Bilirubin:- Part 1 – Total Bilirubin (Direct and Indirect Bilirubin), Classification of Jaundice
Total Bilirubin (Direct and Indirect)
Sample for Bilirubin
- This test is done on the serum of the patient.
- A random sample can be taken, and no need for a fasting sample.
- Fasting samples may be advised to avoid lipemic serum.
- From infants, the blood may be collected from the heel.
- The sample is stable at 4 °C for 3 days and when protected from light.
Precautions for the estimation of Total Bilirubin (Indirect/direct)
- Avoid hemolysis.
- Avoid shaking the tube, which may lead to inaccurate results.
- Do not expose the tube to light. Exposure to light like the sun or even artificial light may decrease the value.
- If there is a delay in the test, keep the sample away from the light (artificial or sunlight) and refrigerate it.
- The sensitivity to light is temperature-dependent; for optimal storage, keep the sample in a colored bottle and at a low temperature.
Purpose of the test (Indications) for Total Bilirubin (Direct and Indirect Bilirubin)
- For the diagnosis of jaundice.
- To differentiate different types of jaundice.
- For the follow-up of a patient with treatment.
- To assess the progress of the disease.
- This test is done to evaluate liver functions.
- This is done in a patient with hemolytic anemia in adults.
- It is also done to evaluate hemolytic anemia in newborns.
Definition of Total Bilirubin (Direct and Indirect Bilirubin):
- Bilirubin is the orange-yellow pigment derived from the catabolism of hemoglobin (Heme).
- The majority of the bilirubin comes from the old RBCs.
- Bilirubin is the basic end product of Hemoglobin metabolism.
- Bilirubin is the end product of heme degradation.
- In circulation, there are 4 types (fractions) of bilirubin:
- Unconjugated bilirubin is also called indirect bilirubin or α-bilirubin.
- Monoglucoronide (monoconjugated) bilirubin, also called β-bilirubin.
- Diglucoronide bilirubin (diconjugated), also called direct bilirubin or γ-bilirubin.
- A fraction of the bilirubin is irreversibly bound to the protein called δ-bilirubin.
- Raised Bilirubin is an indicator of liver dysfunction.
Pathophysiology of the Bilirubin
Sources of the bilirubin:
- Most of the daily bilirubin production is from senescent red blood cells.
- 1 to 2 x 108 RBCs are destroyed per hour in the human body.
- An average person weighing 70 kg will have a roughly turnover of 6 grams of hemoglobin/day.
- 1 gram of the hemoglobin = 35 mg of bilirubin.
- The total daily production of bilirubin is 250 to 300 mg/day.
- Rest is from the hepatic hemoproteins and immature destruction of the RBCs in the bone marrow.
Metabolism of the bilirubin:
- The Bilirubin metabolism can be described in three stages:
- The first stage is RBCs’ breakdown, forming heme and biliverdin, bilirubin IXα by the mononuclear phagocytic system.
- The second stage is the conjugation of bilirubin, which occurs in the hepatocytes.
- The third stage is the bile secretion into the gall bladder and intestine.
The first stage:
Bilirubin metabolism takes place in the mononuclear phagocytic system:
- Hemoglobin from RBC is released and gives rise to Heme and Globin.
- Heme from the RBC gives rise to protoporphyrins.
- Now protoporphyrins by microsomal heme oxygenase form Biliverdin IXα.
- Biliverdin reductase enzyme transforms it into bilirubin IXα.
- Globin is degraded to its components like amino acids, reused or recycled.
- Microsomal heme-oxygenase enzyme degrades the heme into iron Fe++, which will be oxidized to Fe+++ form and reutilized again.
- Under the influence of the above enzyme, iron-free porphyrins are formed.
- O2 molecule is added, which will lead to CO formation, which is exhaled through the lungs.
- The biliverdin IX-α (green color) is formed under the bilirubin reductase enzyme’s action.
- The heme is reduced by NADPH to NADP and forms the bilirubin IX-α (yellow pigment).
- The plasma albumin takes this unconjugated bilirubin to the liver. Albumin acts as the carrier protein.
- Albumin delivers the bilirubin into the hepatocytes and goes back to blood circulation.
- Unconjugated bilirubin (indirect bilirubin) is not water-soluble but is fat-soluble.
- This unconjugated bilirubin combines with albumin, where the albumin is the carrier protein that carries this bilirubin to the hepatocytes.
- Its solubility increases when it combines with albumin (noncovalent binding to the albumin).
Albumin as a carrier:
- Albumin has one high-affinity site and one low-affinity site for bilirubin.
- In 100 ml of the plasma:
- Around 25 mg of the bilirubin combine with a high-affinity site.
- Excess of the bilirubin combined with a low-affinity site is easily detachable.
- Antibiotics and some drugs compete with bilirubin for high-affinity sites.
- Bilirubin bound to the high-affinity site on albumin is carried to the liver.
- Bilirubin is removed from the albumin by the hepatocytes’ sinusoidal side by a carrier-mediated saturable system in the liver.
- This facilitated transport system has an enormous capacity, even in pathological conditions.
- The second stage is the conjugation process in the hepatocytes:
- Bilirubin is nonpolar and is bound to the lipids in the cells.
- The bilirubin conjugation occurs in the hepatocytes, and the bilirubin becomes water-soluble.
- The bilirubin is taken to the hepatocytes, where albumin remains in the blood, and bilirubin goes into hepatocytes from the sinusoidal surface.
- Bilirubin is tightly but reversible in the hepatocytes and binds to the cytosolic binding protein, ligandin, and Z protein.
- Bilirubin undergoes the conjugation process in the hepatocytes by combining glucuronic acid (UDPGA=Uridine diphosphate glucuronic acid) with smooth endoplasmic reticulum enzyme glucuronyltransferase.
- The uridine diphosphate glucose (UDP-glucose) will form glucuronic acid.
- The main conjugation process occurs in the hepatocytes with the help of glucuronic acid and glucuronyl transferase enzyme.
- The microsomal enzyme bilirubin UDP-glucuronyl transferase catalyzes the formation of bilirubin monoglucuronide.
- Then there is the formation of the bilirubin diglucuronide.
- ∼90% of the bilirubin diglucuronide is secreted into the bile.
- ∼10% of bilirubin monoglucuronide is secreted into the bile.
- The third stage is the secretion of conjugated bilirubin into bile and the intestine:
- The secretion of the conjugated bilirubin (bilirubin diglucuronide, direct reacting bilirubin) occurs by an active transport mechanism.
- This secretion of bilirubin in the bile is a rate-limiting process.
- Mainly the conjugated bilirubin (90%) is secreted into the bile.
- This direct reacting conjugated bilirubin reaches the small intestine (terminal ileum) and large intestine.
- Most of it is metabolized in the intestine and excreted in feces.
The Conjugated bilirubin fate in the intestine:
- When bilirubin reaches the terminal ileum and the large intestine, glucuronide is removed by specific bacterial enzymes β-glucuronidase.
- The fecal flora reduces this pigment to a group of colorless tetrapyrrole compounds called urobilinogen.
- A small fraction of the urobilinogen in the small and large intestines is reabsorbed and excreted through the liver to form enterohepatic circulation.
- If the excessive formation of bile or liver disease interferes with the intrahepatic cycle, urobilinogen will be excreted in the urine.
- Normally the colorless urobilinogen in the colon is oxidized by the fecal flora and forms the colored compound urobilin.
Various facts about bilirubin:
- The increased amount of bilirubin causes the yellow color of the skin and conjunctiva (Jaundice).
- Because bilirubin is an orange-yellow pigment derived from the metabolism of heme.
- Bilirubin is not absorbed in the intestine; hydrolyzed by the β- glucuronidase enzyme, which comes from:
- Intestinal epithelial cells.
- The intestinal microbial flora reduces this unconjugated bilirubin to urobilinogen (colorless tetrapyrrole compound).
- Analysis of the bilirubin from the sera of various patients with liver diseases shows the following pattern:
- Unconjugated bilirubin = 27%
- Monoconjugated bilirubin = 24%.
- Diconjugated bilirubin = 13%.
- Protein-bound bilirubin = 37%.
- Analysis of the bilirubin from the sera of various patients with liver diseases shows the following pattern:
- The bilirubin, which is not conjugated, attaches to albumin (carrying protein ), called Indirect bilirubin.
- Unconjugated (indirect) bilirubin is water-insoluble and bound to albumin in the blood.
- This unconjugated bilirubin is not seen in the urine.
Total Bilirubin = Direct bilirubin + Indirect bilirubin.
- Without an accelerator (alcohol), mainly conjugated bilirubin is measured (direct reaction).
- Accelerator permits unconjugated bilirubin to react as well, providing total bilirubin.
- Indirect bilirubin is calculated as follows:
- Indirect bilirubin = Total bilirubin – direct bilirubin.
It differs from the degree of hydrogenation of the vinyl side chain and the two-end pyrrole rings.
- Urobilinogen containing 6, 8, and 12 more hydrogen ions atoms, respectively, are called:
- Up to 20% of urobilinogen produced is reabsorbed and enters the enterohepatic circulation.
- Most of the urobilinogen is reabsorbed, taken up by the liver, and excreted in the bile.
- A small fraction of 2% to 5% goes into general circulation and appears in the urine.
- The bilirubin is conjugated with glucuronic acid. This process occurs in the liver and leads to conjugated or direct bilirubin. Now conjugated bilirubin is no more bound to protein.
It is formed in the liver and consists of Bile salts, phospholipids, cholesterol, bicarbonate, bilirubin, and water.
- Primary bile acids are Cholic acid and chenodeoxycholic acid.
- Secondary bile acids are Deoxycholic acid and Lithocholic acid. Intestinal bacteria form these.
- Both primary and secondary bile acids are from the bile acid pool.
- The liver assists in intestinal digestion by secreting 700 to 1200 ml of bile /per day.
- Bile is a bitter taste, alkaline, and yellowish-green in color.
- Bile consists of the following:
- Bile salt (conjugated bile acids).
- Bilirubin is one component of bile transported from the liver, stored in the gallbladder, and delivered to the intestine.
- In the bile:
- Bilirubin diglucuronide is ∼90%.
- Bilirubin monoglucuronide is ∼10%.
- Functions of bile:
- Intestinal fat emulsification.
- Absorption of the fat.
- Bile has two fractions:
- The bile acid-dependent fraction consists of bile acids, cholesterol, lecithin (phospholipids), and bilirubin. The hepatocytes secrete this fraction.
- The bile acid-independent fraction, which is secreted by the hepatocytes and epithelial cells of bile canaliculi, is a bicarbonate-rich aqueous fluid that gives bile alkaline pH.
Bilirubin in various body fluids and tissues:
- Bilirubin is more in the exudate (exudate more icteric) than transudate.
- CSF is more xanthochromic when there is meningitis.
- Basal ganglia are bile stained due to the high unconjugated bilirubin concentration.
- Occular fluids are yellow in deep jaundice.
- Urine, sweat, serum, and milk contain bile pigments.
- The paralyzed parts and edematous parts tend to remain uncolored.
- Skin, ocular sclera, and blood vessels have high elastic tissue, easily becoming icteric.
The difference between direct (Conjugated) bilirubin and Indirect (Unconjugated) bilirubin:
|Characteristics||Conjugated Bilirubin||Unconjugated Bilirubin|
|Structure||Bilirubin diglucuronide||Bilirubin IXα|
|Type of compound||Polar||Non-polar|
|Presence in urine with jaundice||Positive (Loselybound to albumin)||Negative (tightly bound to albumin)|
- Jaundice appears when the bilirubin level is above 2.5 mg/dl.
- When the liver can not conjugate bilirubin in the newborn, and if the level increases, this indirect bilirubin can cross the blood-brain barrier and lead to toxic injury to the brain called Kernicterus.
- If the bilirubin level exceeds 15 mg/dl, start the treatment to avoid brain damage.
- While physiologic jaundice appears after 3 to 4 days and subsides itself.
Types of jaundice:
- Pre-hepatic jaundice. The etiology is before the liver, like increased hemolysis of RBC.
- Hepatic jaundice. Now the causes are in the liver, like hepatitis.
- Post-hepatic jaundice. The cause is after the liver, like gallstones, and cancers, leading to the obstructive type of jaundice.
The Different Types of Jaundice and their causes:
|Type of Jaundice||Causes||Pathophysiology|
Normal bilirubin value
- Total bilirubin = 0.3 to 1.0 mg/dL or 5.1 to 17.0 mmol/L
- Direct bilirubin (conjugated bilirubin) = 0.1 to 0.3 mg/dL or 1.0 to 5.1 mmol/L
- Indirect bilirubin (unconjugated bilirubin) = (total bilirubin minus direct bilirubin level) = 0.2 to 0.7 mg/dL or 3.4 to 11.9 mmol/L
- Total bilirubin in:
- Umbilical cord blood = less than 2 mg/ dl.
- 0 to 1 day = less than 6 mg/ dl
- 0 to 2 days = less than 8 mg/ dl
- 3 to 5 days = less than 12 mg/dl
- after 5 days = less than .2 to 1.0 mg/ dl
- Urine is negative for bilirubin.
- For conversion to SI unit, multiply by 17.1 and report as µmol/L (mmol/L)
Another source: Total bilirubin level
|Age||Premature mg/dL||Full-term mg/dL||Adult mg/dL|
|Cord blood||<2 mg||<2.0|
|0 to 1 day||<8.0||1.4 to 8.7|
|1 to 2 days||<12.0||3.4 to 11.5|
|3 to 5 days||<16.0||1.5 to 12.0|
|5 days to 60 years||0.3 to 1.2|
|60 to 90 year||0.2 to 1.1|
|>90 year||0.2 to 0.9|
Raised Total Bilirubin level is seen in the following:
- Some infections, such as an infected gallbladder or cholecystitis.
- Some inherited diseases, such as Gilbert’s syndrome.
- Although jaundice may occur in some people with Gilbert’s syndrome, the condition is not harmful.
- Diseases cause liver damage, such as hepatitis, cirrhosis, or mononucleosis.
- Diseases that cause blockage of the bile ducts, such as gallstones or cancer of the pancreas.
- Rapid destruction of red blood cells in the blood, such as sickle cell disease or an allergic reaction to blood received during a transfusion (called a transfusion reaction).
- Medicines that may increase bilirubin levels. This includes many antibiotics, some types of birth control pills, indomethacin (Indocin), phenytoin (Dilantin), diazepam (Valium), and flurazepam (Dalmane).
Decreased Total Bilirubin level is seen in:
- Medicines that may decrease bilirubin levels. This includes vitamin C (ascorbic acid), phenobarbital, and theophylline.
Causes of direct hyperbilirubinemia:
- When more than 50% is direct bilirubin.
- Gallbladder tumors.
- Inflammatory scarring or obstruction of extrahepatic ducts.
- It can be resolved by surgery.
- Dubin-Johnson syndrome.
- Rotor syndrome.
- Drugs may cause cholestasis.
Causes of Indirect hyperbilirubinemia:
- When less than 15 to 20 % is direct bilirubin.
- Increased RBC hemolysis ( Erythroblastosis fetalis).
- Sickle cell anemia.
- Crigler-Najjar syndrome.
- Gilbert syndrome.
- Congenital enzyme deficiency.
- Transfusion reactions.
- There is no role in surgery.
Difference between hepatocellular and cholestatic jaundice:
|Laboratory tests/ symptoms||Hepatocellular jaundice||Obstructive/cholestatic jaundice|
|SGOT/ SGPT||Increased (>3 times the normal)||Normal or raised (<3 times the normal)|
|Alkaline phosphatase||Normal or raised (<3 times the normal)||Increased (>3 times the normal)|
Lab diagnosis for jaundice
- Advise liver function tests (LFT), which include:
- Total bilirubin
- Direct and indirect bilirubin.
- Alkaline phosphatase.
- Total proteins.
- Albumin level.
- γ-GT (gamma-glutamyltransferase) level.
- Bilirubin in urine:
- Foam test: This is done to detect the bilirubin in the urine.
- Place 3 to 5 mL of urine in the test tube.
- Cap the tube and shake it vigorously.
- If the foam at the top is white, = Test is negative (no bilirubin in the urine).
- If the foam is orange in color = the Test is positive (bilirubin is present in the urine).
- Precaution: Phenazopyridine also gives orange foam.
- Ictototest for bilirubin in the urine:
- This can detect bilirubin levels in urine as low as 0.05 mg/dL and as high as 0.1 mg/dL.
- Ictotest tablets are commercially available.
- Don’t expose the ictotest tablets to light, heat, or moisture.
Differential diagnosis of urine bilirubin and urine urobilinogen:
|Urine sample||Normal person||Hemolytic disease||Hepatic diseases||Biliary obstruction|
|Urobilinogen||Normal||Present||Present||Absent or low|
Differential diagnoses of various types of jaundice:
|Type of the jaundice||Direct bilirubin||Indirect bilirubin||Stool bilirubin||Stool urobilinogen||Urine bilirubin||Urine urobilinogen||Total cholesterol|
|Viral hepatitis||Increased (early)||Increased (predominant)||Decreased||Decreased||Increased||Normal or increased||Normal or decreased|
|Cholestatic jaundice||Increased||Mild increase||Decreased||Decreased||Increased||Normal or decreased||Increased|
|Hepatitis due to drugs||Increased early||Increased||Decreased||Decreased||Increased||Normal or increased||Normal|
|Extrahepatic biliary obstruction||Increased||Normal or mildly increased||Markedly Decreased||Markedly decreased||Increased||Decreased||Increased mild to marked|
|Cirrhosis||Increased <indirect||Increased >direct||Increased||Normal or increased||Normal or decreased|