Mohd Hairi Bin A.Hamid Bsc.
Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Introduction of Liver Function
Liver is a large glandular organ which consist 2 main lobes. It contains large right and small left lobes. Then, it was divided into a number of smaller lobules. Location: in the upper part of the right side of the abdomen and extends across the midline to the left The liver is responsible by performing a number of special functions that are important for normal life There are several factors that may change the liver functions including: Infections, viruses, alcohol, toxins and drugs, medicines, poisons, clotting conditions and cancer.
The liver is an organ that has Powers of Regeneration. Means, when a large portion of the liver is damaged or removed, the organ will grow back itself.
Functions of the liver Carbohydrate, fat and protein metabolism Metabolism of steroid and sex hormones Production of bile Elimination of bilirubin Drug metabolism Synthesis of plasma proteins and clotting factors Storage of glycogen, minerals and vitamins
Liver Function Test
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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Bilirubin Bilirubin is the main bile pigment that formed from the breakdown of hemoglobin in the red blood cells. The bilirubin will be secreted into the bile by the liver. Normally, only small amount of bilirubin will circulate in the blood. Bilirubin can be divided into 2 groups: INDIRECT and DIRECT BILIRUBIN
Direct Bilirubin Also known as Conjugated Bilirubin. It was soluble in the water; react directly in diazo reaction It is excreted through the biliary tree into the gut. Conjugated bilirubin is further catabolised by intestinal flora into: Urobilinogen Stercobilinogen It forms a major component of bile in faeces. This gives the characteristic orange colour to feces Only a small amount of conjugated bilirubin is passed in the urine. Conjugated Bilirubin is unstable bilirubin. It is due to: Present of alkaline environment of the duodenum & jejunum Present the specific enzymes, for examples Beta glucoronidase Therefore, it will converted to the Unconjugated bilirubin Conjugated bilirubin is increased in conditions affecting the liver and bile duct, includes: Chronic alcoholism & Hepatitis Liver injury @ Liver disease Gallstone stuck in the common bile duct (Marked increased) Cholestasis & Hemolysis
Indirect Bilirubin Also known as Unconjugated Bilirubin or Free Bilirubin Insoluble in water but soluble in fat Therefore, it cannot be excreted in bile or urine Require addition of alcohol in the diazo reaction (indirectly) - Allow color formation. It was travels in plasma and bound to albumin. Then, it was entered the liver cells with the aid of Y & Z carrier proteins And becomes Conjugated with glucoronic acid The reaction is catalysed by an enzyme Glucuronyl Transferase Unconjugated bilirubin is increased when occur: Hemolysis (Ex: Hemolytic Anemia) Liver disease & Cholestasis
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Liver Function Test
Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Bilirubin metabolism Bilirubin Formation Heme is released from the hemolyzed red blood cells Heme is converted to biliverdin by the enzyme Heme Oxygenase Next, biliverdin is converted to Unconjugated bilirubin (water insoluble) by the enzyme Bilirubin reductase It is also known as Free or Indirect Bilirubin The normal reference range is 0.1 1.0 mg/dL
Bilirubin Excretion
Unconjugated bilirubin is bound with albumin and transported into the liver In liver, Unconjugated bilirubin is conjugated to glucoronic acid to form Conjugated Bilirubin @ Direct Bilirubin (Water Soluble) The normal reference range is 0.0 0.4 mg/dL The process required Bilirubin Uridine diphosphate and Glucuronyl Transferase Next, conjugated bilirubin is excreted via bile salts intestine The bacteria in the intestine will break down bilirubin urobilinogen Urobilinogen (10% - reabsorbed) and broke down to urobilin and stercobilin (90%) It will excrete in the feces (90%), in the kidney as urine (10%) Normal Value of Urobilinogen in feces = 40 280 mg/day
Liver Function Test
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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Bilirubin toxic effects Bilirubin is thought to be a toxin due to it is associated with: Neonatal jaundice Irreversible brain damage due to neurotoxicity. Give toxicity to the erythrocyte - by the increased of Unconjugated Bilirubin concentration Unconjugated bilirubin also stimulate microglial activation When activated, neurotoxic species which cause neuronal destruction will highly produced (neurodegenerative diseases) Bilirubin also can cause DNA damage Bilirubin + DNA + Copper Ion = Bilirubin-Cu(ii)-Complex The complex causing DNA cleavage reaction (Produce Hydroxyl Radical) In the presence of light, bilirubin will generate hydrogen peroxide and other peroxides causing DNA damage Causing Brain damage Unconjugated bilirubin failed to conjugated, lack conjugating enzyme Highly Unconjugated bilirubin circulates in the blood. If untreated, can pass via the blood brain barrier causing brain damage.
Jaundice
Jaundice is the symptom NOT the disease It is the yellow discoloration of skin, sclera and mucous membrane Due to increase level of serum bilirubin in the blood (> 3mg/dL) Increased Unconjugated bilirubin showed pre-hepatic or hepatic jaundice Treated medically Increased Conjugated bilirubin showed post-hepatic jaundice Require bile duct surgery @ therapeutic endoscopy.
Liver Function Test
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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Physiological Jaundice Definition: Jaundice that occur when the level of the serum bilirubin increased in the newborn infants (>5 mg/dL) Why: It results from the overproduction of unconjugated bilirubin and their limited ability to conjugate it or excrete it. Reason:Newborn has immature liver, so, not enough produce conjugating enzymes Therefore, high Unconjugated bilirubin circulate in the blood If untreated, the bilirubin can enter the blood brain barrier causing brain damage & bilirubin encephalopathy. Physiological jaundice usually Occur at peaks 48-72 hours & disappears by 1 week Does not present before 24 hours 80% = Preterm infants Population 30 50 % = Term infants in 1st week of life Only 10% will require phototherapy. Cause of Physiological Jaundice Hemoglobin (Hb) In neonate = 18-19g/dL In adult = 11-14g/dL Breakdown of excess RBCs (Hb released) Hb broken into: Globin - a protein that is conserved and utilised Haem - cannot be used Degraded and excreted
Factors that contributed to Physiological Jaundice Hb level is higher than required RBC have shorter lifespan Hepatic Immaturity Reduced Glucuronyl Transferase activity Reduced active uptake of Unconjugated Bilirubin Reduced intracellular transport system Reduced active secretion of Conjugated Bilirubin Large hepatic circulation of bilirubin Drugs (Ex: penicillin) Bruises & Caput Cephalohematoma Hypoxia, Hypoglycaemia, Hypothermia
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Liver Function Test
Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Jendrassik-Grof method Purpose: This method used to measure accurately the total bilirubin, direct bilirubin and indirect bilirubin level in serum or plasma. Principles: Bilirubin reacts with diazotized sulfanilic acid to form azo dye The dye is red in neutral and blue in alkaline solutions A blue azobilirubin is formed in alkaline Fehling solution II Total bilirubin is measure after addition of caffeine, sodium benzoate and sodium acetate (578 nm) Direct bilirubin is measured by using the Schellong and Wende method without addition of alkali (546 nm) Indirect bilirubin is calculated from the difference between the total and direct bilirubin.
Reference Range Total Bilirubin Time 24 h 2nd day 3rd day 4th 6th day >1 month [mg/dL] < 8.8 mg /dL 1.3 11.3 mg /dL 0.7 12.7 mg /dL 0.1 12.6 mg /dL 0.2 1.0 mg /dL 0.1 1.2 mg /dL [mol/L] < 150 mol/L 22 193 mol/L 12 217 mol/L 1.7 216 mol/L 3.4 17 mol/L 1.7 21 mol/L
Neonates
Children Adults Direct Bilirubin
Adults and children Assay Procedure
[mg/dL] 0.2 mg /dL
[mol/L] 3.4 mol/L
Optical path = 1 cm Temperature = 15C 25 C Measurement Against sample blank Direct Bilirubin determination = Wavelength (546 nm) Total Bilirubin determination = Wavelength (578 nm)
Liver Function Test
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Mohd Hairi Bin A.Hamid Bsc. Medical Laboratory Technology (Hons) UniversitI Teknologi Mara, Malaysia (January 2010)
Kernicterus Kernicterus is a condition where very high bilirubin levels in the blood are deposited in the brain tissue causing irreversible damage to the brain. This condition usually occurs among the newborn infants This condition also develop the symptom called neonatal jaundice which is a yellowing of the skin and eyeballs Early diagnosis and treatment of jaundice @ conditions that lead to jaundice may help prevent kernicterus
A baby that have kernicterus usually: Sleepy, lethargy, difficult to wake up Not eat well, Not respond to touching, Not startle from sudden movements Has very high-pitched cry & poor muscle tone
Seem "floppy" and weak muscle stiffness and tension
References Carl A. Burtis, Edward R. Ashwood, Tietz Fundamentals of Clinical Chemistry (5th Edition) (2001), Saunders An Imprint of Elsevier, Pennsylvania Karen M.Escolas (1998), Saunders Manual of Clinical Laboratory Science (1st Edition): Liver Function, W.B Saunders Company.
James K. Friel, RW. Friesen, AC. Miller (2006), Bilirubin: Friend or Foe?, University of
Manitoba, Department of Human Nutritional Sciences, Canada. Surini Yusoff (2006) Bilirubin: Gene Mutations Among Newborn Babies among Malay Population in Kelantan, University of Sciences, Malaysia Blackburn (1995) Hyperbilinemia & Neonatal Jaundice, Neonatal Network retrieved on January 27, 2010 MSDS of Bilirubin Jendrassik-Grf FS (Dec 2008), Diagnostic System International; received on January 27, 2010 Bilirubin (January 25, 2010) retrieved on January 27, 2010 from http://en.wikipedia.org/wiki/Bilirubin Bilrubin retrieved on January 27, 2010 from http://diaglab.vet.cornell.edu/clinpath/modules/chem/BILI.HTM Jaundice (January 25, 2010) retrieved on January 27, 2010 from http://en.wikipedia.org/wiki/Jaundice Kernicterus (May 27, 2008) retrieved on January 27, 2010 from http://www.webmd.com/hw-popup/kernicterus
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Liver Function Test
