Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen, and finally urobilin.

EXPERIMENT 10BILE ANALYSIS IN URINEIllustrate the negative and positive results.IODINE -SMITH’S TESTNegative ResultPositive Result1.Make an outline showing the steps in the degradation of hemoglobin tobilirubin, urobilinogen, and finally urobilin2.Tabulate the sets of results for urinary bilirubin and urobilinogenconsistent with the diagnosis of the bile duct obstruction, liver disease,and hemolytic disorder.

3.Discuss the principle of the reagent strips test for urinary bilirubinincluding possible causes of error.4.Discuss the advantages and disadvantages of performing an ictotestforthe detection of urine Bilirubin.

EXPERIMENT 11DETERMINATION OF UROBILINOGENIllustrate positive and negative testEHRLICH BENZALDEHYDE TESTNegative ResultPositive ResultQUESTIONS1.What is urobilinogen?2.Describe the Watson-Schwartz test used to differentiate amongurobilinogen, porphobilnogen, and Ehrlich reactive compounds.

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A liver function panel always includes tests for bilirubin levels. Let’s review how bilirubin is formed and eliminated from the body, and how it’s measured in a clinical laboratory.

How is bilirubin formed and excreted? 

Bilirubin is a waste product produced by the breakdown of red blood cells. Bilirubin is the end-product of heme metabolism; the liver is the site for bilirubin metabolism.

Hemoglobin is broken down into heme, which is converted to biliverdin, and finally into unconjugated bilirubin (which is not water-soluble). In the bloodstream, unconjugated bilirubin binds with serum proteins—most commonly albumin. The unconjugated bilirubin is then taken up by the liver.

Figure 1. During the breakdown of hemoglobin, heme is released and converted into biliverdin. Biliverdin is converted into unconjugated bilirubin, which is transported in the bloodstream by binding with albumin and taken up by the liver. 

In the liver, the unconjugated bilirubin is bound to glucuronide by the enzyme uridine 5’-diphospho-glucuronosyltransferase (UDP) and becomes conjugated bilirubin. Conjugated bilirubin is then excreted in bile. 

Figure 2. In the liver, unconjugated bilirubin binds to glucuronide by the enzyme uridine 5’-diphospho-glucuronosyltransferase (UDP). The product is conjugated bilirubin, which can be excreted in bile. 

In the intestines, bacterial enzymes hydrolyze conjugated bilirubin to release free, unconjugated bilirubin, which is reduced to urobilinogen. Urobilinogen, bound to albumin, is then excreted in urine. 

Figure 3. In the intestines, conjugated bilirubin is hydrolyzed by bacterial enzymes to produce unconjugated bilirubin and ultimately urobilinogen, which can be excreted in urine after binding to albumin. 

In the intestines, some urobilinogen is converted to stercobilinogen and excreted in the stool. Thus, in normal urine, only urobilinogen is present; in normal stool, only stercobilinogen is present.

Figure 4. Urobilinogen in the intestines can also be converted to stercobilinogen, which is then excreted in the stool.

What happens to bilirubin with cholestatic or obstructive jaundice?

Cholestatic or obstructive jaundice occurs when liver cells are unable to transport bilirubin through the hepatic-bile capillary membrane because of damage in the liver. Obstructive jaundice can also occur when transport through the biliary tract is blocked because of anatomical obstructions such as gallstones or cancer.

In obstructive jaundice, conjugated bilirubin regurgitates into the blood. Because it is water-soluble, bilirubin is excreted into the urine. This is called choluria, or the presence of bile in the urine. 

As well, less conjugated bilirubin is taken up by the intestines in obstructive jaundice. As a result, less stercobilinogen is found in the stool and the stool appears pale.

Table 1. Under normal conditions, only urobilinogen is present in the urine, and stercobilinogen is present in the stool. With obstructive jaundice, conjugated bilirubin is present in the urine (also called choluria) and less stercobilinogen is present in the stool, giving the stool a pale appearance. 

How is bilirubin measured in the lab and what are the normal levels? 

In the clinical laboratory, conjugated bilirubin is measured as direct bilirubin. If we take the total bilirubin and subtract the direct bilirubin, it provides the concentration of unconjugated bilirubin (also referred to as indirect bilirubin).

Figure 5. Indirect or unconjugated bilirubin can be calculated by subtracting direct (e.g., conjugated) bilirubin from the total bilirubin. 

This concludes our review of bilirubin metabolism and how it’s measured in the laboratory. For your reference, here are the normal values of bilirubin in adults:

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What is the process of hemoglobin degradation?

What are the products of hemoglobin degradation?

Where does most hemoglobin degradation occur?

How is heme degraded?