazone
      - AST - aspartate
        
        AST converts L-aspartate to oxaloacetate
        
        Oxaloacetate can be detected
        
        Incubate sample with 2,4-dinitrophenylhydrazine (20 mins)
        
        Stop reaction by adding strong base
        
        Monitor decrease at \(\lambda\)=340nm
        
        Oxaloacetate converted to oxaloacetate hydrazone
      - AST is not specific to liver damage, can be raised by myocardial infarction
      - ALT specific to hepatocyte damage
      - Non-hepatic causes of raised ALT + AST
        
        Coeliac
        
        Haemolysis
        
        Hyperthyroidism
  - - - Measure rate of pNPP to pNP (yellow) in presence of Mg\(^{2+}\) + Zn\(^{2+}\)
      - Change in abosrbance measured at \(\lambda\)=410/480nm (415nm ideal)
      - Change in absorbance proportional to ALP activity
    - - Catalyses transfer of \(\gamma\)-glutamyl group from \(\gamma\)-glutamyl-3-carboxy-4-nitroanilide to glycylglycine
        
        Yellow 5-amino-2-nitrobenzoate produced
      - Chnage in absorbance measured at \(\lambda\)=405nm
      - Change in absorbance directly proportional to GGT activity
    - - It is not specific
        
        Produced in
        
        Hepatocytes
        
        Biliary epithelial cells
        
        Pancreas
        
        Renal tubules
        
        Intestine
        
        Lots of drugs increase GGT despite normal liver function e.g. alcohol, phenytoin, barbiturates
        
        Also associated with CVD + other clinical conditions e.g. pancreatic disease, MI, COPD, renal failure, DM
  - - - Cheap
      - Visualisation of
        
        Fatty liver
        
        Large masses
        
        Large bile ducts
      - Non-invasive
      - Unable to detect inflammation/cirrhosis
    - - Visualise smaller bile ducts + head of pancreas
      - Expensive
      - Invasive
    - - Expensive
      - Invasive
      - Gold standard
      - See hepatic pathology + hepatocytes
- - - - Haemolysis
      - Gilbert's syndrome (decreased conjugation activity)
      - Transient neonatal jaundice (liver enzymes not fully active)
  - - - Normal route of excretion blocked
    - - No bilirubin reaches gut, no urobilin formed, pale stools
  - - - Haemolysis - bilirubin production
      - Unconjugated
      - Causes
        
        Acute/chronic haemolytic anaemia
        
        Neonatal physiological jaundice (transient)
        
        Underlying causes
        
        Inherited RBC disorders
        
        Vit B12 deficiency
        
        Blood type/rhesus factor incompatibility
        
        Drugs
      - Increased urobilinogen in urine + stool important in diagnosis of haemolytic anaemia
    - - Defect in bilirubin metabolism
      - Mixed
      - Conjugation failure
        
        Crigler-Najjar syndrome
        
        Deficiency/decreased activity of UDP-glucuronyl transferase
        
        Neonatal physiological jaundice (transient)
      - Post-conjugation failure
        
        Dubin-Johnson syndrome
        
        Mutation in multidrug transporter prevents transport of conjugated bilirubin into bile
      - Diffuse hepatocellular damage/necrosis
        
        Viral hepatitis
        
        Toxic hepatitis
        
        Cirrhosis
        
        Injured cells lose ability to conjugate bilirubin
        
        Damaged cells provide path for leakage of bilirubin glucuronides back into sinusoids
        
        Incapable of removing all urobilinogen from portal blood - more into circulation
        
        Kidney excretes urobilinogen + conjugated bilirubin into urine in high amounts
    - - Defect in bilirubin excretion
      - Conjugated
      - Obstruction of common bile duct
        
        Stones
        
        Neoplasms
        
        Spasms or strictures
        
        Narrowing
      - Conjugate bilirubin cannot reach intestine
        
        No urobilinogen produced
      - Faeces vary in colour from light brown to chalky white
      - Urine contains no urobilinogen + high conjugated bilirubin
      - Blood contains high levels of conjugated + unconjugated bilirubin - regurgitation + liver cell impairment
      - Obstructive cholestasis
        
        Accompanied by high ALP
        
        Can cause secondary hepatic damage
- - - - Paracetamol (acute overdose)
      - Carbon tetrachloride
      - Alcohol
  - - - Hep A, B, C, D, E + G
        
        B, C, D blood borne
        
        B + C transmitted through unsafe sex
        
        D only infects people with B
        
        A + E transmitted through contaminated water/food - associated with poor sanitation/personal hygiene
        
        Effective vaccines for all except C
        
        Disease progress
        
        Hep A never leads to hepatic failure - full recovery with no permanent damage (2 months)
        
        Hep B + C may lead to hepatic failure
        
        Some cases of Hep B - persistent antigenaemia leading to chronic liver disease
        
        20% of Hep C patients develop cirrhosis over 20-30 years - transplant
        
        Chronic Hep C associated with increased risk of liver cancer
      - Epstein-Barr
      - Cytomegalovirus
    - - Evidence suggesting hepatocellular damage due to host reactions against virally infected cells
      - Plasma biochemical changes reflect pathological processes similar for all hepatitis virus infections
        
        Early acute phase
        
        Plasma aminotransferases rise - concentrations more than 50 x reference limit
        
        Acute illness subsides
        
        Aminotransferase activity returns to normal
        
        May be moderately elevated for longer in protracted/chronic cases
        
        Plasma bilirubin levels rise slowly, peaking at 10-20 x reference limit
        
        ALP + GGT only mildly elevated
  - - - Accumulation of tough, fibrous scar tissue in liver
      - Over time leads to cirrhosis
    - - Portal hypertension
      - Liver failure
      - Liver cancer
    - - Chronic excessive alcohol intake
      - Autoimmune disease
      - Persistence of Hep B virus
      - Metabolic diseases e.g. Wilson's
    - - Trans-abdominal needle biopsy of liver
        
        Painful, hazardous + time-consuming
        
        Subject to inter-observer variability = poor reference standard
      - Indirect markers of fibrosis
        
        Markers released into blood due to inflammation
        
        ALT + AST
        
        Markers synthesised, regulated or excreted by liver
        
        Clotting factors
        
        Cholesterol
        
        Bilirubin
        
        Processes disturbed by impaired liver function
        
        Insulin resistance