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

Carbohydrate metabolism Glycogenesis Glycogenolysis Gluconeogenesis Hypoglycaemia Glucose Intolerance IMPAIRMENT http://static.howstuffworks.com/gif/diabetes-glucose-regulation.gif

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 http://www.medscape.com/pi/editorial/clinupdates/2001/608/art-cu02.fig04.gif

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

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 http://www.biosbcc.net/doohan/sample/images/blood%2520cells/clottingcascade Clotting disorders, ↓ osmotic pressure IMPAIRMENT

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 http://sbsweb.bangor.ac.uk/images/bsx1016/sm_liver_position

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 http://www.aspartame-detox.info/images/brain-01.jpg

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

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

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? http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/18127.jpg

Liver disease? http://upload.wikimedia.org/wikipedia/en/thumb/e/e0/Gollum.PNG/220px-Gollum.PNG

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 http://www.sciencephoto.com/images/download_lo_res.html?id=771500153

Jaundice Icterus (icteric) Accumulation of bilirubin = hyperbilirubinaemia Skin, conjuctiva, mucous membranes Dark urine from renal excretion of bilirubin http://www.modulomedico.com/fotos/imgJaundiceBig.jpg

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

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

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: http://www.np.edu.sg/~dept-bio/biochemistry/aab/topics/asptrans.gif

Alterations in ALT and AST Mild elevations(<100U/L) Fatty liver/non-alcoholic steatohepatitis (fatty degeneration) Chronic viral hepatitis Moderate elevations(100-300 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 500-1500 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

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

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

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

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;481-492 Histological patterns in drug-induced liver disease

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

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

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

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.

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

Drug Induced Liver Disease (DILD) Types of hepatic injury Hepatitis Cholestasis Mixed Fibrosis Granulomatous lesions Neoplasms http://www.health-writings.com/img/mk/drug-induced-liver-disease/drugs_MIC062ML.jpg

Drug induced hepatic failure http://www.path.cam.ac.uk/Normal/AR_Alimentary/LV_Liver/N_AR_LV_02.jpg http://mayoresearch.mayo.edu/mayo/research/nyberg_lab/images/histology.jpg

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

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

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

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

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

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): 1738-1744. 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: 103-119. 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: 947-954.

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

Paracetamol toxicity Toxicity when GSH reserves overwhelmed by N-acetyl-p-benzoquinone imine (NAPQi) ± gastric lavage and activated charcoal Treatment → IV N-acetylcysteine http://drugdiscoveryopinion.com/images/paracetamol_metabolism.jpg

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): 296-302.

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): 296-302. 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): 296-302.

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): 296-302. 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): 296-302.

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

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

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

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: 541-546

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

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)

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

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

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

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

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

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

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

Clinical findings No abnormalities detected

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

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

Clinical findings Jaundice Temperature 38.3oC No abdominal pain or swelling

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

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

Clinical findings No abnormalities detected

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

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

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

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

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

Any Questions?