Liver Function PDF - 2024

LIVER FUNCTION By Abla Abou-zeid Professor of Clinical Pathology Alexandria University Objectives – State the...

LIVER FUNCTION By Abla Abou-zeid Professor of Clinical Pathology Alexandria University Objectives – State the physiologic functions of the liver. – Discuss laboratory tests used to assess liver function. – Illustrate bilirubin metabolism. – Classify types of hyperbilirubinemia and discuss their causes. – Explain the laboratory methods for bilirubin determination. – Discuss the basic disorders of the liver and choose the appropriate laboratory tests which may be used to diagnose them. – Differentiate the various types of hepatitis. Hepatic blood supply: 1) The hepatic artery (25%): which supplies oxygen and nutrients for liver metabolism. It branches off the aorta, about 30% of the total cardiac output flows to the liver. 2) The portal vein (75%): part of the portal hepatic system. The portal vein connects capillary beds between the GIT and the liver. It brings blood rich in nutrients to the liver. The two blood supplies eventually merge into the hepatic sinusoids The hepatic vein: drains deoxygenated blood from the liver to the IVC. It leaves the liver carrying nutrients for the body (made by the liver) and waste products. Normal Liver Function The liver is composed of three systems: Hepatocyte : concerned with most metabolic reactions in the body, e.g. protein synthesis, synthesis of all coagulation factors (except vWF), carbohydrate metabolism, amino acid & nucleic acid metabolism, amino acid and dicarboxylic acid interconversions via transaminases (aminotransferases), lipoprotein synthesis and metabolism, xenobiotic & drug metabolism, storage of iron and vitamins such as A, D & B12,  synthesis of hormones e.g. angiotensinogen & IGF-1, hormone clearance e.g. insulin, parathyroid hormone (PTH), estrogens and cortisol. Metabolism of ammonia to urea. Biliary system: concerned with (a) bilirubin metabolism, a process that involves transport of bilirubin into the hepatocyte, its conjugation to glucuronic acid and its secretion into bile canaliculi, and the enterohepatic system. (b) bile salts metabolism. Reticuloendothelial system: (Kupffer cells) macrophages: (a) the immune system: site of defense against intestinal bacteria and removal of Ag-Ab complexes from the circulation (b) breakdown of hemoglobin from dead RBCs, giving rise to bilirubin, which, together with bilirubin from the spleen, enters the hepatocyte. Liver Functions – Synthetic: proteins, lipids and carbohydrates. – Detoxification: endogenous substances e.g. bilirubin, ammonia & hormones and exogenous substances e.g. drugs & carcinogens. – Excretory: Formation and excretion of bile and excretion of natural and foreign substances in the biliary tract. – Metabolic: carbohydrates, lipids and protein metabolism. – Storage: glycogen & iron. Liver Function Tests LFT are the various lab tests that are used to: – Screen for liver disease; – Identify nature of disease (hepatocellular, cholestatic, infiltrative); – Assess severity and prognosis of liver disease; – Follow up the course of liver disease LFT is a misnomer Limitations of liver function tests – Do not necessarily assess liver function – Lack sensitivity (i.e. may be normal in some liver diseases like cirrhosis) – Lack specificity (i.e. may be abnormal in non-liver disorders ) e.g. serum albumin is  low in nephrotic syndrome and in cirrhosis – Sensitivity is the number of true positives as a percentage of all the results that should have been positive: – The sensitivity of a test reflects the fraction of those with a specified disease that the test correctly predicts. – {TP/(TP + FN)} x 100 – It is the probability that a test result will be positive when the disease is present (true positive rate, expressed as a %). – Specificity probability that a test result will be negative when the disease is not present (true negative rate, expressed as a %). – The specificity of a test is the fraction of those without the disease that the test correctly predicts. – {TN / (TN + FP)} x 100 Non-hepatic causes of abnormal LFT – Increased serum bilirubin: Hemolysis – Ineffective erythropoiesis – Resorption of a large hematoma – Increased liver enzymes: Muscle injury – Alcohol abuse – Myocardial infarction – Increased serum alkaline phosphatase: Pregnancy – Bone disease – Low serum albumin: Poor nutritional status – Proteinuria – Malabsorption – Severe illness causing protein catabolism LFTs can be classified as follows: 1. Tests that assess excretory function: Serum & urine bilirubin, and urobilinogen in urine and stools. 2. Tests that assess synthetic and metabolic functions: Serum proteins, albumin, A/G ratio, PT, and blood NH3. 3. Tests that assess hepatic injury (liver enzyme studies): Serum ALT, AST, or cholestasis: ALP, GGT, and 5’-NT. 4. Tests that assess clearance of exogenous substances: caffeine or lidocaine excretion test. Hepatic Excretory Function – Organic anions are extracted from sinusoidal blood, biotransformed and excreted into bile or urine. – Tests to assess excretory function include: 1. Serum levels of endogenous substances: S. bilirubin & S. bile acids 2.Clearance rates of exogenous compounds (e.g. caffeine, lidocaine) Bilirubin Biochemistry: Daily production: 250 – 300mg Sources: – 85%: Haem from senescent RBCs destroyed in RES – 15%: Red cell precursors (ineffective erythropoiesis), catabolism of haem-containing proteins e.g. myoglobin, cytochromes, peroxidases Formation: 1 mole haem produces 1 mole bilirubin as follows: Haem → protoporphyrin IX microsomal haem oxygenase biliverdin IXa biliverdin reductase bilirubin IXa. Bilirubin Metabolism In macrophages mainly in the spleen, Hb from RBCs is split to give free globin chains and heme. The porphyrin ring of heme is oxidized by microsomal heme oxygenase, producing the straight-chain compound biliverdin. Biliverdin is then reduced to bilirubin by biliverdin reductase. Bilirubin is then transported mainly in the portal system, bound to albumin, to the liver where it enters the hepatocyte on its membrane surface in contact with the sinusoids Albumin dissociates at sinusoidal membrane of hepatocytes & bilirubin alone is transported into the cell by an active carrier-mediated process where it binds to cytosolic proteins: Z & Y. Ligandin improves uptake efficiency by preventing reflux of bilirubin to plasma Inside hepatocyte: bil microsomal bil UDPglucuronyltransferase bil. monoglucuronide (10%) + diglucuronide (90%). Conj. bilirubin is then transported to the canalicular face of the hepatocyte to be secreted, by an energy-dependent mechanism, into the canaliculi; unconjugated bilirubin can’t traverse this membrane. In the intestine: bil. glucuronides β glucuronidase from intestine& bacteria. unconj. bil. reduction by intestinal flora 3 colourless tetrapyrroles : urobilinogen, stercobilinogen & mesobilinogen. 20% of urobilinogen is reabsorbed, and most of it enters an EHC to be re-excreted in bile, 2 – 5% of re-absorbed urobilinogen enters the systemic circ. to be excreted in urine. In the lower intestine, urobilinogens oxidize spontaneously into the bile pigments: urobilin, stercobilin & mesobilin which are the main pigments (orange brown) in stool. Hyperbilirubinemia Hyperbilirubinaemia results in jaundice which becomes clinically manifest when serum bilirubin exceeds 3 mg/dL. I- Unconjugated: A- Pre-hepatic: Acute hemolytic anemia. Chronic hemolytic anemia. B- Hepatic. Decreased hepatic uptake of bilirubin. Conjugation defect. II. Conjugated: A- Hepatic: Impaired secretion of conjugated bilirubin into bile. B- Post-hepatic: Intra-hepatic cholestasis. Post-hepatic cholestasis. 1. Prehepatic: (Unconjugated hyperbilirubinemia) – Hemolysis: Usually, bilirubin levels are not very high. Rarely exceed 5 mg/dL. – Neonatal physiological jaundice – Haemolytic disease of the newborn due to Rh incompatibility. Unconjugated bilirubin is increased, may reach 20 mg /dL, crossing the BBB resulting in kernicterus. 2. Hepatic: A) Decreased hepatic uptake of bilirubin (Unconjugated hyperbilirubinemia) –Gilbert's syndrome, hepatitis, cirrhosis. B) Conjugation defect, e.g.: (Unconjugated hyperbilirubinemia) – Crigler- Najjar syndrome, (Gilbert). – Neonatal physiological jaundice due to defect in the enzyme system responsible for conjugation. It is corrected after a few days of life. – Diffuse hepatocellular damage e.g. viral hepatitis, toxic hepatitis, cirrhosis C) Impaired secretion of conjugated bilirubin into bile (Conjugated hyperbilirubinemia) – e.g. Dubin- Johnson syndrome, Rotor syndrome and in diffuse hepatocellular damage e.g. viral hepatitis, toxic hepatitis, PBC, PSC. 3. Post hepatic: (Conjugated hyperbilirubinemia) Cholestasis may be intra or extra- hepatic, both lead to conjugated hyperbilirubinemia which is excreted in urine. Intrahepatic cholestasis: Occurs in acute hepatocellular damage due to hepatitis, cirrhosis and carcinoma. Post hepatic cholestasis: Due to obstruction of the biliary tree, mostly by gall bladder stones, carcinoma or biliary atresia.  Biliary obstruction due to cholelithiasis results in the elevation of total bilirubin, with > 90% being direct bilirubin. In most patients, there is a concomitant ↑ in ALP: levels are variable but are frequently > 300 IU/L. Drugs commonly associated with cholestatic injury are oral contraceptives, anabolic steroids, oral anti-diabetics, phenothiazines, and erythromycin. Septicemia and TPN are also associated with conj. hyperbilirubinemia. Neonatal Jaundice A) Unconjugated hyperbilirubinaemia 1. Physiological jaundice of the newborn Peak bilirubin: within 1 – 7 days of birth, remains high for 2 wks. Value usually < 5 mg/dL, with 90% unconj. bilirubin Hyperbil. > 5 mg/dL on first day, or > 10 – 12 mg/dL later is pathological Contributing factors: ↑bilirubin load due to short lifespan of RBCs Ineffective erythropoiesis ↓bil. hepatic uptake &conjugation d.t. immaturity of conjugation enzymes glucuronidase in meconium →↑deconjugation to a more reabsorbed form exposure of breast-fed infant to pregnanediol which inhibits conjugation Phase one Term infants - jaundice lasts for about 10 days with a rapid rise of serum bilirubin up to 12 mg/dL. Preterm infants - jaundice lasts for about two weeks, with a rapid rise of serum bilirubin up to 15 mg/dL. Phase two - bilirubin levels decline to about 2 mg/dL for two weeks, eventually mimicking adult values. Preterm infants - phase two can last more than one month. Exclusively breastfed infants - phase two can last more than one month. 2. Haemolytic Disease of the Newborn – Antigens of fetal RBCs entering into maternal circulation provoke the development of maternal Abs which on passing into the fetal circulation produce hemolysis of fetal RBCs. – The first born baby escapes the disease. A normal first pregnancy sensitizes the mother's blood which provokes hemolytic disease in subsequent infants. – Jaundice appears in the first 2 days of life, liable to kernicterus – Coombs test, Rh typing HDN happens most often when an Rh negative mother has a baby with an Rh positive father. If the baby's Rh factor is positive, like his or her father's, this can be an issue if the baby's red blood cells cross to the Rh negative mother. This often happens at birth when the placenta breaks away. Direct Coomb's Test This is the test that is done on the newborn's blood sample, usually in the setting of a newborn with jaundice. The test is looking for "foreign" antibodies that are already adhered to the infant's red blood cells (rbcs), a potential cause of hemolysis. To detect HDN, the presence of maternal anti-Rh IgG must be identified. In vivo, these antibodies destroy Rh D-positive fetal RBCs, but in vitro, they do not lyse cells or even cause agglutination, making them difficult to identify. Therefore, the Coombs test is used. This test uses antibodies that bind to anti-D antibodies. To find out whether a pregnant Rh D-negative mother has been sensitized to the Rh D antigen, an indirect Coombs test is done. If anti-D is not found in the mother's serum, it is likely that she has not been sensitized to the Rh D antigen. The risk of future sensitization can be greatly reduced by giving all unsensitized mothers anti-D Ig, which "mops up" any fetal RBCs that may have leaked into the maternal circulation, reducing the risk of first-time exposure to the D antigen. Usually, Rh D-negative mothers receive on injection of anti-D Ig at about 28 weeks gestation, which is about the time when fetal RBCs start to express the D antigen, and mothers receive another dose at about 34 weeks, a few weeks before labor begins during which the risk of fetomaternal hemorrhage is high. A final dose of anti-D Ig is given after the baby has been delivered. Direct Coombs test: diagnoses HDN The direct Coombs test detects maternal anti-D antibodies that have already bound to fetal RBCs. First, a sample of fetal RBCs is washed to remove any unbound antibody (Ig). When the test antibodies (anti-Ig) are added, they agglutinate any fetal RBCs to which  maternal antibodies are already bound. This is called the direct Coombs test because the anti-Ig binds "directly" to the maternal anti-D Ig that coats fetal RBCs in HDN. Indirect Coombs test: used in the prevention of HDN The indirect Coombs test finds anti-D antibodies in the mother's serum. If these were to come into contact with fetal RBCs they would hemolyse them and hence cause HDN. By finding maternal anti-D before fetal RBCs have been attacked, treatment can be given to prevent or limit the severity of HDN. For this test, the mother's serum is incubated with Rh D-positive RBCs. If any anti-D is present in the mother's serum, they will bind to the cells. The cells are then washed to remove all free antibodies. When anti-Ig antibodies are added, they will agglutinate any RBCs to which maternal antibodies are bound. This is called the indirect Coombs test because the anti-Ig finds "indirect" evidence of harmful maternal antibodies, requiring the addition of fetal RBCs to show the capacity of maternal anti-D to bind to fetal RBCs. 3. Breast Milk Hyperbilirubinaemia – Pregnanediol in milk inhibits bilirubin conjugation. – β-glucuronidase in breast milk → ↑deconjugation → ↑reabsorption – Appears after the first week of life and lasts for 2 weeks – 2 months In its most common isomeric (trans-) form, bilirubin is highly insoluble in water, and most of it is transported bound to albumin, with only a small fraction of free bilirubin. Light can cause photoisomerization of bilirubin, from a trans-form to a more compact cis-form, making it much more water soluble allowing it to be excreted in urine. This forms the basis for phototherapy in treatment of neonatal (unconjugated) hyperbilirubinemia. B) Conjugated Hyperbilirubinaemia 1. Biliary atresia 2. Idiopathic neonatal hepatitis: Jaundice appears in the first 2 weeks Conjugated bilirubin > 30% of the total Cholestasis, aminotransferases > 400 U/L, ↑ PT  Inherited Disorders of Bilirubin Metabolism 1. Gilbert's Syndrome: Defect: Reduced hepatic uptake of bilirubin Familial, autosomal recessive. Occurs in 3 – 5% of the population. Mild hyperbilirubinaemia: ≤ 3 mg/dL, all of which is unconjugated; levels can increase further with fasting but rarely exceed 5 mg/dL. No bilirubinuria. Other tests of liver function are normal. 2. Crigler-Najjar Syndrome (type 1): Defect: complete absence of UDP glucuronyltransferase. Autosomal recessive. Very high unconj. bil, often reaches > 20 mg/dL & results in kernicterus & death in the first year of life. 3. Crigler-Najjar Syndrome (type 2): Defect: partial deficiency of UDP glucuronyltransferase. Autosomal dominant. Patients may survive into adulthood. Unconj. hyperbilirubinaemia (5 – 20 mg/dL). 4. Lucy-Driscoll Syndrome: Defect: circulating inhibitor of bilirubin conjugation. Mild unconj. hyperbilirubinaemia (5 mg/dL) Disappears after 2 – 3 wks of life. 5. Dubin Johnson 6. Rotor Defect Rare. Autosomal Defective canalicular excretion. impaired recessive. Defective hepatocellular storage of conjugated canalicular excretion. bilirubin that leaks into plasma causing hyperbilirubinemia Hyperbilirubinemia Conjugated. Mild. Serum T. bil: 2-5 mg/dL Urinary Normal Increased Coproporphyrin I, it is coproporphyrins transported from the hepatocyte back into the circulation and excreted in urine. Liver biopsy Dark pigment in No pigment. Cytosolic inclusion bodies hepatocytes & Kupffer within hepatocytes. cells Investigation of Jaundice  Analytical Methods for Bilirubin Sampling: A) Serum or plasma Fasting (to avoid lipaemia) No haemolysis (low results with diazo methods) Protect from light (to avoid photo-oxidation of both conj. and unconj bil.) Best storage: in the dark at low temp Stability: 2 days at RT, 7 days at 2 – 8°C, 3 months at -20°C in the dark. B) Urine: Fresh specimen, if not, store at 2 – 8°C in the dark up to 24 h. Analytical Methods: – Chemical (diazo reaction) – Direct spectrophotometry – Chromatograpgy (HPLC is reference method) – Reflectance photometry Reflectance photometry – Bilirubin has also been quantified using bilirubinometry in neonates. (carotenoids in adult serum cause strong positive interference in adults). – Bilirubinometry involves the measurement of reflected light from the skin using two wavelengths that provide a numerical index based on spectral reflectance. – The photometer determines the optical densities of bilirubin, hemoglobin, and melanin in the subcutaneous layers of the infant’s skin. Mathematical isolation of hemoglobin and melanin allows measurement of the optical density created by bilirubin I. Photometric methods based on the diazo reaction: – diazo reagent (diazotized sulphanilic acid) + bilirubin → 2 azodipyrroles (reddish purple at neutral pH, blue at acid or alk. pH) – Direct reacting bilirubin reacts immediately with diazo reagent, while indirect reacting bilirubin reacts only after the addition of an "accelerator" which displaces bil. from its binding sites on albumin and makes it water soluble by disrupting its internal H2 bonds. – Direct bil. = β, γ and δ bil. (mono-conj., di-conj. & bilirubin bound tightly to albumin respectively) – Indirect bil. = unconj. α bil a) Van den Bergh and Muller Method: – Accelerator: ethanol b) Malloy and Evelyn Method: – Accelerator: Methanol – Disadv: Hb interference, protein precipitation by methanol → turbidity, long reaction time. c) Jendrassik and Grof Method: (Method of choice in routine) – Accelerator: caffeine/ sodium benzoate solution. – Principle: Total bil: serum + caffeine/benz, then add diazotized sulphanilic acid 10 min add ascorbic acid (stops reaction) + alk. tartrate (alk pH) + dil. HCL → green blue (600 nm). – Direct bil: Serum + dil HCl + diazotized sulphanilic acid. Stop reaction by ascorbic acid, then add alk. tartrate and caffeine/benzoate which shifts abs. peak to 600 nm. (NB. Read against sample blank tubes) Advantages of Jendrassik-Grof method over the Malloy-Evelyn method: – Not affected by pH changes – Not affected by protein concentration of the sample – Sensitive at low bilirubin concentrations – Has minimal turbidity and a relatively constant serum blank – Not affected by Hb up to 7.5 g/dL – II. Direct Spectrophotometry: (Differential spectrophotometry) – Absorbance of bilirubin in serum at λ 454 nm is proportional to its concentration. Correction for oxyHb if present is done by differential reading at λ 454 (abs. due to bil + Hb) and at λ 540 nm (abs. due to Hb only) and subtracting abs540 from abs454. – Calib: Bil solution, multilayered glass filter,or methyl orange calibrators – Disadvantage: Only applicable to newborns where conc. of carotenes and other  pigments are v. low and do not interfere. – Sample: capillary blood (heel or fingertip. Avoid hemolysis) A reagent blank refers to a small positive error in test results that comes from the reagents themselves. To measure the reagent blank, use good quality deionized water in place of your sample and run the test as usual, adding reagents and waiting any timed steps. Then subtract this value from your sample results. A sample blank refers to using the sample for zeroing an instrument during a test procedure. A sample blank can correct for potential error from existing color or turbidity in the sample before reagents are added. When zeroing the instrument on a sample blank, only the color that develops from reaction with the reagents is measured. Because background color and turbidity can vary from sample to sample, a sample blank is most commonly used to zero the instrument.