1.DR. RAJENDER KUMAR PG 1ST YEAR BIOCHEMISTRY

2. INTRODUCTION Bilirubin (formerly referred to as haematoidin) is the yellow breakdown product of normal haeme catabolism. Bilirubin is a linear tetrapyrrole M.W. 585 Da. Having molecular formula C33H36N4O6. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, the background straw-yellow color of urine. the brown color of faeces (via its conversion to stercobilin), and the yellow discoloration in jaundice. Bilirubin is the end product of haemoglobin and serve as a diagnostic marker of liver and blood disorders. 3. HISTORY Discovered by virchow in 1849, in blood extravasates , named hematoidin Term bilirubin coined by Stadeler in 1864 In 1874 Tarachanoff demonstrated direct assoc to Hb In 1942 Fisher and Plieninger synthesized bilirubin IX alpha and proposed its structure Bilirubin is nonpolar, and is insoluble in plasma. Therefore it binds by noncovalent bonds to plasma albumin. This form is called: unconjugated or indirect bilirubin. 4. HAEME SYNTHESIS HAEME DEGRADATION LIVER / BONE MARROW GLYCINE + SUCCINYL CO A CATALYSED BY - AMINOLAVULINIC ACID - AMINOLAVULINATE SYNTHASE - LEAD PORPHOBILINOGEN HYDROXYMETHYLBILANE UROPORPHYRINOGEN III COPROPORPHYRINOGEN III PROTOPORPHYRINOGEN IX PROTOPORPHYRIN IX - LEAD INHERITED ENZYME DEFICIENCY IN LIVER LEADS TO PORPHYRIA HAEME RATE LIMITING STEP LIVER / SPLEEN ERYTHROCYTES ERYTHROID CELLS HAEMOGLOBIN,CYTOCHROME HAEME PROTEINS, MYOGLOBIN AMINO ACIDS HAEME CO, FE3+ HAEME OXIGENASE BILIVERDIN NADPH + H+ BILIRUBIN BILIVERDIN REDUCTASE CONJUGATED BILIRUBIN UDP GLUCURONYL TRNSFERASE 3 O ALBUMIN NADP+ UNCONJUGATED BILIRUBIN 2 UDP GLUCURONATE 2 UDP 5. STRUCTURE OF HAEME HAEME 1. Haeme consists of protoporphyrin IX , Containing 4 pyrrole rings linked togethor by methenyl bridge 2. Iron incorporated is in ferrous form Fe2+ 3. Haeme synthesis occurs in all cells 6. FORMATION OF BILIRUBIN The breakdown of haeme produces bilirubin (an insoluble waste product) and other bile pigments. Bilirubin must be made water soluble to be excreted. This transformation occurs in 5 steps;- 1. Formation 2. Plasma transport 3. Liver uptake 4. Conjugation 5. Biliary excretion One gram of hemoglobin yields 35 mg of bilirubin. The daily bilirubin formation in adult human is about 250 mg. 7. 1.1FORMATION Bilirubin is formed in the monocytic macrophages of the spleen and bone marrow and in hepatic Kupffer cells, from haeme by opening of the ring at the carbon bridge. This cleavage is catalyzed by the microsomal enzyme haem-oxygenase of RE cells forming biliverdin IX. NADPH is used as the reducing agent, molecular oxygen enters the reaction, carbon monoxide(CO) is produced and the iron is released from the molecule as the ferric ion (Fe3+). CO acts as a cellular messenger and functions in vasodilatation. Biliverdin IX is then reduced to bilirubin IX by cytosolic enzyme biliverdin reductase. This is unconjugated bilirubin, which is not soluble in water, due to intramolecular hydrogen bonding and they also shield the central CH 2 , which thus becomes inaccessible for the diazo- reagent. 7 8. Cleavage at non- sites is possible; it is probably non-enzymic and occurs only to a minor extent. This results in the formation of other isomers. The IX isomer is present in neonatal urine and in meconium. Because intramolecular hydrogen bonds cannot be formed in these isomers, they are more hydrophilic, and appear in urine or bile as an unconjugated pigment. Phototherapy, leads to the formation of another group of more hydrophilic derivatives of the natural UCB IX, such as the 4E,15Z ; 4Z,15E and 4E,15E photoisomers, which can be excreted in bile without conjugation. 1.2 FORMATION 9. STRUCTURE OF BILIRUBIN Structure of naturally occuring unconjugated bilirubin IX 4Z,15Z Formation of bilirubin. 10. STRUCTURE OF BILIRUBIN Overall structure of bilirubin was established by Xray crystallography. Bilirubin assumes a ridge tiled configuration stabilised by six intramolecular hydrogen bonds. Two additional festures have been noted:- 1. Z-Z (trans) conformation for double bonds b/t carbon 4 & 5 and 15&16. 2. An involuted hydrogen bonded structure in which propoinic acid- carboxylic acid groups are hydrogen bonded to nitrogen atoms of the pyrrole rings.these bonds stabilizes Z-Z configuration of bilirubin and prevents its interaction with polar groups. when exposed to light Z-Z configuration is converted to E-E & other configuration like E-Z, Z-E. 11. ISOMERS OF BILLIRUBIN 12. 2. PLASMA TRANSPORT From R.E. cells bilirubin enters the circulation binds to albumin.It cannot pass through the glomerular membrane into the urine. Albumin binding weakens under certain conditions (eg, acidosis), and some substances (eg, salicylates, certain antibiotics) compete for the binding sites. Depending on the pH of the plasma, bile or urine UCB can be present as uncharged diacid, as a monoanion or as a dianion. The uncharged diacid is by far the dominant species at low and physiological pH (>80%). The ionized fractions become more important in an alkaline pH, because the pKa values have been determined to be 8.12 and 8.44, respectively, for the first and for the second anion. 13. 3. LIVER UPTAKE Entry into the hepatocytes appears to be partly passive and partly mediated by organic anion transporter proteins (OATP 1B1 has the highest binding affinity). In the hepatocytic cytosol, UCB is mostly bound to glutathione-S-transferase A (ligandin), and a small part is bound to the fatty acid-binding protein. As in serum, this binding keeps the free fraction low. (which is potentially toxic). 14. 4. CONJUGATION Bilirubin is conjugated in hepatocytic microsomes in an ester linkage with sugar moieties donated by uridine diphosphate The conjugation is catalyzed by UDP- glucuronyltransferase. Sugar moieties are coupled to the COOH of the propionic acid side chain of UCB, resulting in mono or diconjugated bilirubin. The esterification disrupts the intramolecular hydrogen bonds, thereby opening the molecule and rendering the (CB) more water-soluble. Conjugation also decreases the binding to albumin or to intracellular proteins, and prevents intestinal re-absorption. The bilirubin conjugates formed in the hepatocytes are excreted in bile against a concentration gradient and mediated by the canalicular membrane transporter multidrug resistance-related protein 2 (MRP2) also termed ABC-C2, belonging to the adenosine triphosphate (ATP)-binding cassette family. 15. 15 Structure of Conjugated Bilirubin 16. 5.1 BILIARY EXCRETION The conjugates are incorporated into mixed micelles (with bile acids, phospholipids and cholesterol) and pass with the bile into the intestine, where reductive breakdown into urobilinogen occurs by intestinal or bacterial enzymes. A minor part undergoes deconjugation mainly the intestinal and bacterial enzyme - Glucuronidase, forming UCB and reabsorbed through enterohepatic circulation. In neonates, the bacterial flora is not yet developed, deconjugation will thus prevail and this adds to the enhanced serum bilirubin levels observed in neonatal jaundice. Bile salts incorporate bilirubins in micelles and protect them from deconjugation. Normally, 95% of bile salts are re- absorbed in the terminal ileum. 17. 1. Intestinal bacteria acts on bilirubin diglucuronide leading to: a) Removal of glucuronides (by b-glucuronidases enzymes). b) Reduction of bilirubin to colorless compounds called: Urobilinogens 2. A small fraction of urobilinogens are reabsorbed from intestine to the liver again and re-excreted in the bile (95%), forming the enterohepatic urobilinogens cycle. some part of urobilinogen is excreted in urine (5%)in the form of urobilin (is responsible for the yellow colour of urine)which is the product of oxidation of urobilinogen. 3. The remainder travels down the digestive tract and is converted to stercobilinogen. This is oxidized to stercobilin. which is excreted and is responsible for the color of faeces 17 5.2 BILIARY EXCRETION 18. BILIRUBIN PRODUCTION Heme Heme oxygenase Biliverdin reductase Hemoglobin (80 to 85%) Erythroid cells Heme proteins myoglobin, cytochromes (15 to 20%) Biliverdin Bilirubin NADPH + H+ NADP+ apoferritin ferritin indirect unconjugated pre-hepaticalbumin GlobinAmino acid H2O O2 met CO NADPH + H+ NADP+ fp fp Fe3+ 19. BILIRUBIN PROCESSING Bilirubin-albumin ligandin Bilirubin diglucuronide hepatocyte UDP-GLUCURONYL TRANSFERASE ligandin-Bilirubin bile (gall bladder) Intestine albumin 2 Udp-glucuronate 2 UDP 20. Bilirubin diglucuronide Intrahepatic urobilinogen cycle 15% Stercobilinogen Bacterial enzymes Bilirubin b-glucuronidases enzyme2 glucuronate Bacterial enzyme Urobilinogen 8H bile Urobilin (5%) kidneys urine Stercobilin faeces INTESTINE 21. TYPES OF BILIRUBIN The decomposition of hemoglobin in the body results in the formation of the Z,Z-isomer of bilirubin. Phototherapy results in the conversion of the Z,Z-isomer of bilirubin into the E,E-isomer. Two terminal rings of bilirubin favour lactam form. A cyclic system containing the grouping CONH is called a lactam, and the isomeric form, COH & bond;NH, a lactim. However both the forms are available and are interchangeable. Four bilirubin fractions have been isolated from serum:- 1. Unconjugated bilirubin (-bilirubin) 2. Monoconjugated bilirubin (-bilirubin) 3. Diconjugated bilirubin (-bilirubin) 4. A fraction irreversibly bound to protein (-bilirubin) 22. 22 Unconjugated Bilirubin and Conjugated Bilirubin 23. DELTA-BILIRUBIN (BD) Delta-bilirubin arises through a non-enzymatic covalent coupling reaction between glucuronated bilirubin and albumin this fraction is found in patients with hepatic and posthepatic icterus or with Dubin-Johnson syndrome Delta-bilirubin is not excreted by liver or kidneys, but is slowly metabolized with a half-life of 20 days. It can constitute up to 90% of the total bilirubin in the convalescence phase of cholestatic disorders. Free bilirubin (Bf):- Free bilirubin, i.e., unconjugated and not bound to albumin, represents a significant component of the neurotoxicity of bilirubin, which is made responsible for the bilirubin encephalopathy of the neonate (resulting in kernicterus) The toxic effect is thought to occur even at a concentration of 0.005 mg/dL . 24. Bilirubin Fraction in Different Methods HPLC PEAK IRREVERSIBLY BOUND TO PROTEIN DICONJUGATED MONOCONJUGATED VITROS METHOD DIAZO METHOD UNCONJUGATED TOTAL BILIRUBIN INDIRECT BILIRUBIN T-D ? DIRECT BILIRUBIN TOTAL BILIRUBIN UNCONJUGAT ED BILIRUBIN Bu CONJUGATED BILIRUBIN Bc DELTA BILIRUBIN = T-Bn NEONAL BILIRUBIN=Bu+Bc INDRECT DIRECT BILIRUBIN =T-Bu ? In Diazo method reactivity with direct reaction varies 25. Physiological Role Of Bilirubin In Our Body Studies have found higher levels of bilirubin in elderly individuals are associated with higher functional independence. Studies have also revealed that levels of serum bilirubin are inversely related to risk of certain heart diseases. An uncoupler is a compound that impedes ATP generation but has no effect on electron transport chain.The energy released by electron transport is mostly dissipated Some portion of it generates heat. Bilirubin levels well above physiological range can act as an physiological uncoupler and thus helps maintain body temperature in infants. Bile pigments such as biliverdin naturally possess significant anti-mutagenic and antioxidant properties. Biliverdin and bilirubin have been shown to be potent scavengers of peroxyl radicals. 26. Physiological Role Of Bilirubin In Our Body They have also been shown to inhibit the effects of polycyclic aromatic hydrocarbons, heterocyclic amines, and oxidantsall of which are mutagens. In vitro experiments showed that biliverdin and bilirubin competitively inhibited HIV-1 proteases at low micromolar concentrations, reducing viral infectivity. In1950s, bilirubin was reported to protect against the oxidation of lipids such as linoleic acid and vitamin A. In the late 1980s, Ames and colleagues demonstrated that the antioxidant effect of bilirubin exceeds that of vitamin E toward lipid peroxidation. Higher bilirubin levels, 0.9 mg/dL were associated with lowered risk of myocardial infarction and other cardiovascular disease events. 27. Diagnostic Importance of Bilirubin Clinically hyperbilirubinemia appears as jaundice or icterus. Jaundice can usually be detected when the serum bilirubin level exceeds 2.0 to 2.5 mg/dl. When the level of bilirubin is between 1 to 2 mg/dl ,it is known as latent jaundice. unconjugated plasma bilirubin that is not bound to albumin can cross the blood brain barrier.In conditions such as neonatal jaundice or type-I or type-II Crigler-Najjar syndrome extremely high concentrations (>20mg/dl) of unconjugated bilirubin can accumulate, and the resulting diffusion of bilirubin into the central nervous system can cause encephalopathy and permanent impairment of nervous function. 28. Clinico-pathological classes of Jaundice 1. Pre-hepatic:- Excessive destruction of red blood cells increases bilirubin formation. However, with normal hepatic function, the liver is able to remove the excess bilirubin fairly rapidly so this jaundice is usually mild. As the amounts of bile pigments entering the gut are greater than normal, the amount of urobilinogen reabsorbed from the gut is also increased, this raises its levels in the urine. There is often also a small increase in the serum conjugated bilirubin. 2. Hepatic:-This type of jaundice results either because of defective uptake or conjugation. In neonate because of immature hepatic enzyme glucuronyl transferase and lack of intestinal bacteria preventing conversion of bilirubin into urobilinogen, unconjugated hyperbilirubinemia results. 29. Clinico-pathological classes of Jaundice Glucuronyl transferase inhibition by variety of agents such as chloramphenicol, novobiocin, vitamin K, breast milk , pregnanediol, free fatty acids, hypothyroidism. Flavaspidic acid, used in the treatment of tape worm infestation, competes with bilirubin for binding to ligandin, may cause unconjugated Hyperbilirubinemia. Damaged liver cells are less able to transfer bilirubin from the blood into the bile. Hepatic jaundice may also be caused by conditions which lead to intrahepatic obstruction. 30. Clinico-pathological classes of Jaundice 3.Post hepatic:- A. Familial defects in hepatic excretory function- I. Dubin-Johnson syndrome-The serum contains more diconjugated than monoconjugated bilirubin. II. Rotor syndrome- Serum conjugated bilirubin has more monoconjugates than diglucuronide conjugates. B. Acquired defects of hepatic excretory function- I. Drug induced and post operative. Obstruction of the bile ducts after they have left the liver, sometimes referred to as extrahepatic Cholestatic jaundice.. As there is no bilirubin in the gut none is reabsorbed into the blood to be excreted by the kidneys as urobilinogen.This means the urine contains little or none of this pigment.However as the levels of bile pigment in the blood continue to rise it is excreted in the urine, once a renal threshold is reached, causing dark colored urine. II. Hepatitis and Cirrhosis. 31. Role of bilirubin in Neonate 1.Physiologic Jaundice:- Newborns produce bilirubin at a rate of approximately 6 to 8 mg per kg per day.The average total serum bilirubin level usually peaks at 5 to 6 mg per dL (86 to 103 mol per L) on the third to fourth day of life and then declines over the first week after birth. Bilirubin elevations of up to 12 mg per dL, with less than 2 mg per dL of the conjugated form, Infants with multiple risk factors may develop an exaggerated form of physiologic jaundice in which the total serum bilirubin level may rise as high as 17 mg per dL. 2.Jaundice and breast feeding:- Total serum bilirubin levels vary from 12 to 20 mg per dL (340 mol per L) and are non pathologic. Substances in maternal milk, such as -glucuronidases , and nonesteriied fatty acids, may inhibit normal bilirubin metabolism. 3.Pathologic Jaundice:- Jaundice is considered pathologic if it presents within the first 24 hours after birth. a rapidly rising total serum bilirubin concentration (increase of more than 5 mg per dL per day), and a total serum bilirubin level higher than 17 mg per dL in a full-term newborn and elevation of the serum conjugated bilirubin level to greater than 2 mg per dL. 32. Predominantly Unconjugated Hyperbilirubinemia I. Overproduction A. Hemolysis (intra and extravascular) B. Ineffective erythropoesis II. Decreased hepatic uptake III. Decreased bilirubin conjugation (decreased hepatic glucuronyl transferase activity) A.Hereditary transferase deficiency 1. Gilberts syndrome 2. Crigler-Najjar type II (moderate transferase deficiency) 3. Crigler Najjar type I (absence of transferase) B. Neonatal jaundice ( transient transferase deficiency) C. Acquired transferase deficiency 1. Drug inhibition (chloramphenicol pregnanediol) 2. Breast milk jaundice (transferase inhibition by pregnanediol and fatty acids in breast milk) 3. Hepatocellular disease (hepatitis, cirrhosis) D. sepsis 33. Predominantly Conjugated Hyperbilirubinemia I. Impaired hepatic excretion A. Hereditary disorders 1. Dubin-Johnson syndrome 2. Rotor syndrome 3. Recurrent (benign) intrahepatic cholestasis 4. Cholestatic jaundice of pregnancy B. Acquired disorders 1. Hepatocellular disease (e.g.,viral or drug induced hepatitis,cirrhosis) 2. Drug induced cholestasis (e.g.,oral contraceptives,androgens,chlorp romazine) 3. Alcoholic liver disease. 4. Sepsis 5. Postoperative state 6. Parenteral nutrition 7. Biliary cirrhosis II. Extrahepatic Biliary obstruction 1. Gallstones 2. Biliary malformation . 3. Infection 4. Malignancy 5. Hemobilia (trauma,tumor) 6. Sclerosing cholangitis 7. Malignancy 8. Inflammation (pancreatitis) 34. BILIRUBIN LAB VALUES Bilirubin form Normal value Total (elderly, adult, child) 0.2 to .8 mg/dL (newborn) .8 to 12.0 mg/dL Critical value (adult) >12 mg/dL Critical value (newborn) >15 mg/dL Pre-hepatic,unconjugated, indirect 0.2 to 0.7 mg/dL Post-hepatic, conjugated, direct 0.1 to 0.4 mg/dL Fecal urobilinogen 40 to 280 mg/day Urine 0.0 to 0.02 mg/dL 35. Specimen Collection and Storage Serum or plasma A fasting morning sample collected Excessive hemolysis (serum hemoglobin >300mg/ dL) should be avoided because it may falsely lower the bilirubin value Specimens should be protected from direct exposure to either artificial light or sunlight as soon as they are drawn assay should be carried out within 2 hours of sample collection. If a longer delay is unavoidable, refrigerate the sample The sensitivity to light is temperature-dependent; for optimal stability, storage of specimens in the dark and at low temperatures is essential. point of care direct spectrophotometric measurement of bilirubin in neonates heel prick samples may be taken directly in hematocrit capillary tubes. 36. Methods Of bilirubin estimation A. Non invasive :- I. Clinical method using cephalocaudal progression II. Icterometer simple colored Perspex which has grades of yellow color compaired with body skin III. Transcutaneous billirubinometer :- using the principle of skin reflectance . B.Invasive :-I. Capillary billirubin estimationbased on the principle of spectrophotometry. II. Filter paper with billirubinometer. III. Laboratory estimation1. Diazo reaction 2. Bilirubinometera. Direct spectrophotometry b. The Jendrassik-Grof method. c. The Malloy-Evelyn method 3. Reflectance spectrophotometry 4. high pressure liquid chromatography 5. Peroxidase method method 6. Peroxidase Diazo method 7. Simple colorimetric method 8. New enzymatic assay 37. CLINICAL METHODS I.using cephalocaudal progression II. Icterometer Gossett Icterometer CRAMMERS METHOD 38. Transcutaneous billirubinometer These meters work by directing light into the skin and measuring the intensity of specific wavelength that is returned. The meter analyzes the spectrum of optical signal reflected from subcutaneous tissues.These optical signals are converted to electrical signal by a photocell. Signals are analyzed by a microprocessor to generate a serum bilirubin value. The major skin components, which impart the spectral reflectance in neonate, are (i) melanin, (ii) dermal maturity, (iii) hemoglobin, and (iv) bilirubin.Hyperemia at the test site may affect the results. The available meters can be divided into 2 categories: 1.Multi wavelength Spectral Reflectance meters (Bilicheck) TM. 2.Two wavelength (460,540 nm) Spectral Reflectance meters (Minolta, Bili-test). Transcutaneous bilirubinometers can serve as a screening tool, However, this cannot serve as a substitute for TSB estimations. 39. Transcutaneous billirubinometer Transcutaneous billirubinometer the dual optical path system Multi wavelength Spectral Reflectance meters 40. Diazo Reaction Ehrlich in 1883 treated bilirubin in urine with diazo reagent and found that a red blue coloured pigment was formed. Introduction of the diazo reaction for serum bilirubin by van den Bergh in 1918 led to its widespread adoption for quantitating the pigment in serum.In an aqueous solution, Ehrlich's diazo reagent reacts with the direct bilirubin in the serum to form a pink to reddish-purple colored compound (azobilirubin). read at one minute. In a 50% methyl alcohol solution, Ehrlich's diazo reagent reacts with the total bilirubin in the serum to form a pink to reddish-purple colored compound. (Read at 30 minutes.) Malloy & Evelyn used diazo reagent for the colorimetric estimation of bilirubin which was read at 540 nm. These methods require preliminary step of protein precipitation in alcoholic solution at about pH 4.This leads to low & poorly reproducible results because of co precipitation of bilirubin esters along with protein. 41. . 42. Jendrassik and Grof doumas et al Reference Method In 1938 Jendrassik & Grof modified the diazo method by using caffeine benzoate as an accelerator. PRINCIPLE:- Bilirubin reacts with diazotized sulfanilic acid in the presence of caffeine benzoate-acetate & converted to azopigments The reaction occurs at the methylene carbon atom between rings B and C , with the formation of one molecule each of azopigment & hydroxypyromethenecarbinol.A second molecule of azopigment is formed from the reaction between this carbinol & diazo reagent. The tartrate buffer makes the mixture alkaline and converts the red acid bilirubin to a green coloured compound which shows peak absorbance at 598 nm. At this wavelength the absorbance due to haemoglobin or carotene is minimal. Ascorbic acid is used to stop the coupling reaction, and to eliminate interference by haemoglobin. 43. Salient features Ease of standardization, high precision, excellent linearity, minimum interference from haemoglobin, rapid colour development constancy of the molar absorptivity of the azopigment in various protein matrices, and adaptability to various instruments for automated chemical analysis. Suitable for neonatal sera. Measures all 4 bilurubin fractions ( T.bil values with HPLC corelate well with Jendrasic and Grof method) Colour Stability - with BIL in HSA or BSA, the color of the azopigment is stable for at least 30 min. With biirubin in serum, there is a small increase in absorbance with time in both tests and sample blanks, caused by a gelatinous precipitate, slowly formed in both tests and sample blanks. 44. For direct bilirubin estimation Dilution of serum with dilute HCl is done allowing it to stand for 5 minutes before adding the diazo reagent , this keeps the unconjugated bilirubin from reacting as direct. Ascorbic acid is added to destroy excess diazo reagent, which would react with unconjugated bilirubin when alkaline tartrate is added. The caffeine reagent suppresses the absorptivity of the azopigment at 598nm by about 12%. Omission of the caffeine reagent in this assay has been responsible for the paradox of sometimes having direct bilirubin values exceeding those of total bilirubin. Interference by hemoglobin is far stronger than in the method for total bilirubin.The negative bias is due to oxidation of conjugated bilirubin to the diazo negative biliverdin and to the destruction of the azopigment by H2O2 produced by the conversion of hemoglobin to methemoglobin at the low pH of the reaction medium; the negative bias is substantially reduced by addition of potassium iodide to the serum diluent (HCl). 45. Other Diazo Reaction Powell(1944) added conventional diazo reagent to the plasma followed by sodium benzoate urea solution. In this method a plasma blank test is carried out in which HCL replaces diazo reagent. Colour densities were read in EEL colorimeter at Ilford filter 625.This method was used for plasma containing bilirubin conc. upto 5mg/dl. Method of King & Coxon(1950) modified in a manner as suggested by Perryman et al(1957)-Ammonium sulphamate(0.15%) was incorporated in diazo reagent and few crystals of sodium azide were added after the addition of ethanol (85%v/v).Readings were made at 530nm and 425nm,and the azo pigment extinction was then corrected for interference by haeme pigments by means of a simple formula. Method of Lathe & Ruthven Synthetically prepared taurobilirubin was used as a model for direct bilirubin. It was found that extinction and absorption maximum values depend on pH, alcohol & albumin. 46. Method used in our deptt. Principle:- Van Den Berg devised colorimetric estimation Reagents required: Diazo reagent;10 ml of Diazo A + 0.3 ml of Diazo B DiazoA;1g sulphanillic acid & 15ml of Conc. HCl in water Diazo B; 0.5 g of sodium nitrite/100 ml in water. This solution must be prepared fresh as is unstable. Diazo blank; 0.1 N HCl Methanol. Standard stock: An artificial standard is used i.e. methyl red 290mg/100ml glacial acetic acid that gives an optical density (0.32) equal to 8 mg/dl of bilirubin. Methyl red is an azo dye with an absorbance curve very similar to azobilirubin and it can therefore be used as a standard. Original standard is not used because it is photosensitive and thus unstable, so cannot be obtained easily and is costly. 47. Procedure:- take 2 test tubes and label them test and control. TEST CONTROL Distilled Water 1.8 ml 1.8 ml Serum 0.2 ml 0.2 ml Diazo reagent 0.5 ml -------- Diazo blank -------- 0.5 ml Methanol 2.5 ml 2.5 ml Keep the test tubes in dark. Readings taken at 1 minute is direct/conjugated bilirubin, at 30 minutes is total bilirubin. Unconjugated bilirubin= Total bilirubin conjugated bilirubin. Calculations:- S. Bilirubin (mg%)=[ODt ODc/ODs ]X 8 48. Enzymatic methods Bilirubin oxidase from the fungus species Myrothecium verrucaria. BOX is a 52 kDa enzyme with one copper ion attached and with a maximum activity at pH 8.0 Near pH 8 in presence of sodium cholate and sodium dodecylsulphate , all 4 fractions are oxidized to biliverdin which is oxidized to purple and finally colorless products Decrease in absorbance at 425 / 460 nm is proportional to conc of total bilirubin. Direct bil is measured at pH 3.7 4.5 , when enzyme oxidizes conjugated and delta bil but not unconjugated bil At pH 10 enzyme activity is selective for the two glucuronides , delta fraction is not oxidized , and about 5% of unconjugated bil is oxidized. 49. Peroxidase method- The peroxidase method obtains the unbound conjugated bilirubin (bc ) and unbound unconjugated bilirubin ( bu) using horse radish peroxidase (HRP)-catalyzes oxidation of bc and bu by a peroxide (usually hydrogen or ethyl hydrogen peroxide) to colorless products at sample dilutions of about 1:40. Bilirubin bound to albumin (A:bc and A:bu) is protected from oxidation. The unbound bilirubin concentration is calculated by dividing the reaction velocity (rate of change in bilirubin light absorption at 460 nm) by Kp z [HRP], where Kp (min21) is the first-order rate constant for the oxidation of bilirubin by peroxide in an albumin- free solution containing 1 mg/ml HRP. 50. Peroxidase Diazo method Add a small volume of HRP and peroxide to an aliquot of sample (usually 25 ml) and allowing the reaction to proceed 0 or t min before initiating the diazo test. The sulfanilic acid denatures the HRP, stopping the bilirubin oxidation reaction. Since the bilirubin oxidation products are diazo negative , only nonoxidized bilirubin will form diazo derivatives. The volumes of HRP and peroxide added determine the sample dilution at which the unbound bilirubin is measured. For example, if 25 ml of HRP and 10 ml of peroxide are added to 25 ml of sample, the unbound bilirubin is measured at a dilution of 1:2.4. The reaction would be stopped at the selected times with 0.5 ml of sulfanilic acid solution and the diazo test completed by adding 25 ml of nitrite followed by 0.5 ml of diluted methanol. 51. High Pressure Liquid Chromatography HPLC methods allow for relatively rapid separation and quantification of the four bilirubin fractions. The bilirubin mono and diglucuronide conjugates are converted to mono and dimethyl esters by treatment with alkaline methanol. Unconjugated bilirubin is not affected by the reaction and is extracted into chloroform with the methyl ester derivatives. The pigments can then be separated and quantified by high performance liquid chromatography(HPLC) or thin layer chromatography and detected spectrophotometrically in the effluent. Use of an internal standard and calibration of the method with crystalline reference bilirubin and bilirubin methyl esters permit direct measurement of the individual pigment fractions in the sample 52. HPLC In earlier methodological studies the mono- and diglucuronide were first converted into the stable methyl ester by methanol, and then extracted together with the Bu with chloroform. The separation then proceeded either by normal-phase HPLC on a silica gel column or with reversed-phase HPLC on a C18 column. This procedure also permits separation of the C8 and C12 isomers of the monomethyl ester. Since serum proteins are denatured and completely removed, measurement of Bd is not possible with this method 53. Lauff et al. developed a HPLC method in which the serum is pretreated with a saturated sodium sulfate solution. This method precipitates mainly proteins that are larger than albumin, while albumin itself, together with Bd, remains in solution. The serum is then transferred to a reversed-phase column, which is eluted, according to decreasing polarity, with a linear gradient from a diminishing, acid, aqueous phase based on phosphoric acid and an increasing isopropanol-based phase. The Bd is eluted most rapidly through the column. Then the other bilirubin fractions follow in the order bilirubin-diglucuronide, bilirubin-monoglucuronide and Bu .Since the precipitation with sodium sulfate is not completely selective, there is a risk that variable proportions of albumin will also precipitate leading to a loss of Bd. 54. Direct Spectrophotometery Direct photometric measurements are based on direct measurements of serum at the wavelength of 455nm, which is the absorption maximum of bilirubin. oxyhaemoglobin absorbs light at the wavelength of 455nm. 55. reflectance spectrophotometry 56. Simple colorimetric method for the estimation of plasma biliverdin A new colorimetric method for the assay of biliverdin in biological fluids is described. The method, based upon the reaction of biliverdin with barbituric acid, offers improved sensitivity and selectivity when compared to direct spectrophotometric measurements. Using this method biliverdinaemia was observed in two patients with obstructive jaundice of malignant origin 57. New enzymatic assay New enzymatic assay- for total (TBil) and direct bilirubin (DBil), the principle of which involves measuring the decrease in absorbance at 450 nm produced by bilirubin oxidase from Myrothecium verrucaria. Since TBil and DBil are oxidized at pH 7.2 and 3.7, respectively, the degree of bilirubin oxidation is measurable in each case. An analysis of bilirubin by high-performance liquid chromatography, before and after the enzymatic reaction with bilirubin oxidase, verified the specificity of the enzyme. The results obtained using this method varied linearly with TBil and DBil concentrations up to at least 250 mg/L and 150 mg/L, respectively. Reducing substances, commonly used anticoagulants and hemoglobin showed no apparent interference. The degree of day-to- day precision (CV) for TBil and DBil ranged from 1.2% (206.2 mg/L) to 10.6% (3.5 mg/L) and from 1.8%(84.3 mg/L) to 12.4% (2.1 mg/L), respectively. Values measured using this new method correlated well with those obtained by Malloy-Evelyn's method and the slide method employing the Kodak Ektachem analyzer 58. CSF BILIRUBIN CSF Bilirubin is an important test for excluding subarachnoid haemorrhage. The Gold Standard test is scanning spectrophotometer New Diazonium ion Sample volume 3 uL Limit of detection 1.7 umol/L Haemolysis no interference up to 1000 mg/dL The bilirubin assay is resistant to haemolysis improving clinical sensitivity compared to spectrophotometry. 59. Linearity, Precision, and Accuracy Linearity the relation is linear and that there is adherence to Beers law up to an absorbance of at least 1.92 A. Precision -The within-run precision, calculated from duplicate determinations is excellent (CV 0.1%). Accuracy- 60. Interference is negligible by- 1.oxyhemoglobin(up to 2 gIL), mechanism oxyhb in acidc conditions produces acid hematin and H2O2. H2O2 oxidizes bilirubin to biliverdin due to pseudoperoxidase activity of acid hematin 2.ascorbic acid (up to 20 mg/L), (with ingestion of 1 to 3 g, ascorbic acid concentration in serum does not exceed 35 mg/L) 3.zinc (at physiological concentrations) 4. L-dopa and a-methyldopa can interfere 5. A propranolol metabolite, a conjugate of 4- hydroxypropranolol that is normally excreted in the urine, accumulates in the plasma of undialysed patients with chronic renal failure and interferes with the widely used diazo reaction to give falsely raised bilirubin concentrations, which may cause confusion clinically and lead to unnecessary investigations. The Bilirubinometer, available in most neonatal units, provides a simple way of avoiding this source of error. 61. WHO STANDARD OPERATING PROTOCOL Principle is same as reference method Modifications- 1. half reaction volumes are used 2. waveleanght used is 607 nm 3. simplified procedure is used for total and direct bil estimation for ease in routine use 4. Commercially available bilirubin is used to make standard by dissolving in pooled non icteric human sera(tested ve for HIV) with the aid of of a small amount of dimethyl sulphoxide (DMSO) and NaOH. The exact value of bil in it has to be determined against a primary standard 62. Precautions Storage of standard Aliquot small volumes of standard into screw-capped vials and store in the freezer on the same day it is prepared. Stable for 2 months at -200C. Do not refreeze leftover standard after use Hazardous materials This method uses sulphanilic acid and sodium hydroxide. Avoid contact with eyes, skin and mucous membranes Samples with bilirubin concentrations higher than 20mg/dl should be diluted with an equal volume of distilled water and the result obtained should be multiplied by 2 63. QUALITY CONTROL Include one internal QC in every batch of samples analysed every day irrespective of the number of samples in a batch. Since bilirubin is analysed in a single batch in a day in an intermediate laboratory, it will not be possible to analyse several QC samples and calculate within- day precision. However, even if a single QC sample is analysed in a day, this value can be pooled with the preceding 10 or 20 values obtained in the previous days and between-day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e, 10%. Once a week it is good to analyse another QC serum from either a low QC or high QC pool. 64. Method used in our deptt. Reagents required: Diazo reagent: 10 ml of Diazo A + 0.3 ml of Diazo B Diazo A: 1g sulphanillic acid and 15ml of Conc. HCl/L in water. Diazo B: 0.5 g of sodium nitrite/100 ml in water. This solution must be prepared fresh as is unstable Diazo blank: 0.1 N HCl, Methanol Standard stock: An artificial standard is used i.e. methyl red 290mg/100ml glacial acetic acid. working standard 1ml of stock , 5ml glacial acetic acid,14.4 gm hydrated Na acetate in 1L water gives an optical density (0.32) equal to 8 mg/dl of bilirubin. Methyl red is an azo dye with an absorbance curve very similar to azobilirubin and it can therefore be used as a standard. Original standard is not used because it is photosensitive and thus unstable, so cannot be obtained easily and is costly. 65. DPD method in an acid medium with 2,5-dichlorophenyl- diazonium-tetrafluoroborate (DPD), bilirubin forms an azopigment that can be measured photometrically at 540 to 560 nm. The release of the Bu from the albumin is achieved with the detergent Triton X which also helps to avoid protein precipitations There is good agreement between the measurement results of the Jendrassik-Grof principle and the DPD method however, the latter is much less laborious 66. Enzymatic methods bilirubin oxidase (BOX, EC 1.3.3.5) from the fungus species Myrothecium verrucaria MT-1 has permitted the development of an enzymatic method for measuring total bilirubin. BOX is a 52 kDa enzyme with one copper ion attached to every enzyme molecule, and with a maximum activity at pH 8.0 Near pH 8 in presence of sodium cholate and sodium dodecylsulphate , all 4 fractions are oxidized to biliverdin which is oxidized to purple and finally colorless products Decrease in absorbance at 425 / 460 nm is proportional to conc of total bil Results of total bil are slightly lower than diazo methods Direct bil is measured at pH 3.7 4.5 , when enzyme oxidizes conjugated and delta bil but not unconjugated bil At pH 10 enzyme activity is selective for the two glucuronides , delta fraqction is not oxidized , and about 5% of unconjugated bil is oxidized 67. HPLC In earlier methodological studies the mono- and diglucuronide were first converted into the stable methyl ester by methanol, and then extracted together with the Bu with chloroform. The separation then proceeded either by normal-phase HPLC on a silica gel column or with reversed-phase HPLC on a C18 column. This procedure also permits separation of the C8 and C12 isomers of the monomethyl ester. Since serum proteins are denatured and completely removed, measurement of Bd is not possible with this method 68. Lauff et al. developed a HPLC method in which the serum is pretreated with a saturated sodium sulfate solution. This method precipitates mainly proteins that are larger than albumin, while albumin itself, together with Bd, remains in solution. The serum is then transferred to a reversed-phase column, which is eluted, according to decreasing polarity, with a linear gradient from a diminishing, acid, aqueous phase based on phosphoric acid and an increasing isopropanol-based phase. The Bd is eluted most rapidly through the column. Then the other bilirubin fractions follow in the order bilirubin-diglucuronide, bilirubin- monoglucuronide and Bu .Since the precipitation with sodium sulfate is not completely selective, there is a risk that variable proportions of albumin will also precipitate leading to a loss of Bd. 69. In another method for differentiation of bilirubin with HPLC, serum diluted with acetic acid is filtrated through a 0.45 mm filter, transferred to a polyacrylic-ester column and finally eluted with a linear pentasulfonic acid acetonitrile gradient. Five bilirubin fractions can be detected in the eluate; the additional fifth fraction in comparison to the both methods described above is the (Z,E) or (E,Z) photoisomer of bilirubin. Disadvantage is the high cost of the procedure and the use of microfilters through which possible losses of Bd may arise. Additionally, the solvent acetonitrile is a carcinogenic substance 70. However,the accuracy and precisionof HPLC in the measurement of serum TBIL are at present inadequate because- (a) Calibration is performed with Bu with the ssumption that the molar absorptivity of the other three bilirubin species are identical to that of Bu although in fact they are not known (b) errors in the measurements of the fractions are cumulative (c) Some BIL may be lost during the complex pre treatment of samples. 71. DETERMINATION OF BILIRUBIN IN INFANTS BY DIRECT SPECTROPHOTOMETRYBilirubin in human serum exhibits an absorption maximum near 460nm.. Because carotenes and other pigments are virtually absent in the newborns blood, hemoglobin is the only pigment in serum that interferes with the bilirubin measurement This interference is eliminated by analysis of a two-component system Turbidity (lipemia) will cause a negative bias if the secondary wavelength is shorter than the primary, and a positive bias if it is longer. This is because light scattering (and hence absorbance) varies inversely with the wavelength 72. Spectrophotometric methods following dilution serum sample is diluted with caffeine benzoate solution With the use of a photometer with monochromatic light (spectral band width of 2 nm) the method does not need to be calibrated. The bilirubin concentration is calculated from the following formula: c (micromol/l) =f X 21.26 X(E 465 nmE 528 nm) where f is volume of the test/volume of the sample. Interferences through oxyhaemaglobin and turbidity are well corrected However, since the method cannot be automated, its use remains restricted to situations where sample numbers are low. 73. Spectrophotometric measurement without dilution (bilirubinometer) Since the molar extinction coefficient of the haemoglobin is identical at 454 nm and 540 nm, the bilirubin concentration can be calculated from the difference Bilirubin, mg/dL = (A454 - A540) X 1.19 X 51 To obtain sufficiently accurate absorbance readings required by this method, a spectrophotometer with a bandpass of less than 8 nm should be used A haematocrit capillary tube serves as cuvette; it is filled with (capillary) blood and centrifuged in a special centrifuge. 74. Direct spectrophotometry in whole blood The direct, spectrophotometric measurement of total bilirubin in whole blood is possible with the kits ABL 730, 735, 830 Flex, 835 Flex and 837 Flex (Radiometer), Rapidlab 1200 (Siemens Medical Solutions) or cobas b 221 (formerly OMNI S) (Roche Diagnostics). The measurement principle is identical for all kits in the Co-oximetry module, bilirubin is determined in the haemolysed (ABL, cobas) or not-haemolysed (Rapid lab) sample together with the haemoglobin fractions by means of a multi-wavelength measurement (ABL, 128 wavelengths in the range 478672 nm; cobas, 512 wavelengths in the range 460 660 nm, Rapidlab 256 wavelengths in the range 500680 nm).. The bilirubin concentration is calculated from the results of the measurements of absorption with the help of a multi-component analysis. 75. Measurements with carrier-bound reagents (dry chemistry) Multilayer film technology (Vitros, Ortho-Clinical Diagnostics) The required reagents are applied to a thin film, that together with a carrier layer forms a slide with a reaction zone of about 1 cm2. A serum or plasma sample is applied and low-molecluar components and water diffuse into the underlying layer, the latter thereby dissolving the reagents enclosed in the slide. After completion of the indicator reaction, the pigment formed is measured reflectometrically through the transparent carrier layer. 76. Determination of total bilirubin by the diazo- procedure (TBIL slide) The slide contains as reagents - a stabilized diazonium salt (4-(N-carboxy- methylaminosulfonyl)- benzoldiazoniumhexafluorphosphate) and diphyllin and Triton X-100 as accelerators. All bilirubin fractions react quantitatively to a pigment, which is bound to a mordant and measured at 540/460 nm. The range of measurement reaches from 0.1 mg/dL to 27 mg/dL 77. Measurement of unconjugated and conjugated bilirubin with the help of direct spectrophotometry (BuBc slide) The reagent-carrying layer of the slide contains caffeine, sodium benzoate and surfactants, which cause a dissociation of bilirubin and albumin. The dissociated, unconjugated bilirubin migrates together with the bilirubin glucuronides through a barrier layer that retains proteins and with these delta-bilirubin too. After binding to a mordant the glucuronides show an absorption maximum at 420 nm, whereas that of unconjugated bilirubin is at 460 nm. At 400 nm the molar extinction effects are virtually equal. Using a two-wavelength measurement and an elaborate calculation it is possible to determine the concentrations of unconjugated and conjugated bilirubin separately. Since haemoglobin is held back by the barrier layer, the test is practically not influenced by haemolysis 78. Transcutaneous measurement Uses reflectance photometry Measurement is required at mulltiple (8) sites Bili check provides results within +/- 2 mg /dl of diazo procedure Underestimates bil when conc is greater than 10 mg/dl Provides instaneous information and spares heel sticks Aids in predicting those babies who require folow up by hour- specific normogram 79. Reference Ranges In adults and infants older than 1 month, the normal reference interval for total serum bilirubin is total- 0.2 to 1.2mg/dL (3.4 to 20.5mmol/L) conjugated bilirubin - 0 to 0.2mg/dL(0 to 3.4mmol/L) 80. URINE BILIRUBIN Presence indicates conjugated hyperbilurubinemia Dipstick methods can detect conc as low as 0.5 mg/dl Fresh sample is required In case of delay store at 2-8 C for max 24 hrs, protected from light Strip is immersed in specimen for 1 sec and read at 60 sec Use diazo reaction Ictotest tablet is semi quantitative method Medications that color urine red or give red color in acidic media , e.g. phenazopyridine give false +ve Large quantities of vit C and nitrite interfere 81. Urine Urobilinogen Ehrlichs test Principle: Urobilinogen reacts with p-dimethylamino-benzaldehyde in chloroform to form a pink coloured aldehyde complex. Reagents: - Ehrlichs reagent - Saturate sodium acetate - Chloroform Procedure: 5 ml Urine + 5 ml Ehrlichs reagent Mix and allow to stand for 10 min + 5 ml saturated sodium acetate and mix + 5 ml chloroform shake vigourously and allow layers to separate Appearance of pink colour in the chloroform layer indicates presence of urobilinogen Colour is easily detected when viewed from top of the test tube 81 82. and faeces Laboratory determination of fecal or urine urobilinogen is based on Ehrlichs reaction, which uses para-dimethylaminobenzaldehyde to form a red color. Ascorbic acid may be added to the sample to maintain urobilinogen in its reduced state. The Specimen The test requires fresh sample; urobilinogen may be oxidized to urobilin on standing. Interferences Urobilin in the sample is reduced by alkaline ferrous hydroxide to urobilinogen. Sodium acetate further reduces other chromogens, which may interfere with Ehrlichs reagent. Bilirubin may interfere with the reaction; significant amounts of bilirubin must be precipitated with barium chloride and removed by filtration. Reference Ranges Urine 0.54.0 Ehrlich units/day Feces 75400 Ehrlich units/day Increased in prehepatic / hepatic jaundice . Absent in post hepatic jaundice 83. Free bilirubin (Bf) Free bilirubin, i.e., unconjugated and not bound to albumin, represents a significant component of the neurotoxicity of bilirubin, which is made responsible for the bilirubin encephalopathy of the neonate (resulting in kernicterus) The toxic effect is thought to occur even at a concentration of 0.005 mg/dL . Up to now, there has been no really reliable method for measuring free bilirubin in plasma . 84. Mechanisms and Causes Unconjugated hyperbilirubinemia Mechanism Examples Suggestive Findings Increased bilirubin production Common: Hemolysis Less common: Resorption of large hematomas, ineffective erythropoiesis Few or no clinical manifestations of hepatobiliary disease; sometimes anemia, ecchymoses Serum bilirubin level usually < 3.5 mg/dL (< 59 mol/L), no bilirubin in urine, normal aminotransferase levels. Decreased hepatic bilirubin uptake Common: Heart failure Less common: Drugs, fasting, portosystemic shunts Decreased hepatic conjugation Common: Gilbert syndrome Less common: Ethinyl estradiol , Crigler-Najjar syndrome, hyperthyroidism 85. Conjugated hyperbilirubinemia Hepatocellular dysfunction Common: Drugs, toxins, viral hepatitis Less common: Alcoholic liver disease, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis,steatohepatitis, Wilson disease Aminotransferase levels usually > 500 U/L Intrahepatic cholestasis Common: Alcoholic liver disease, drugs, toxins, viral hepatitis Less common: Infiltrative disorders (eg, amyloidosis, lymphoma, sarcoidosis, TB), pregnancy, primary biliary cirrhosis, steatohepatitis Gradual onset of jaundice, sometimes pruritus If severe, clay-colored stools, steatorrhea If long-standing, weight loss Alkaline phosphatase and GGT usually > 3 times normal Aminotransferase levels < 200 U/L Extrahepatic cholestasis Common: Common bile duct stone, pancreatic cancer Less common: Acute cholangitis, pancreatic pseudocyst, primary sclerosing cholangitis, common duct strictures caused by previous surgery, other tumors manifestations possibly similar to those of intrahepatic cholestasis or a more acute disorder (eg,common bile duct stone or acute pancreatitis) Alkaline phosphatase and GGT usually > 3 times normal Aminotransferase levels < 200 U/L Other, less common Hereditary disorders (mainly Dubin- Johnson syndrome and Rotor syndrome) Normal liver enzymes 86. Some Drugs and Toxins That Can Cause Jaundice Mechanism Drugs or Toxins Increased bilirubin production Drugs that cause hemolysis (In G6PD deficiency), such as sulfa drugs and nitrofurantoin Decreased hepatic uptake Chloramphenicol , probenecid , rifampin Decreased conjugation Ethinyl estradiol Hepatocellular dysfunction Acetaminophen (high dose), amiodarone , isoniazid , NSAIDs, statins, many others, many drug combinations. Amanita phalloides mushrooms, carbon tetrachloride, phosphorus Intrahepatic cholestasis Amoxicillin/clavulanate, anabolic steroids, chlorpromazine , pyrrolizidine alkaloids (eg, in herbal preparations), oral contraceptives, phenothiazines