Urinalysis and Body Fluids CRg Unit 3Chemical Examination of UrinePart 5, Bilirubin and UrobilinogenChemical Exam of Urine - bilirubin• Bilirubin and Urobilinogen - presence in urine may be the 1st indication of liver disease •Bilirubin formation (overview)•Formed by hemoglobin degradation•RBCs breakdown @ 120 days• Iron, proteins & amino acids are recycled, •Protoporphyrin (heme) eventually becomes bilirubin.•Liver’s reticuloendothelial cells (Kupffer) & liver parenchymal cells (hepatic cells) Chemical Exam of Urine - bilirubin• Free bilirubin (insoluble, indirect, unconjugate)• Formed first• Circulates in blood bound to protein - albumin - which adds to its size - keeping it from being excreted• **Water insoluble – cannot be excreted by kidneyChemical Exam of Urine• Insoluble bilirubin• Goes to liver – converted to water soluble bilirubin by the liver parenchymal (hepatocytes) cells• conjugated with glucuronic acid• Forms bilirubin diglucuronide(also called direct bilirubin, conjugated bilirubin, or water soluble bilirubin)Chemical Exam of Urine• Conjugated or direct bilirubin - also called bilirubindiglucuronide or direct bilirubin• Most excreted through bile duct into the intestine• Small amounts of conjugated bilirubin regurgitate back from the bile duct and into the blood system (not more than 0.2 – 0.4 mg/dL).• Get filtered through the glomerulus and excreted in the urine whenever the plasma level is increased.• Normally, no detectable amounts are present in urine• Majority goes down bile duct – into bile – into intestineChemical Exam of Urine• Urobilinogen• Formed in intestine as a result of the bacterial action on the conjugated / direct bilirubin• The conjugated bilirubin is degraded to form stercobilinogenand urobiliniogen.• The urobilinogen formed is absorbed by intestine and returned to liver by portal circulation•- where it is filtered out to again be pushed down into the intestines.• Most of which will be returned to intestine again• A small amount,@ 1%, escapes the liver clearance and will be excreted into urine.• Urobilinogen / stercobilinogen in the intestine / bowel will be reduced again (by bacteria) to form urobilin.•Bacteria continue to act on it to reducing it - forming urobilin- feces normal brown colorChemical Exam of Urine• Jaundice• Condition when serum bilirubin becomes greater than the liver can handle, and there is an abnormal collection of bilirubin in the tissues giving them a yellow color- eyes, skin, urine, serum etc. has a very distinctive yellow color.WikipediaJaundice – typesWikipediaJaundice• Hemolytic jaundice• Excessive hemolysis of red cells (sickle cell anemia, cold agglutinins, malaria, burns, mechanical heart valves, etc.• Liver functions normal – conjugates and eliminates bilirubin• Too much bilirubin produced – liver can't clear blood resulting in a build up of indirect, insoluble bilirubin• No bilirubin found in urine -why? • Increased urobilinogen found in urine • the liver is occupied with conjugating indirect bilirubin• can’t reprocess the urobilinogen that is coming from the intestines.• So the urobilinogen goes into the urineJaundice• Hemolytic jaundice clinical picture• Negative urine bilirubin•- the serum level of unconjugated bilirubin is increased, but it is not soluble• Increased urine urobilinogen• Increased fecal urobilinogenJaundice• Obstructive jaundice• Causes some type of blockage (Gall stones, Tumor, Edema)• Liver conjugates but can't excrete• Conjugated bilirubin regurgitated into blood • Conjugated (direct) bilirubin found in urine• No urobilinogen found in urine• No urobilin in feces• Obstructive jaundice clinical picture• Positive urine bilirubin• Negative urine urobilinogen• Negative-trace fecal urobilinogen•stools are a grayish, chalky, clay color.Jaundice• Hepatocellular jaundice• Malfunction of liver cells ie. viral hepatitis, cirrhosis, etc.• Both urobilinogen and bilirubin(direct)found in urine• Hepatocellular jaundice clinical picture• Positive urine bilirubin• Normal fecal urobilinogen• Increased urine urobilinogenJaundiceChemical Exam of Urine• Testing for Bilirubin• Review questions:• If specifically interested in detecting urine bilirubin / urobilinogen, what special collection and handling requirements are necessary? & Why?• What physical characteristics would you expect a sample containing bilirubin to have?• What about its microscopic sediment?Chemical Exam of Urine - bilirubin• If specifically interested in detecting urine bilirubin / urobilinogen what special collection and handling requirements are necessary? & Why?• Because bilirubin is easily destroyed in light and air, great care should be taken to protect it from these factors. The specimen should also be transported and tested ASAP. Testing specific for urobilinogen requires collection from 1-3 PM.• What physical characteristics would you expect a sample containing bilirubin to have?• Dark, amber color. Yellow foam.• What about its microscopic sediment?• Bilirubin stained sediment, bilirubin crystals, others?Testing for bilirubinMost current tests for bilirubin are based on a diazo reaction.• Ictotest – bilirubin confirmatory test• Reagents• Diazo• Sulfanilic acid – provides suitable acid environment• Napthylamines• Procedure overview: • Add drops of urine to a special asbestos mat• Bilirubin, if present in the urine, remains on outer edge of mat. • Place a tablet on top, • Add drops of water to the tablet, • Allow it to spill over onto the mat,• Look for a purple color development on the mat.Urine bilirubin confirmatory testIctotest color reactions.Most current tests for bilirubin are based on a diazo reaction.•Ictotest•Reagents•Diazo•Sulfanilic acid - provides suitableacid environment•Napthylamines•More sensitive•As little as 0.05mg/dL•More specific•Less interference•Generally easier to readUrine dipstick bilirubinBilirubin color chartacid Bilirubin + diazide azobilirubinSensitivity = 0.5 mg/dLUrine bilirubin: false positive• Technique errors– Reading after the prescribed time• atypical color reactions produced by:– Indican– Metabolites of etodolac (Lodine) – Chlorpromazine (Thorazine) – Metabolites of phenazopyridine• Confirm results with IctotestUrine bilirubin: false negative• Large amounts of