1. Liver function Organ = metabolism Gland = secretions
Haemotological regulation
Phagocytosis and antigen presentation
Plasma protein synthesis
Removal of circulating hormones
Removal of antibodies
Removal /storage of toxins
Metabolic regulation
Carbohydrate metabolism
Lipid metabolism
Amino acid metabolism
De-toxification and excretion
Bilirubin metabolism
Storage - vitamins [A, D, B12], Cu, Fe
Bile production

2. Carbohydrate metabolism
Glycogenesis
Glycogenolysis
Gluconeogenesis
Hypoglycaemia
Glucose Intolerance
IMPAIRMENT

3. Lipid Metabolism Lipoprotein synthesis Oxidation of fats
Synthesis of cholesterol, phospholipids
Catabolism of steroids
IMPAIRMENT
Altered lipid profiles
2O Hyperaldosteronism →↑BP ↓K ↑pH
2O Hypercorticolism = Cushing’s disease
Gonadal dysfunction → ∆ oestrogen:testestorone

4. Amino acid metabolism Deamination and transamination of amino acids
Removal of ammonia
Synthesis of amino acids
IMPAIRMENT
Hepatic encephalopathy
Hypoproteinaemia
Source: seqcore.brcf.med.umich.edu/ mcb500/aametov.gif

5. Synthesis of hormones and plasma proteins…….
Insulin-like growth factor (IGF-1) → stimulated by pituitary growth hormone
Thrombopoietin → platelet production
Angiotensinogen (α-2-globulin) → hydrolysed by renin to angiotensin [renin-angiotensin-aldosterone system (RAAS)]
Heparin
Albumin
Extrinsic pathway clotting factors →I (fibrinogen), II (Prothrombin), IV, V, VI, and VII
Clotting disorders, ↓ osmotic pressure
IMPAIRMENT

6. Bile Acids e.g. taurocholic
Bile production ….
Water
Bile Acids e.g. taurocholic
and glycocholic acid
Electrolytes
Cholesterol
Phospholipids
Bilirubin
IMPAIRMENT
Malabsorption of fats and fat soluble vitamins
Jaundice

7. Detoxification – refer to Biochemistry and PK done in years 2/3.
Phase I = modify
Phase II = conjugate
IMPAIRMENT
Reduced drug metabolism, reduced protein binding of drugs

8. Filtration – Reticuloendothelial system
Blood filtration, phagocytosis of bacteria and other particulate matter
Exposure to bacteria and other particles
IMPAIRMENT
Source : i/15204loa.jpg

9. Symptoms of liver disease
Most symptoms non specific - anorexia, malaise, fatigue, fever
↓ general health
cirrhotic habitus = wasted extremities plus protuberant abdomen with ascites
Generalised pruritus (itchiness) – due to retention of bile salts
Xanthelasma (fat build up under skin surface), xanthomas
Pale stools – lack of bile
Which of these is most specific?
akimichi.homeunix.netXanthoma disappearance Document 272 x 324 pixels - 55k - gif

10. Disorders of coagulation/circulation
↑ bleeding and bruising
↑ prothrombin time (PT) → extrinsic clotting pathway (Prothrombin ratio (PR) and international normalized ratio (INR) are derived measures of PT).
Thrombocytopaenia (↓platelets)
Dysfibrinogenaemia (altered fibrinogen function)
Portal hypertension → endothelial stretching and shear stress → ↑ NO → systemic vasodilation = hyperdynamic circulation
Hepatopulmonary syndrome = pulmonary vasodilation → ↑blood flow (ventilation-perfusion mismatch) → arterial desaturation
Cyanosis and clubbing → enlargement of distal fingers and toes. Due to vasodilation?

11. Liver disease?

12. Other changes ↑ parotid salivary gland → fatty infiltation
Gynecomastia (mammary gland development in males), testicular atrophy, impotence
Amenorrhoea (absence of menstrual period)
Erythema (redness of skin) – build up of unmetabolised wastes in body

13. Jaundice Icterus (icteric)
Accumulation of bilirubin = hyperbilirubinaemia
Skin, conjuctiva, mucous membranes
Dark urine from renal excretion of bilirubin

14. Serum chemistry/diagnostic testing
Bilirubin
Breakdown product of haemoglobin
Globin = protein
Heme = iron containing → biliverdin (green bruising) → bilirubin → yellow (bruising/bile)
Bilirubin → conjugated with glucuronides in liver
Excreted in bile
Can be measured in the unconjugated (indirect) or conjugated (direct) form
Relatively insensitive indicator of liver disease
Source:web.indstate.edu/thcme/ mwking/hemedegradation.jpg

15. From Beckett - Lecture Notes : Clinical Biochemistry 7th ed 2005
From Swaminathan – Handbook of Clinical Biochemistry 2004

16. Aminotransferases (ALT and AST)
Transaminase enzymes (aminotransferases) → reversible
transfer of an amino group between two a-keto acids.
Alanine aminotransferase = liver cytosol (ALT)
Aspartate aminotransferase = liver and other tissues (AST)
Reasonably sensitive indicators of liver disease, ALT >> AST
Source:

17. Alterations in ALT and AST
Mild elevations(<100U/L)
Fatty liver/non-alcoholic steatohepatitis (fatty degeneration)
Chronic viral hepatitis
Moderate elevations( U/L)
Acute or chronic hepatitis
Alcoholic hepatitis
Mild/moderate inflammation
High elevated(>300 U/L)
Acute viral hepatitis
Hepatic necrosis → drugs or toxins
Ischemic hepatitis /circulatory shock. VALUES CAN BE U/L
Values >3000 U/L- toxic necrosis, or severe hypoxia
AST/ALT ratio
Significant overlap between different conditions BUT
> 2 suggestive alcoholic liver disease (if ALT < 500 U/L)
< 1 viral hepatitis can ↑ratio as fibrosis and cirrhosis develop

18. Alkaline phosphatases (ALP)
ALP = hydrolase →removes phosphate groups
Present in bile canaliculi, bone and placenta
↑ sensitivity for hepatobiliary disease
↓ specificity for hepatobiliary disease
Due to its numerous isoenzymes, its presence in non-hepatic tissues, and its sensitivity to drug induction

19. Increases in liver ALP
Cholestasis, cholecystitis, cholangitis, cirrhosis, hepatitis, fatty liver, liver tumour, liver metastases, drug intoxication
Drugs e.g. verapamil, carbamazepine, phenytoin, erythromycin, allopurinol, ranitidine
↑ → enhanced synthesis rather than hepatocytic leakage
↓ ≠ clinically significant
If source ↑ ALP is not clear check other liver enzymes

20. Gamma glutamyl transpeptidase (GGT)
GGT → hydrolysis of gamma-glutamyl peptide bonds
Biliary enzyme → obstruction of biliary tract + damage to biliary capillaries
Easily induced (alcohol, drugs) → disproportionately ↑ in alcoholic liver disease
Can be elevated in other diseases e.g. CHD, MI, COPD, pancreatitis, renal disease

21. Patterns of enzyme alterations
ALP
ALT/ALP ratio
Hepatocellular
≥ 2x upper limit of normal
Normal
≥ 5
Cholestasis (bile flow obstruction)
≥ 2x ULN
≤ 2
Mixed disease ↑
2-5
Ramachandran and Kakar J Clin Pathol 2009;62; Histological patterns in drug-induced liver disease

22. ERCP = Endoscopic Retrograde Cholangiopancreatography From Beckett - Lecture Notes : Clinical Biochemistry 7th ed 2005

23. From Beckett - Lecture Notes : Clinical Biochemistry 7th ed 2005

24. Other tests Haematology (anaemia, RBC parameters)
Clotting tests (PT) NOTE – these are vitamin K-dependent clotting factors
Lipid tests (total cholesterol, HDL, LDL, triglycerides)
Biochemistry (albumin, glucose)
Serology
-Hepatitis virus
-Antimitochondrial antibody (present in >90% of biliary cirrhosis cases)
-Antinuclear factor- inflammatory marker
-Antismooth muscle antibody-inflammatory marker
-Alpha fetoprotein-hepatic carcinoma marker
Functional tests
-Clearance tests (caffeine, bromosulphthalein), Elimination tests (galactose)
Imaging procedures
-Abdominal radiographs
-US, NMR, CT
-Direct biliary visualisation → contrast studies
Liver Biopsy
-Percutaneous
-Surgical

25. Drug Induced Liver Disease (DILD)
> 1000 drugs → DILD
↑Hz drug withdrawal from the market R x
Reference Hughes et al. Use of laboratory test data: a process guide and reference for health care professionals. 2nd Ed, PSA, 2009.

26. Drug Induced Liver Disease (DILD)
Consider wherever altered liver function tests
However
Broad range of drugs
Wide variation in hepatic injury caused

27. Drug Induced Liver Disease (DILD)
Types of hepatic injury
Hepatitis
Cholestasis
Mixed
Fibrosis
Granulomatous lesions
Neoplasms

28. Drug induced hepatic failure

29. Drug Induced Liver Disease (DILD)
Other changes (functional)
Enzyme induction and inhibition
Dietary /other deficiencies e.g. cysteine, Vit K
Steatosis = fatty change
Phospholipidosis

30. Drug Induced Liver Disease (DILD)
Intrinsic
Dose related
Occurs within few days of use
Related to drug or toxic metabolite of drug
Known/reported/expected
Idiosyncratic
Unexpected
Variable latency
Not usually dose related

31. Intrinsic - usually dose related:
Drug
Reason
Paracetamol
Hepatocellular necrosis
Amiodarone
Chronic steatosis- due to total dose over time
Cyclosporine
Cholestasis due to toxic serum levels
Methotrexate
Elevated aminotransferase and fibrosis after single large dose
Niacin
Vascular injury after prolonged administration
Oral Contraceptives
Hepatic tumour after prolonged administration
Tetracycline
Steatosis after large total dose and renal dysfunction

32. Idiosyncratic - usually hypersensitivity
Drug
Reason
Isoniazid, diclofenac, nefazodone, trazodone, venlafaxine etc
Hepatocellular necrosis
CPZ, oestrogen, macrolides
Cholestasis
Phenytoin, sulphamethoxazole
Immune reaction
Diltiazem, sulphonamides
Granulomatous Hepatitis
Didanosine, tetracycline, valproic acid
Steatosis
Methotrexate
Fibrosis
Amoxycillin/Clavulonic Acid
Cholestatic injury

33. Factors affecting DILD
Age
Gender
Genetic factors
Nutritional status
Renal function
Dose and duration
Alcohol
Cigarette smoking
Other conditions- Hep C, HIV, RA

34. Paracetamol toxicity
Paracetamol = #1 drug → calls to poisons information centres in Australia and NZ. 1
Paracetamol = #1 drug overdose → hospital presentation and admission. 2,3
Hepatic failure and death → uncommon outcomes
#1 cause of acute fulminant hepatic failure in Western countries.4
Buckley N, Eddleston M. Paracetamol (acetaminophen) poisoning. Clin Evid 2005; (14):
Dart RC, Erdman AR, Olson KR, et al. Acetaminophen poisoning: an evidence-based consensus guideline for out-of-hospital management. Clin Toxicol (Phila) 2006; 44: 1-18.
Linden CH, Rumack BH. Acetaminophen overdose. Emerg Med Clin North Am 1984; 2:
Ostapowicz G, Fontana RJ, Schiødt FV, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 2002; 137:

35. Phases of paracetamol hepatotoxicity
PHASE 1 (0-24 H)
Asymptomatic
Anorexia
Nausea or vomiting
Malaise
Subclinical rise in serum transaminases levels begins at about 12 hours postingestion
PHASE 2 (18-72 H)
Right upper quadrant abdominal pain, anorexia, nausea, vomiting
Continued rise in serum transaminases levels
Admitted to hospital
PHASE 3 (72-96 H)
Centrilobular hepatic necrosis with continued abdominal pain
Jaundice
Coagulopathy
Hepatic encephalopathy
Nausea and vomiting
Renal failure
Fatality
PHASE 4 (4 D TO 3 WK)
Complete resolution of symptoms
Complete resolution of organ failure

36. Paracetamol toxicity
Toxicity when GSH reserves overwhelmed by N-acetyl-p-benzoquinone imine (NAPQi)
± gastric lavage and activated charcoal
Treatment → IV
N-acetylcysteine

37. Management plan- dose thresholds for NAC treatment
≠ serum readings <4 hours of poisoning
Frank F S Daly, John S Fountain, Lindsay Murray, Andis Graudins and Nicholas ABuckley(2008).Guidelines for the management of paracetamol poisoning in Australia and New Zealand — explanation and elaboration. A consensus statement from clinical toxicologists consulting to the Australasian poisons information centres. MJA 2008; 188 (5):

38. Management Plan
Source- Frank F S Daly, John S Fountain, Lindsay Murray, Andis Graudins and Nicholas ABuckley(2008).Guidelines for the management of paracetamol poisoning in Australia and New Zealand — explanation and elaboration. A consensus statement from clinical toxicologists consulting to the Australasian poisons information centres. MJA 2008; 188 (5):
Source: Frank F S Daly, John S Fountain, Lindsay Murray, Andis Graudins and Nicholas A Buckley(2008).Guidelines for the management of paracetamol poisoning in Australia and New Zealand — explanation and elaboration. A consensus statement from clinical toxicologists consulting to the Australasian poisons information centres. MJA 2008; 188 (5):

39. Management Plan cont…
Source- Frank F S Daly, John S Fountain, Lindsay Murray, Andis Graudins and Nicholas ABuckley(2008).Guidelines for the management of paracetamol poisoning in Australia and New Zealand — explanation and elaboration. A consensus statement from clinical toxicologists consulting to the Australasian poisons information centres. MJA 2008; 188 (5):
Source: Frank F S Daly, John S Fountain, Lindsay Murray, Andis Graudins and Nicholas A Buckley(2008).Guidelines for the management of paracetamol poisoning in Australia and New Zealand — explanation and elaboration. A consensus statement from clinical toxicologists consulting to the Australasian poisons information centres. MJA 2008; 188 (5):

40. Management Plan cont… Monitoring of liver function
Post –discharge patient education
Referral to Psychologist if needed

41. Kava (Piper methysticum)
Kava = plant native to South Pacific Islands
Western society → herbal remedy anxiety
Kavalactones = active components
-6 KL ~ 95% of the activity
Anxiolytic effect ~ benzodiazepines
-few side effects
-limited cognitive and motor impairment
Kava → hepatotoxicity e.g. hepatitis, cirrhosis, fulminant liver failure, death
Banned in many countries
Australia → voluntary recall 2002

42. Isolated Perfused Rat Liver (IPRL)
PUMP
Perfusate
Kavalactone
95% O2/5% CO2
Taurocholic acid
Krebs-Henseleit (KH) buffer
Inferior Vena Cava
Hepatic Portal Vein
So the model currently used in the laboratory is known as the isolated perfused rat liver model (IPRL) which allows simulation of in vivo conditions and standardization of experimental conditions. It is set up as follows.
So in this model the hepatic portal vein and inferior vena cava of rats are cannulated to isolate the liver within a contained circuit
The perfusate flowing the system will contain the kavalactone being studied either, kavain or methysticin, or none (if it is control) and oxygen/carbon dioxide is being bubbled through it for oxygenation. Taurocholic acid will also be in the perfusate to maintain bile flow and these components will be dissolved in an appropriate Buffer.
The perfusate will be pumped around the system for up to 2 hours. Periodically samples of the liver effluent and tissue will be taken for biochemistry and microscopy.
NOTE:
Think about dose of kavain and methysticin used
Think about the advantages and disadvantages of this system
lies between 8
and 10mmHg and thus remains within
physiological limits, i.e. 8–12mmHg (Gores
et al. 1986). A higher perfusion pressure
potentially causes barotrauma of the liver
and injury to the sinusoidal endothelial
cells, resulting in leakage of large particles
into the space of Disse due to an enlargement
of the sinusoidal fenestrations.
37°C

43. 10 µg/mL Kavain for 2 hours is hepatotoxic in IPRL studies
Light Microscopy
Scanning Electron Microscopy
This study was completed in our lab using the IPRL model.
If we look at the control light microscopy image you can see the hepatic triad (consisting of hepatic artery, hepatic portal vein and bile duct) as well as distinct sinusoids (micro-capillaries of the liver) which contain kupffer cells and natural killer cells.
However following kavain treatment you can see disruption of the hepatocytes, with not distinct sinusoids and severe vacuolisation. What you also notice in this scanning electron microscopy image is the lifting of the endothelial lining of the sinusoids and enlargement of the liver fenestry. This demonstrates that the major kavalactone, kavain induces significant and direct hepatocellular alterations in isolated perfused rat liver.
Control Liver
Following Kavain Treatment
Fu (2008) World J Gastroenterology 14:

45. Membranes and electron
dense structures
Distorted cell nuclei
Autophagosome
Whirled ER surrounding
mitochondrion

46. Advice for case studies
Common things occur commonly.
Uncommon things don’t.
When you have eliminated the impossible, whatever remains, however improbable, must be the truth.
Sherlock Holmes - The Sign of the Four (1890)

47. Case Study #1 – history and signalment
68 YO male retired labourer
Lethargy but no pain
BW ↓19kg in the last 3 months
Eating normally up to last 3 weeks
Dark urine and pale stools
‘Moderate’ drinker throughout lifetime

48. Clinical findings Jaundice Weakness
Palpable, non-tender mass in upper RHS abdominal quadrant

49. Serum chemistry Bilirubin predominantly conjugated ↑↑ ALP and GGT
Analyte
Result
Reference Range
Units
Albumin 32
36-47
g/L
ALP
632
40-125
U/L
ALT 55
10-40
Total bilirubin 90
2-17
µmol/L
GGT
200
10-55
Bilirubin predominantly conjugated
↑↑ ALP and GGT
Predominantly cholestasis
Pancreatic tumour obstructing common bile duct

50. Case Study #2 – history and signalment
21 YO female student
Flu-like symptoms for 2 days
Condition deteriorating – dark urine, vomiting
Recently returned from long holiday in Asia

51. Clinical findings Jaundice Temperature 38.6oC (36.5-37.5 oC)
Liver enlarged and tender

52. Serum chemistry Bilirubin predominantly conjugated ↑↑ ALT
Analyte
Result
Reference Range
Units
Albumin 40
36-47
g/L
ALP
190
40-125
U/L
ALT
560
10-40
Total bilirubin
110
2-17
µmol/L
GGT 60
10-55
Bilirubin predominantly conjugated
↑↑ ALT
Predominantly hepatocellular
Any other tests?
Serology - high Hepatitis A titre

53. Case Study #3 – history and signalment
48 YO male gardener
Fatigue
BW ↓8 kg over last 4 months
Denies any history of hepatitis, alcohol use, family history of liver disease, exposure to hepatotoxins

54. Clinical findings
No abnormalities detected

55. Serum chemistry Any other tests? PT = 18 s (10-13 s)
Analyte
Result
Reference Range
Units
Albumin 20
36-47
g/L
ALP 56
40-125
U/L
ALT 16
10-40
Total bilirubin 13
2-17
µmol/L
GGT 24
10-55
Any other tests?
PT = 18 s (10-13 s)
IM dose 10 mg Vitamin K returned PT to 12 s < 48 hours
Serology and endoscopy → Coeliac disease → interfering with Vit K and protein absorption

56. Case Study #4 – history and signalment
13 YO male
Muscle pain and feeling hot for 2 days
Eaten little over this time

57. Clinical findings Jaundice Temperature 38.3oC
No abdominal pain or swelling

58. Serum chemistry Bilirubin 90% unconjugated Any other tests?
Analyte
Result
Reference Range
Units
Albumin 45
36-47
g/L
ALP
180
40-125
U/L
ALT 30
10-40
Total bilirubin 60
2-17
µmol/L
GGT 35
10-55
Bilirubin 90% unconjugated
Any other tests?
Haematology – RBC and reticulocyte count normal
Gilbert’s syndrome = most common hereditary hyperbilirubinaemia
Familial autosomal dominant in 2-3% men
↓ activity of the glucuronyltransferase → ↓ bilirubin conjugation
Exposed by caloric restriction while ill with cold
↑ ALP but GGT normal ??
Raised ALP from bone turnover with onset of puberty

59. Case Study #5 – history and signalment
36 YO female lawyer
Very stressful job
Working 12 hour days striving for promotion
Feeling run down
Annual blood test

60. Clinical findings
No abnormalities detected

61. Serum chemistry Any other tests?
Analyte
Result
Reference Range
Units
Albumin 42
36-47
g/L
ALP
130
40-125
U/L
AST
180
8-42
IU/L
ALT 70
10-40
Total bilirubin 15
2-17
µmol/L
GGT
380
10-55
Any other tests?
Haematology – leukopaenia (↓WBC), macrocytosis (↑MCV)
Toxic effects on bone marrow?
Finally admits drinking one bottle wine per day over lunch and after work
Quit job. Quit drinking
3 months later – ALT 28 IU/L, GGT 50 IU/L, ALP 54 IU/L

62. Case Study #6 – history and signalment
26 YO male
Admitted to hospital with 5-day history of excruciating abdominal pain radiating up back
Patient reports persistent nausea and vomiting

63. Clinical findings Vital stable signs on admission No fever
Extreme reaction to abdominal palpation

64. Serum chemistry Any other tests? Blood test normal
Analyte
Result
Reference Range
Units
Albumin 34
36-47
g/L
ALP
110
40-125
U/L
ALT 24
10-40
Total bilirubin 16
2-17
µmol/L
GGT 23
10-55
Any other tests?
Blood test normal
Serum amylase normal
US and CT normal
Requested ongoing opiates for pain control
Opiates withdrawn → patient checked out → continue search for opiates elsewhere

65. Summary Variable interspecies anatomy
Histological structure open to interpretation
Metabolic functions (glucose, protein, fat)
Glandular function (produces bile)
Jaundice = hyperbilirubinaemia
Bilirubin is a product of heme catabolism
Pre-hepatic (unconjugated)
Hepatic (unconjugated + conjugated – glucuronic acid)
Cholestatic/obstructive → intra/post-hepatic (conjugated)
Diagnosis – symptoms, laboratory, imaging, biopsy

66. Any Questions?