Delta Bilirubin : Characteristics, Synthesis, Clinical Significance and Measurement

Delta Bilirubin (Bδ) is a conjugated bilirubin covalently bound with albumin when conjugated bilirubin accumulates in the bloodstream due to impaired biliary excretion. This is nontoxic and excreted neither in urine nor in bile. This binding occurs in conditions like cholestasis or liver disease, where bilirubin can't be properly excreted.  Due to its attachment to albumin, delta bilirubin has a longer half-life than other types of bilirubin and remains in the blood for an extended period, making it a useful marker for chronic liver dysfunction or prolonged hyperbilirubinemia even after other bilirubin levels normalize.

Clinical utility of measuring Unconjugated bilirubin comes in the management of infants with routine physiologic jaundice, those with increased bilirubin production and those with defective conjugation, and the accuracy of measurement specifically relates to assessing risk for bilirubin encephalopathy. Serum conjugated bilirubin levels reflect the reflux of bilirubin from hepatocytes and excretion of bile after conjugation.

Thus the clinical utility of measuring Conjugated bilirubin is as an indicator of cholestatic liver disease. Indeed, an elevated Conjugated bilirubin (or direct bilirubin) is the indication associate with the diagnosis of Biliary Artesia (BA). Delayed clearance of delta-Bilirubin from the blood circulation may interfere with interpretation of true conjugated Bilirubin level. Delta bilirubin is directly related to the chronicity and severity of cholestasis in congenital hepatobiliary anomalies. It serves as a valuable biomarker for diagnosis, prognosis, and management in conditions such as biliary atresia, choledochal cysts, and Alagille syndrome.

Characteristics of Delta Bilirubin

1. Biochemical Properties

• Chemical Structure: Delta bilirubin retains the properties of conjugated bilirubin but is structurally altered due to its binding to albumin. This complex is important for its solubility and transport in the bloodstream.
• Physical Appearance: Like other forms of bilirubin, delta bilirubin is typically yellowish, contributing to the jaundice seen in hyperbilirubinemia.

2. Half-Life and Stability

• Extended Half-Life: Delta bilirubin has a notably longer half-life compared to free and conjugated bilirubin, primarily due to its attachment to albumin, which protects it from rapid metabolism and excretion. This extended presence in the blood means delta bilirubin can be detected for weeks following an acute episode of jaundice.
• Metabolic Fate: Delta bilirubin is eventually cleared from circulation as albumin is metabolised, leading to gradual reduction in delta bilirubin levels.

Synthesis of Delta Bilirubin

Delta bilirubin synthesis occurs not in the liver, but in the bloodstream when there is an accumulation of conjugated bilirubin that binds covalently to albumin. This process only occurs under specific pathological conditions involving liver or bile duct impairment, and the resulting delta bilirubin remains in circulation due to its stable bond with albumin. The presence of delta bilirubin can provide a clinical marker for recent hepatobiliary issues, even as total bilirubin levels normalize.

The synthesis process of delta bilirubin consists of three steps, production of bilirubin, and conjugation of bilirubin in the liver and subsequent formation of delta bilirubin in the blood stream. Process flow is as follows-

1. Production of Bilirubin:

• Heme Catabolism: Bilirubin originates from the breakdown of hemoglobin, which is a heme-containing protein found in red blood cells. When red blood cells are recycled, heme is released and converted to biliverdin through the action of the enzyme heme oxygenase.
• Conversion to Unconjugated Bilirubin: Biliverdin is then reduced to unconjugated bilirubin (indirect bilirubin) by the enzyme biliverdin reductase. Unconjugated bilirubin is lipophilic (fat-soluble) and cannot be excreted by the kidneys, so it binds to serum albumin for transport in the bloodstream to the liver.

2. Conjugation in the Liver:

• Uptake by Hepatocytes: In the liver, unconjugated bilirubin is taken up by hepatocytes (liver cells) through specific transporter proteins.
• Conjugation Process: Within hepatocytes, unconjugated bilirubin undergoes conjugation, which involves the addition of glucuronic acid via the action of the enzyme uridine diphosphate-glucuronosyltransferase (UGT1A1). This reaction transforms unconjugated bilirubin into conjugated bilirubin (direct bilirubin), which is water-soluble.
• Bile Excretion: The conjugated bilirubin is then excreted into bile and transported to the intestines, where it is further processed and ultimately eliminated from the body through feces.

3. Formation of Delta Bilirubin:

• Impaired Biliary Excretion: Under pathological conditions, such as cholestasis (bile flow obstruction), there is an accumulation of conjugated bilirubin in the bloodstream due to the liver's inability to excrete it into bile. This can occur due to:

• • Intrahepatic conditions (e.g., hepatitis, cirrhosis).
  • Extrahepatic conditions (e.g., gallstones, tumors obstructing bile ducts).

• Binding to Albumin: When levels of conjugated bilirubin in the bloodstream exceed the liver's capacity for processing and excretion, some of it binds to albumin, resulting in the formation of delta bilirubin. This form of bilirubin is not solely a product of conjugation but rather a complex between conjugated bilirubin and albumin.

Clinical Significance of Delta Bilirubin

Elevated delta bilirubin might have an intimate association with elevated serum total bilirubin in certain post hepatic inflammation or in an obstruction state. Hence, measurement of elevated delta bilirubin along with total bilirubin may provide us with a prospective diagnostic parameter for assessing effective biliary drainage along with duration or chronicity of jaundice, which already has been found among the subjects with obstructive jaundice.

Thus, elevated delta bilirubin percentage may help to differentiate the diagnosis of post hepatic jaundice (obstructive jaundice) from hepatic or pre-hepatic jaundice (hemolytic jaundice).

The major causes of pathological jaundice include blood incompatibilities and diseases, hereditary syndromes, several forms of hepatitis, cirrhosis of the liver and other liver diseases, bile duct blockage along with infections and certain drug medications.

• Indicator of Chronic Liver Disease: Elevated levels of delta bilirubin can indicate chronic conditions such as hepatitis, cirrhosis, or prolonged cholestasis. Its persistence in the blood is especially significant in evaluating patients with a history of liver disease.
• Diagnostic Utility: Although delta bilirubin is not routinely measured in standard bilirubin tests, its levels can provide important insights:

• • Differentiation of Causes of Jaundice: High levels of delta bilirubin can help differentiate between different causes of jaundice, particularly distinguishing cholestatic conditions from hemolytic processes.
  • Prognostic Indicator: In patients recovering from liver disease, monitoring delta bilirubin can be useful to determine the resolution of biliary obstruction or liver function. Persistent elevations can suggest ongoing liver dysfunction.

• Clinical Impact of Delta Bilirubin : Delta bilirubin, a bilirubin-albumin complex that forms during prolonged cholestasis, plays a critical role in the diagnosis, monitoring, and management of various hepatobiliary disorders, especially biliary atresia, choledochal cysts,Neonatal Cholestasis, Alagille syndrome and Congenital hepatobiliary anomalies. Its persistence in circulation due to the extended half-life of albumin provides unique clinical insights. Clinical impact of delta bilirubin in specific conditions:
  1. Biliary Atresia : Biliary atresia is characterized by progressive fibrosis and obliteration of the extra hepatic bile ducts, leading to severe cholestasis. Persistent elevation of delta bilirubin is a hallmark of chronic cholestasis in biliary atresia. It helps differentiate biliary atresia from transient neonatal cholestasis. Elevated delta bilirubin levels after the Kasai portoenterostomy indicate incomplete bile drainage, ongoing bile duct damage, or liver fibrosis. Persistence of delta bilirubin can predict the likelihood of progressive liver dysfunction and need for liver transplantation. High delta bilirubin levels correlate with more severe liver damage and poorer long-term outcomes.
  2. Choledochal Cysts: Congenital dilations of the bile ducts lead to bile stasis, recurrent infections, and chronic cholestasis. Elevated delta bilirubin levels reflect prolonged bile stasis in choledochal cysts, especially in cases of delayed diagnosis or complications like cholangitis. Persistently elevated delta bilirubin after cyst excision may indicate residual bile duct obstruction, incomplete surgical correction, or chronic liver damage.Chronic elevations may suggest progression to liver fibrosis or cirrhosis due to prolonged pre-surgical damage.
  3. Neonatal Cholestasis: Neonatal cholestasis includes a spectrum of conditions causing impaired bile flow in the first weeks of life, ranging from transient idiopathic causes to structural anomalies. Elevated delta bilirubin helps distinguish transient cholestasis (e.g., due to sepsis or prematurity) from more chronic and severe conditions like biliary atresia or genetic/metabolic disorders. Delta bilirubin contributes to jaundice even after resolution of the underlying cause, aiding in identifying cases needing further investigation. Persistent delta bilirubin suggests incomplete resolution or residual liver damage.
  4. Alagille Syndrome: A genetic disorder characterized by intrahepatic bile duct paucity, associated with multi-system involvement. Delta bilirubin contributes to prolonged jaundice in Alagille syndrome, reflecting chronic cholestasis due to reduced bile ducts. High delta bilirubin levels correlate with more severe bile flow impairment and liver dysfunction. Persistently elevated delta bilirubin indicates the development of advanced liver disease, such as cirrhosis, and guides consideration for transplantation.
  5. Congenital Hepatobiliary Anomalies: These include structural or functional abnormalities of the liver and bile ducts, such as biliary hypoplasia, intrahepatic bile duct malformations, or ductal plate anomalies. Delta bilirubin is a reliable indicator of chronicity in conditions like bile duct hypoplasia or agenesis. Elevated delta bilirubin post-surgical or medical treatment indicates ongoing cholestasis or hepatic injury, guiding the need for further interventions. Persistent elevation reflects significant bile flow impairment and progression to fibrosis or cirrhosis.

  Delta bilirubin is a crucial clinical marker across various congenital hepatobiliary conditions, providing insights into disease chronicity, treatment response, and long-term prognosis. Its role is especially significant in biliary atresia, choledochal cysts, neonatal cholestasis, Alagille syndrome, and other congenital anomalies, where it guides diagnosis, monitoring, and therapeutic planning.

• Impact of delta bilirubin on Infants: Normally, Infants younger than 28 days, delta-Bilirubin level was less than 2% of total Bilirubin. In case of older infants and children having hyper bilirubinemic condition, delta-Bilirubin level was about 35%. High delta-Bilirubin that is greater than 50% of total bilirubin in newborns was associated with Intra and/or Extra-hepatic cholestasis, Biliary cirrhosis, Biliary Artesia, and Hepatitis.

Among older infants and children, a proportion of low delta-Bilirubin (less than 10%) was found in Hemolytic anemias, Sepsis, Shock, and other Non-hepatic jaundice. In several cases, when low delta-Bilirubin was accompanied by increased conjugated bilirubin, the prognosis was very poor.

Measurement of Delta Bilirubin

The measurement of serum bilirubin is probably the single most valuable diagnostic parameter in the clinical evaluation of infants with liver disease. The majority of circulating serum bilirubin consists of Unconjugated bilirubin (Bu) and Conjugated bilirubin (Bc). First is unconjugated bilirubin (Bu), which is insoluble in water and is tightly bound to albumin and other plasma proteins. The other is conjugated bilirubin (Bc), which consists of an array of sugar ester (mainly glucuronide) to form mono & di conjugates of bilirubin. Delta bilirubin can be calculated using the following formula:

Delta bilirubin = Total bilirubin − (Indirect bilirubin + Direct bilirubin)

1. Routine Techniques for Delta Bilirubin Quantification

• Total bilirubin in the blood includes unconjugated (indirect), conjugated (direct), and delta bilirubin. Most automated systems used in clinical chemistry laboratories measure total bilirubin directly by reacting the serum with a diazo reagent. However, this method does not separate delta bilirubin from conjugated bilirubin, so an elevated total bilirubin result might include a high delta bilirubin component, particularly in patients with cholestasis or liver disease.
• Conjugated bilirubin (also known as direct bilirubin) is measured by methods that use solvents or surfactants to selectively react with the water-soluble forms of bilirubin (conjugated and delta). Because delta bilirubin is covalently bound to albumin, it is often included in the direct bilirubin measurement. This can lead to a “direct bilirubin” result that may overestimate the true level of freely circulating conjugated bilirubin.
• Calculation of Delta Bilirubin : Delta bilirubin is often calculated rather than directly measured by subtracting the unconjugated (indirect) bilirubin level from the direct bilirubin level.

Formula: Delta Bilirubin=Total Bilirubin−(Conjugated Bilirubin+Unconjugated Bilirubin)

2. Specialized Techniques for Direct Delta Bilirubin Quantification

• In cases requiring precise measurement, high-performance liquid chromatography (HPLC) can separate delta bilirubin from other bilirubin fractions. This is often used in research settings or specialized labs for in-depth studies of bilirubin metabolism.
• Gel electrophoresis can also be used to distinguish delta bilirubin, as it separates bilirubin bound to albumin from free bilirubin fractions.

Laboratory measurement of delta bilirubin involves specific analytical techniques designed to separate and quantify this bound form of bilirubin. Since delta bilirubin is covalently bound to albumin, it cannot be easily removed or measured by conventional bilirubin assays. Accurate measurement requires a method that differentiates it from other forms of bilirubin (unconjugated and conjugated) to provide a complete picture of the patient’s bilirubin profile, especially in cases of chronic liver disease or prolonged jaundice.

 Frequently Asked Questions (FAQs) about Delta Bilirubin

1. What is delta bilirubin?

Delta bilirubin is a form of bilirubin that is covalently bound to albumin in the blood. It forms when there is an accumulation of conjugated bilirubin due to impaired bile excretion, such as in liver disease or biliary obstruction.

2. How is delta bilirubin different from other types of bilirubin?

Unlike unconjugated or conjugated bilirubin, delta bilirubin is bound to albumin, which gives it a much longer half-life. This allows it to remain in the bloodstream even after the underlying condition causing jaundice is resolved.

3. What causes an increase in delta bilirubin levels?

Delta bilirubin levels increase in conditions where conjugated bilirubin is unable to be excreted properly. Common causes include:

• • Cholestasis (bile flow blockage)
  • Biliary Artesia (BA)
  • Liver diseases such as hepatitis or cirrhosis
  • Biliary obstructions like gallstones or tumors etc.

4. Why is delta bilirubin clinically significant?

Delta bilirubin’s persistence in the bloodstream provides information about chronic or past liver dysfunction. It helps distinguish between recent and resolved liver issues, as elevated levels can indicate residual liver damage even after acute symptoms resolve.

5. How is delta bilirubin measured?

Delta bilirubin is typically calculated indirectly by measuring total and direct (conjugated) bilirubin and subtracting the unconjugated (indirect) bilirubin. Specialized techniques like high-performance liquid chromatography (HPLC) or gel electrophoresis can be used for direct measurement.

6. What does a high delta bilirubin level mean?

High delta bilirubin suggests prolonged or ongoing liver dysfunction. It is often seen in chronic liver conditions and indicates that bilirubin metabolism was impaired for a significant duration, resulting in residual albumin-bound bilirubin in the blood.

7. How does delta bilirubin affect jaundice?

Delta bilirubin contributes to the persistence of jaundice in patients even after acute liver issues are addressed. Due to its long half-life, patients may appear jaundiced for weeks after the resolution of the primary cause of bilirubin elevation.

8. What are normal levels of delta bilirubin?

There are no set “normal” ranges specifically for delta bilirubin, as it is generally considered part of the direct bilirubin measurement. Elevated delta bilirubin is typically inferred if total bilirubin remains high with resolved conjugated bilirubin.

9. How does delta bilirubin impact treatment decisions?

Delta bilirubin is used primarily for monitoring rather than direct treatment decisions. Persistently elevated levels can suggest that liver function has not fully recovered, which may prompt additional testing or ongoing monitoring in chronic liver disease patients.

10. Can delta bilirubin be reduced through treatment?

Delta bilirubin naturally decreases as liver function improves and albumin-bound bilirubin is metabolized. Treating the underlying liver or biliary condition is key; once resolved, delta bilirubin levels will gradually fall over a few weeks.

11. Why isn’t delta bilirubin measured routinely?

Delta bilirubin measurement is not routine because it is often calculated indirectly and doesn’t provide immediate therapeutic implications. However, it is valuable for understanding bilirubin dynamics in patients with chronic or complex liver conditions.

12. Does delta bilirubin indicate irreversible liver damage?

Not necessarily. Delta bilirubin indicates prolonged bilirubin retention but does not confirm irreversible damage. Persistent levels may, however, suggest ongoing liver issues that require further evaluation.

 References :

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