Clinical Chemistry - 10 Liver Function Tests

CLINICAL CHEMISTRY LIVER FUNCTION TESTS OUTLINE • The Liver o Liver o Functions of the Liver • Tests for Liver Synthetic Function o Total Protein o Serum Protein Electrophoresis o Albumin o Albumin/Globulin Ration o Prothrombin Time • Test for Conjugation and Excretion Function o Bilirubin o Delta Bilirubin o Jaundice • Inherited Disorders of Bilirubin Metabolism o G ilbert’s Syndrome o Crigler-Najjar Syndrome o Dubin-Johnson Syndrome and Rotor Syndrome o Lucey-Driscoll Syndrome • Bilirubin Methodology o Bilirubin Assay o Clinical Significance o Urobilinogen • Tests for Detoxification Function o Enzymes Tests o Ammonia THE LIVER LIVER • The chief metabolic organ o Most biochemical/metabolic reactions in the body occur in the liver • A large organ divided unequally into two lobes by a falciform ligament • Lobules – functional unit of the liver o 2 major type of cells: ▪ Hepatocytes (80%) - functional hepatic cells ▪ Kupffer cells – hepatic macrophage (phagocytes) • 2 blood supply (receive 1500 mL per minute): o Hepatic artery (25%) - supply oxygenated blood to liver o Portal vein (75%) - supply deoxygenated blood to liver ▪ Supply majority of blood in liver FUNCTIONS OF THE LIVER 1. Synthetic Function o Liver secretes plasma proteins, carbohydrates, lipids, lipoproteins, ketone bodies, enzymes o Synthesis/production ▪ Albumin = 12 g/day (most abundant plasma protein) 2. Conjugation Function o Metabolism of bilirubin o Bilirubin = 200-300 mg/day 3. Detoxification and Drug Metabolism o Protects the body from potentially toxic substances absorbed from intestine & toxic metabolic by-products ▪ Toxic substances include drug metabolites, etc. 4. Excretory and Secretory Function o Excretion of bile involves elimination of bile acids or salts, pigments, cholesterol 5. Storage Function o Storage site for all fat-soluble & water-soluble vitamins o Storage site for glycogen (stored form of energy) ▪ complex carbohydrate composed of multiple glucose molecules TESTS FOR LIVER SYNTHETIC FUNCTION • Quantitates severity of hepatic dysfunction • Includes albumin and vitamin K dependent clotting factors for assessment o Albumin - included due to synthesis in liver o Clotting factors - proteins which are all produced in liver except for calcium ▪ vitamin K dependent for production TOTAL PROTEIN • Assessment of nutritional status and presence of severe disease involving the liver, kidney and bone marrow • Included in liver synthetic function because albumin comprises large function of total protein content • Sample : serum o Plasma contains fibrinogen and anticoagulant ▪ Not preferred o Fibrinogen in plasma = increase total protein (0.2-0.4 g/dL) o Anticoagulant = decrease total protein due to dilutional effect. • Interference : hemolysis, ictericia • Reference value (serum): 6.5-8.3 g/dL (CF to g/L: 10) o Serous fluid (serous cavities) : ▪ <3.0 g/dL – Transudates ▪ >3.0 g/dL - Exudates METHODS FOR TOTAL PROTEIN • Kjeldahl Method • Biuret Method • Folin-Ciocalteu (Lowry) Method • UV Absorption Method • Refractometry • Turbidimetry and Nephelometry • Salt Fractionation • Coomasie Brilliant Blue Dye • Ninhydrin • Serum Protein Electrophoresis SERUM PROTEIN ELECTROPHORESIS (SPE) • Principle : Migration of charged particles in an electric field o (+) charged substance move toward cathode (-) o (-) charged migrate toward anode (+) • The width of protein band in a particular fraction depends on the concentration of the protein in that fraction. o Thicker protein band in a fraction on the electrophoretic field, there is an increase in protein

• Isoelectric Property of Proteins o Proteins are amphoteric in anture o Amphoteric – either positive or negative depending on pH condition ▪ Proteins contain amino acids in which its structures include central C atom, carboxyl group, ammonia group, R group • Carboxyl group - provide (+) charge • Amino group - provide (-) charge o No charge at isoelectric point ▪ pH in which a substance has 0 net charge o The acidic and basic amino acids content of proteins determines its net charge. ▪ Positively- charged substance → cathode ▪ Negatively- charged substance → anode o Buffer : Barbital (Veronal) – pH 8.6 ▪ Adjust pH of electrophoretic media to basic ▪ Major serum proteins are negatively-charged at pH 8.6 (move toward anode + ) NORMAL SPE PATTERN • Separates proteins into 5 fractions: 1. Albumin band (1st band) – fastest band o Reference value : 3.5-5.0 g/dL (53-65%) o Include albumin (major plasma protein) o Farthest from origin (most anodal) o High spike indicates high concentration 2. α 1 Globulin – 2nd fastest band o α 1 -antitrypsin as the major contributor (90%) o Increased in response to inflammation (non-specific) ▪ Most proteins are positive acute phase reactant o Reference value : 0.1-0.3 g/dL (2.5-5%) 3. α 2 Globulin band – 3rd fastest band o Reference value : 0.6-1.0 g/dL (7-13%) 4. β Globulin band – 4th fastest band o Transferrin - major contributor (90%) o Reference value : 0.7-1.1 g/dL (8-14%) 5. γ Globulin band – slowest band o Include immunoglobulins o Closest to point of origin o Reference value : 0.8-1.6 g/dL (12-22%) • Measured using densitometry MULTIPLE MYELOMA • Cancer of plasma cells • Plasma cells produces immunoglobulins • Increase in immunoglobulin due to autonomous production of plasma cell due to cancer • Electrophoresis shows a gamma spike (monoclonal gammopathy) o Monoclonal gammopathy - 1 abnormal protein band HEPATIC CIRRHOSIS • Decreased albumin due to decreased hepatic synthesis. • Increased γ-globulin is also present • Electrophoresis shows abnormality in 3 bands (Albumin, β- globulin, γ-globulin) with characteristic β-γ bridging o IgA can connect β-globulin and γ-globulin bands NEPHROTIC SYNDROME • Decrease in albumin and other low-molecular weight proteins due to increased renal excretion in the urine. o Glomerulus fails to filter proteins • Increased in α 2 - macroglobulin, β-lipoprotein, complement proteins, and haptoglobin. • Electrophoresis shows α 2 -globulin band spike o May include slight increase in β-band (from β-lpp)

α 1 -ANTITRYPSIN DEFICIENCY • Seen in juvenile cirrhosis • Electrophoresis shows α 1 -globulin band flat curve • α 1 -Antitrypsin comprises 90% of the proteins found in the α 1 -globulin band - causes band flat INFLAMMATION • Inflammation can cause increase of positive acute phase reactants. • Most positive acute phase reactants are found in the α 1 , α 2 , and β globulin bands • Electrophoresis shows spikes in α 1 , α 2 , & β globulin bands CLINICAL SIGNIFICANCE • Increased Total Protein o Malignancy - autonomous production of globulins o Multiple myeloma - cancer of plasma cells which produce increased immunoglobulins (gamma spike) o Waldenströms macroglobulinemia - blood cancer with increase of IgM (gamma spike) • Decreased Total Protein o Hepatic cirrhosis - decreased synthetic function o Glomerulonephritis - increased protein secretion o Nephrotic syndrome - increased protein secretion o Starvation - decreased hepatic synthesis due to low amino acid reserves/source ALBUMIN • Concentration is inversely proportional to the severity of hepatic disease. o Low conc. = more severe • Low albumin due to decreased synthesis • Plasma albumin declines if severe hepatocellular disease lasts more than 3 weeks. • Low total protein + low albumin = hepatic cirrhosis and nephrotic syndrome o Hepatic cirrhosis - decreased protein synthesis o Nephrotic syndrome - increased protein secretion • Can be measured directly based on its dye-binding property METHODS FOR ALBUMIN DETERMINATION Method Principle Salt Precipitation Globulins are precipitated (from salt) Albumin in supernatant is quantitated by biuret reaction Dye binding Methyl orange Nonspecific for Albumin HABA More specific but with many Interferences (salicylates, bilirubin) BCG (Bromcresol green) Sensitive Overestimates low albumin levels Most commonly used dye BCP (Bromcresol purple) Specific, Sensitive and Precise HYPERALBUMINEMIA • Increased albumin levels • Severe dehydration • Prolonged tourniquet application o Causes pseudohyperalbuminemia due too poor specimen collection. HYPOALBUMINEMIA • Reduced synthesis o Chronic liver disease - impair hepatic synthetic function o Malabsorption syndrome - non-absorption of necessary nutrients/substances for protein synthersis o Malnutrition and muscle wasting disease o Analbuminuria ▪ Hereditary absence of albumin or inability to synthesize albumin. • Increased catabolism o Massive burns o Widespread malignancy o Thyrotoxicosis - increased conc. thyroid hormones (bound to albumin) • Increased loss o Nephrotic syndrome (20-30g/day) ▪ Increased albumin excretion due to inability of glomerulus to restrict passage of proteins from the blood o Massive burns o Protein-losing enteropathy o Orthostatic albuminuria - posture-related BISALBUMINEMIA • Presence of two albumin bands instead of a single band in electrophoresis • It is the presence of albumin with unusual molecular characteristics in the blood. • Associated w/ excess amount of therapeutic drugs in serum

ALBUMIN/GLOBULIN RATIO • Determined to validate if globulin is higher that serum • Inverted A/G ratio : o Occurs if globulin is greater that albumin o Seen in cirrhosis, multiple myeloma and waldenström’s macroglobulinemia ▪ Cirrhosis - ↓ albumin from ↓ synthesis ▪ Multiple Myeloma an d Waldenström’s Macroglobulinemia - increased gamma globulins • Reference value : 1.3-3.1 g/dL (CF to g/L: 10) • Not analytically measured and are computed • Formula : PROTHROMBIN TIME • Vitamin K Response Test • It differentiates intrahepatic disorder from extrahepatic disorder o Intrahepatic disorder = prolonged prothrombin time o Extrahepatic disorder = normal prothrombin time • Some clotting factors are vitamin K dependent • Prolonged prothrombin time after vitamin K administration indicates loss of hepatic capacity to synthesize the proteins. • Acute/Chronic Hepatitis – prolonged prothrombin time signifies massive cellular damage. • Vitamin K is administered intramuscularly (10mg for 1-3 days) TEST FOR CONJUGATION AND EXCRETION FUNCTION • Involves measurement of Bilirubin BILIRUBIN • End product of hemoglobin metabolism • Principal pigment in bile • Also formed from destruction of heme-containing proteins such as myoglobin, catalase and cytochrome oxidase TYPES OF BILIRUBIN • Bilirubin 1 o Unconjugated Bilirubin o Water Insoluble o Non-polar Bilirubin o Indirect bilirubin o Aka Hemobilirubin ▪ Found in blood o Slow reacting o Prehepatic bilirubin o Colorless bilirubin • Bilirubin 2 o Conjugated Bilirubin o Water soluble ▪ Can be in urine o Polar Bilirubin o Direct bilirubin o Aka Cholebilirubin ▪ Found in liver o One-minutes/Prompt Bilirubin o Post-hepatic/ Hepatic/ Obstructive/ Regurgitative Bilirubin • When RBCs reach senescence (after 120 days), RBCs are destroyed in spleen extravagating Hgb • Hgb are degraded into components (iron, heme, globin) o Iron - bind to transferrin o Transferrin will transport globin to bone marrow or other iron stores where protein will be broken down into its amino acid components • UDGPT - Uridine diphosphate glucuronyl transferase • B2 will be bound to bile and transported to intestines • Intestinal flora - normal bacteria found in the intestines • Urobilinogen can be reabsorbed o Can be re-conjugated into the liver to form B2 o Can be excreted through urine • Plasma concentration of bilirubin increases upon birth and reaches its on the 5th day. o Increased bilirubin with newborns • The intracellular conjugation of glucoronic acid onto two sites of the bilirubin molecule confers negative charge to it, making conjugates bilirubin soluble in aqueous phase • Only small amounts of conjugated bilirubin circulates in the blood due to minor leaking of hepatocytes • If bilirubin synthesis > liver clearance rate = increased bilirubin level • Reference value : o Conjugated bilirubin : 0-0.2 mg/dL (0- 3 μmol/L) ▪ Hypobilirubinemia do not exist o Unconjugated bilirubin : 0.2-0.8 mg/dL (3- 14 μmol/L) o Total bilirubin : 0.2-1.0 mg/dL (3- 17 μmol/L) DELTA BILIRUBIN • Conjugated bilirubin bounded to albumin • Has longer half-life than other forms of bilirubin • Formed due to prolonged elevation of conjugated bilirubin in biliary obstruction • Helps in monitoring the decline of serum bilirubin following cholecystectomy (removal of gallbladder) • It reacts w/ diazo reagent (reagent for bilirubin measurement) • Reference value : <0.2 mg/dL o Computed : Delta bilirubin = TB – DB + IB o Not calculated on neonatal patients (≤14 days)

JAUNDICE • Hyperbilirubinemia characterized by yellow discoloration of the skin, sclera, and mucus membranes. • Jaundice do not develop on fetus as placenta normally removes bilirubin • Hyperbilirubinemia (adults): most commonly caused by choleslithiasis (gallbladder stones) • Bilirubin >2.0 mg/dL (3.0-5.0 mg/dL) • Icterus – more commonly termed for serum or plasma with yellow discoloration due to hyperbilirubinemia. o Visible icterisia : bilirubin >25 mg/L PRE-HEPATIC JAUNDICE • Hemolytic jaundice • Unconjugated hyperbilirubinemia o Increase in unconjugated bilirubin in blood • Caused by too much red blood cell destruction o Too much RBC destruction increase Hgb extravagated which increase degradation into heme that will be broken down into bilirubin o Seen in: ▪ Hemolytic disease of the newborn • Fetus do not develop jaundice as placenta normally removes bilirubin ▪ Hemolytic anemia ▪ Malaria • Laboratory findings : o Indirect Bilirubin : increased o Direct Bilirubin : normal o Urobilinogen : normal o Urine bilirubin : Negative • Indirect Bilirubin – can cross the blood brain barrier (BBB) resulting to kernicterus o BBB - separates the brain from other parts o Kernicterus – bilirubin deposition in the brain causing severe motor dysfunction and retardation POST-HEPATIC JAUNDICE • Obstructive jaundice • Caused by failure of bile to flow to the intestine or impaired bilirubin excretion • Laboratory diagnosis : o Indirect bilirubin : normal (heme = normal) o Direct bilirubin : increased (excreted in blood) o Urobilinogen : decreased (no B2 degraded) o Urine bilirubin : positive (B2 is water soluble) o Alkaline phosphatase : increased ▪ Enzyme found in liver ▪ #1 marker of post-hepatic jaundice ▪ Increased due to increased excretion rate HEPATIC JAUNDICE • Hepatocellular combined jaundice • Caused by disorders of bilirubin metabolism, transport defects, hepatocellular injury or destruction. • Laboratory findings : o Indirect bilirubin : increased o Direct bilirubin : increased o Urobilinogen : decreased (transport defects) o Urine bilirubin : positive INHERITED DISORDERS OF BILIRUBIN METABOLISM • Gilbert’s Syndrome • Crigler-Najjar Syndrome (Type I & II) • Dubin-Johnson Syndrome • Rotor Syndrome • Lucey-Driscoll Syndrome GILBERT’S SYNDROME • Bilirubin Transport Deficit • Characterized by impaired cellular uptake of bilirubin due to genetic mutation in UGT1A1 gene (chromosome #2) o UGT1A1 gene - encodes for production of UDGPT • Affected individuals may have no symptoms but may have mild icterus and predisposed acetaminophen toxicity. o Acetaminophen - metabolized in liver with UDGPT • Laboratory findings : o Indirect Bilirubin of < 3.0 mg/dL (1.5-3.0 mg/dL) ▪ Rarely increased 4.5 mg/dL CRIGLER-NAJJAR SYNDROME • Conjugation deficit • Chronic non-hemolytic unconjugated hyperbilirubinemia • Treatment : phototherapy • Laboratory result : elevated indirect bilirubin o Due to inability to conjugate bilirubin to form conjugated bilirubin cause by UDGPT deficiency • Crigler-Najjar Syndrome Type I o Complete deficiency of UDGPT o Laboratory result : ▪ Indirect bilirubin : >25 mg/dL ▪ Direct bilirubin : none o Symptoms : kernicterus, colorless bile • Crigler-Najjar Syndrome Type II o Partial deficiency of UDGPT o Laboratory results: ▪ Indirect bilirubin : 5-20 mg/dL o Only small amount of direct bilirubin is produces. DUBIN-JOHNSON SYNDROME AND ROTOR SYNDROME • Bilirubin Excretion Deficit • Defective excretion of direct bilirubin into the canaliculi caused by hepatocyte membrane defect. o Cell membrane defect inhibits bilirubin excretion • Characterized of an intense dark pigmentation of the liver (“black liver”) due to accumulation of lipofuscin pigment • Rotor Syndrome – similar with Dubin-Johnson Syndrome without the “black liver” o Cause is unknown • Laboratory findings : o Elevation in direct bilirubin & total bilirubin (2-5 mg/dL) ▪ B1 is still converted into B2 but not excreted to intestines which eventually forms delta bilirubin o Delta Bilirubin : increased LUCEY-DRISCOLL SYNDROME • Familial form of unconjugated hyperbilirubinemia caused by a circulating inhibitor of bilirubin conjugation • UDGPT is inhibited • Laboratory findings : o Indirect bilirubin : increased (2-3 weeks of life)

BILIRUBIN METHODOLOGY • Sample : serum o Should be stored immediately in dark after specimen collection ▪ Bilirubin is a photosensitive analyte o If sample is prepared and stored properly : ▪ Room temperature : 2 days ▪ Refrigerator (4 ̊C): 1 week ▪ Freezer (-20 ̊C): indefinite o If unprotected from light : ▪ Bilirubin is reduced by 30-50% per hour o Fasting sample is preferred • Interferences : o Lipemia : increased bilirubin concentration ▪ May interfere with spectrophotometry o Hemolysis : decrease reaction of bilirubin with diazo reagent (decreased bilirubin) • Unconjugated bilirubin is slow reacting: o Addition of accelerants allows measurement of total bilirubin (after 15 minutes of reaction time) o Without accelerants, only direct bilirubin can be measured BILIRUBIN ASSAY • Principle : Van den Berg Reaction – diazotization of bilirubin to form azobilirubin • Only measure conjugated bilirubin and total bilirubin. • Unconjugated bilirubin = Total bilirubin – conjugated bilirubin • Two Methods : o Evelyn and Malloy Method o Jendrassik and Grof Method EVELYN AND MALLOY METHOD • Principle : Van den berg reaction • Reagents : o Coupling accelerator : Methanol ▪ May also precipitate proteins increasing turbidity which can interfere with the test o Diazo reagents : ▪ Diazo A – 0.1% sulfanilic acid + HCl ▪ Diazo B – 0.5% sodium nitrite ▪ Diazo blank – 1.5% HCl • Performed at pH 1.2 • Final reaction : pink to purple azobilirubin (with maximal absorption at 560 nm) JENDRASSIK AND GROF METHOD • Most commonly used method • More sensitive than Evelyn and Malloy method • Popular technique for discreet analyzers • Main reagent : Diazo reagent • Accelerator : Caffeine Sodium Benzoate • Buffer : Sodium acetate • Ascorbic acid : immediately added after addition of accelerator to terminate the accelerator and destroys excess diazo reagent • Alkaline tartrate solution : provides alkaline pH after addition of ascorbic acid • Final reaction : blue azobilirubin (measured at 600nm) • CLINICAL SIGNIFICANCE • Increased Direct Bilirubin o Gilbert’s Syndrome o Criggler-Najjar Syndrome o Hemolytic anemia o Hepatocellular disease o Lucey-Driscoll Syndrome o G-6-PD deficiency ▪ Also presents increased RBC destruction • Increased Indirect Bilirubin o Biliary obstruction (gall stones) o Pancreatic (head) cancer o Dubin-Johnson Syndrome o Alcoholic and viral hepatitis o Biliary atresia o Hepatocellular disease UROBILINOGEN • Colorless end product of bilirubin metabolism that is oxidized by intestinal flora to urobilin (brown pigment) • Either excreted in urine and feces, or reabsorbed in to the portal blood and returned to the liver. • Absence of this substance in urine or stool denotes complete biliary obstruction o Biliary obstruction - cannot allow excretion of bilirubin in intestines which converts B2 to urobilinogen • In the collection of sample: avoid light exposure • Specimen : 2-hr freshly collected urine or freshly collected stool. • Method : Ehrlich’s method o Reagent : p-dimethyl aminobenzaldehyde o Also used in urine reagent strip for urobilinogen • Reference value : o Urine : 0.1-1.0 Ehrlich Unit/2 hour or 0.54 Ehrlich Unit/day urine o Stool : 75-275 Ehrlich Unit/100g of feces BROMSULFONTHALEIN (BSP) DYE EXCRETION TEST • Rarely used • Requires administration of BSP dye • Test for hepatocellular function and potency of bile duct • 2 Methods of Administration : o Rosenthal White Method o Mac Donald Method • Rosenthal White Method o Double Collection Method (twice blood collection) o BSP Dose : 2mg/Kg body weight o Specimen collection : ▪ 5 minutes after BSP administration ▪ 30 minutes after BSP administration o Reference value : ▪ After 5 minutes: 50% dye retention ▪ After 30 minutes: 0% dye retention • Mac Donald Method o Single Collection Method o BSP Dose : 5 mg/Kg body weight o Specimen collection : after 45 minutes post BSP o Reference value : After 45 mins: +/- 5% dye retention

TESTS FOR DETOXIFICATION FUNCTION • Involves enzyme and ammonia test ENZYMES TESTS • It is used to assess the extent of liver damage. • Indicator of cell injury in early or localized liver disease (hepatocellular/functional disease) • Enzymes are intracellular • Hepatic enzymes : o Alanine aminotransferase (ALT) o Aspartate aminotransferase (AST) o 5’ nucleotidase o Gamma glutaryl transferase o Lactate dehydrogenase o Ornithine Carbamoyl Transferase AMMONIA • 2nd detoxification marker • 2nd marker for cirrhosis (2 nd to albumin) • Diagnostic marker of hepatic failure • Produced by the catabolism of amino acids and by bacterial metabolism in the intestine. • Neurotoxin – can be converted to glutamate which can compromise Kreb’s cycle leading to coma o Kreb’s cycle - an important cycle for producing ATP as energy source • Also released from metabolic reactions in skeletal muscle • Detoxified into urea (liver), then eliminated by the kidneys (urine) • Not useful for renal studies o Ammonia is not freely filtered by kidneys o Requires conversion to urea to be excreted via urine • Reference value : 19-60 ug/dL METHODOLOGY • Preferred specimen : o Arterial blood ▪ Heparin and EDTA ▪ Kept in ice water during transport o Serum collection requires fist clenching and the use of tourniquet ▪ May promote hemoconcentration that may cause hemolysis • Specimen precautions : o Smoking – increased by 100-200 ug/L/cigar ▪ Smoking or exposure to cigar leads to increased ammonia. ▪ Patient & phlebotomist should not smoke/be exposed to smoke prior to collection o Exercise o Probing of vein o Under filing of tube o Hemolysis • Common methods : Berthelot & Glutamate Dehydrogenase • Methods : o Digestion Method o Nesslerization Reaction o Berthelot Reaction o Glutamate dehydrogenase DIGESTION METHOD • Kjeldahl Method • Nitrogen ion in a protein-free filtrate of the specimen is converted to ammonia using hot concentrated sulfuric acid in the presence of catalyst • Catalyst : copper sulfate, mercury, and selenium DIRECT COLORIMETRIC METHOD • Nesslerization Reaction o Yellow end-color: N2 (low to moderate) o Orange-brown end-color: N2 high • Berthelot Reaction GLUTAMATE DEHYDROGENASE METHOD • Inverse colorimetric enzymatic reaction • Decrease in absorbance at 340nm as NADPH is consumed (oxidized) • Measures consumption of NADPH •