BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

CHAPTER 13. HEME METABOLISM AND IRON METABOLISM

IV. Diagnostic Significance of Determining Bilirubin Concentration in Human Biological Fluids

Currently, to determine the bilirubin content in Blood serum (plasma), the diazo-reaction-based method for serum bilirubin analysis proposed by Van den Bergh in 1916 is widely used.

Under normal conditions, the total bilirubin concentration in plasma ranges from 0.3 to 1 mg/dL (1.7 to 17 µmol/L), with 75% of the total bilirubin present in the unconjugated form (indirect bilirubin). Clinically, conjugated bilirubin is referred to as direct bilirubin because it is Water-soluble and rapidly reacts with the diazo reagent to form a pink-colored compound, which constitutes the direct Van den Bergh reaction. Unconjugated bilirubin is hydrophobic; therefore, it circulates in Blood Plasma as a complex with albumin and does not react with the diazo reagent until an organic solvent, such as ethanol (which precipitates albumin), is added. Unconjugated bilirubin that interacts with the azo dye only after protein precipitation is called indirect bilirubin.

When bilirubin levels exceed the normal range, the condition is termed Hyperbilirubinemia. Depending on which type of bilirubin is elevated in the plasma—unconjugated or conjugated—hyperbilirubinemia is classified as either unconjugated or conjugated.

In patients with hepatocellular pathology accompanied by a prolonged elevation in conjugated bilirubin concentration, a third form of plasma bilirubin is detected in the blood, in which bilirubin is covalently bound to albumin and thus cannot be removed by standard Methods. In some cases, up to 90% of the total blood bilirubin content may be present in this form.

A. Jaundice

Hyperbilirubinemia can be caused by either an increased production of bilirubin that exceeds the Liver's capacity to excrete it, or by liver damage resulting in impaired secretion of bilirubin into the Bile in normal amounts. Hyperbilirubinemia is also observed in cases of biliary tract obstruction.

In all such cases, the blood bilirubin level rises. Upon reaching a certain concentration, bilirubin diffuses into Tissues, imparting a yellow color to them. The yellowing of tissues due to bilirubin deposition is known as jaundice. Clinically, jaundice may not manifest until the plasma bilirubin concentration exceeds the upper limit of normal by more than 2.5 times, i.e., rising above 50 µmol/L.

1. Hemolytic (Prehepatic) Jaundice

It is known that the liver's capacity to form glucuronides and secrete them into bile is 3 to 4 times greater than their formation under physiological conditions. Hemolytic (prehepatic) jaundice is the result of intensive erythrocyte hemolysis. It is caused by excessive bilirubin production that exceeds the liver's excretory capacity. Hemolytic jaundice develops when the reserve capacity of the liver is exhausted. The primary cause of prehepatic jaundice is hereditary or acquired hemolytic anemias. In hemolytic anemias caused by Sepsis, radiation sickness, erythrocyte glucose-6-phosphate dehydrogenase deficiency, thalassemia, incompatible blood transfusions, or sulfonamide poisoning, The amount of Hemoglobin released from erythrocytes per day can reach up to 45 g (compared to a norm of 6.25 g), which significantly increases bilirubin production. Hyperbilirubinemia in patients with hemolytic jaundice is caused by a significant elevation (103 – 171 µmol/L) in the blood concentration of albumin-bound unconjugated bilirubin (indirect bilirubin). The hepatic formation and intestinal influx of large amounts of bilirubin glucuronides (direct bilirubin) lead to increased formation and excretion of urobilinogens in feces and urine, resulting in a more intense coloration (Fig. 13-16).

Class="center">Fig. 13-16. The bilirubin-urobilinogen cycle in hemolytic jaundice. 1 — Hb Catabolism proceeds at an accelerated rate; 2 — blood concentration of indirect bilirubin is approximately 10 times higher; 3 — albumin is released from the bilirubin-albumin complex; 4 — The rate of the glucuronidation reaction increases, but remains lower than the rate of bilirubin production; 5 — bilirubin secretion into bile is elevated; 6, 7, 10 — increased urobilinogen content in feces and urine imparts a more intense coloration to them; urobilinogen is reabsorbed from the intestine into the blood (8) and returns to the liver via the portal vein (9).

One of the main diagnostic features of hemolytic jaundice is an elevated blood level of unconjugated (indirect) bilirubin. This makes it easy to differentiate from obstructive (posthepatic) and hepatocellular (hepatic) jaundice.

Unconjugated bilirubin is toxic. Hydrophobic, lipophilic unconjugated bilirubin easily dissolves in Membrane Lipids and thereby penetrates Cell/35.html">Mitochondria, uncoupling Respiration and Oxidative Phosphorylation within them, disrupting Protein Synthesis AND the flux of potassium ions across The Cell and organelle membranes. This adversely affects the Central Nervous system, causing a series of characteristic neurological symptoms in patients.

Neonatal Jaundice

A frequent variant of hemolytic jaundice in newborns is "physiological jaundice," which is observed During the first days of a child's life. The cause of the elevated blood concentration of indirect bilirubin is accelerated hemolysis coupled with functional insufficiency of liver Proteins and Enzymes responsible for the uptake, conjugation, and secretion of direct bilirubin. In newborns, not only is The activity of UDP-glucuronosyltransferase reduced, but the Synthesis of the second substrate for the conjugation reaction, UDP-glucuronate, also appears to be insufficiently active.

It is known that UDP-glucuronosyltransferase is an inducible enzyme (see Chapter 12). Newborns with physiological jaundice are administered the drug phenobarbital, the inducing effect of which was described in Chapter 12.

One of the most concerning complications of "physiological jaundice" is bilirubin encephalopathy. When the concentration of unconjugated bilirubin exceeds 340 µmol/L, it crosses the blood-Brain barrier and causes brain damage.

2. Hepatocellular (Hepatic) Jaundice

Hepatocellular (hepatic) jaundice is caused by damage to hepatocytes and bile capillaries, such as in acute viral infections, chronic hepatitis, and toxic hepatitis.

The cause of the elevated blood bilirubin concentration is damage and Necrosis of a portion of the liver Cells. Bilirubin is retained in the liver, a process facilitated by a sharp decline in metabolic activity within the damaged hepatocytes. These cells lose their ability to normally perform various biochemical and physiological processes, specifically The transport of conjugated (direct) bilirubin from the cells into the bile against a concentration gradient. Hepatocellular jaundice is characterized by the fact that, instead of the normally predominant bilirubin diglucuronides, the damaged liver cells predominantly produce monoglucuronides (Fig. 13-17).

Fig. 13-17. Impairment of the bilirubin-urobilinogen cycle in hepatocellular jaundice. The rate of the bilirubin glucuronidation reaction in the liver is reduced (4), leading to an elevated blood concentration of indirect bilirubin; due to hepatic parenchymal damage, a portion of the bilirubin glucuronide formed in the liver enters the bloodstream (12) and is subsequently excreted from the body via the urine (10). The urine of patients contains urobilins and bilirubin glucuronides. The remaining numbers correspond to the stages of bilirubin METABOLISM shown in Fig. 13-16.

As a result of hepatic parenchyma destruction, the generated direct bilirubin partially enters the systemic Circulation, leading to jaundice. Bile excretion is also impaired. Less bilirubin reaches the intestine than under normal conditions.

In hepatocellular jaundice, the blood concentration of both total bilirubin and its two fractions—unconjugated (indirect) and conjugated (direct)—increases.

Since less bilirubin glucuronide enters the intestine, the amount of urobilinogen produced is also reduced. Consequently, the feces are hypocholic, i.e., less pigmented. Urine, conversely, has a more intense color due to the presence not only of urobilins, but also of conjugated bilirubin, which is highly water-soluble and excreted in the urine.

3. Obstructive (Posthepatic) Jaundice

Obstructive (posthepatic) jaundice develops when bile flow into the duodenum is disrupted. This can be caused by bile duct obstruction, for instance, in cholelithiasis, tumors of the Pancreas, Gallbladder, liver, or duodenum, Chronic Pancreatitis, or postoperative stricture of the common bile duct (Fig. 13-18).

Fig. 13-18. Impairment of the bilirubin-urobilinogen cycle in obstructive jaundice. Due to gallbladder obstruction, bilirubin glucuronide is not secreted into the bile (5); the absence of bilirubin in the intestine leads to pale, clay-colored stools (6); soluble bilirubin glucuronide is excreted by The Kidneys in the urine (10). There are no urobilins in the urine; bilirubin glucuronide produced in the liver enters the blood (12), resulting in an increased level of direct bilirubin. The remaining numbers correspond to the stages of bilirubin metabolism in Fig. 13-16.

In complete obstruction of the common bile duct, conjugated bilirubin as part of the bile does not enter the intestine, although hepatocytes continue to produce it. Since bilirubin does not reach the intestine, its catabolite products, urobilinogens, are absent in the urine and feces. The stool is clay-colored (acholic). Because normal pathways of bilirubin excretion are blocked, it leaks into the bloodstream, which is why patients exhibit elevated blood concentrations of conjugated bilirubin. Soluble bilirubin is excreted in the urine, giving it a saturated orange-brown color.

B. Cytology/practical/136.html">Differential Diagnosis OF Jaundice

When diagnosing jaundice, one must keep in mind that in practice, a single type of jaundice in its "pure" form is rarely observed. More commonly, a combination of types occurs. For example, in severe hemolytic jaundice accompanied by an increase in indirect bilirubin concentration, various Organs inevitably suffer, including the liver, which can introduce elements of parenchymatous jaundice—i.e., an elevation of direct bilirubin in the blood and urine. In turn, parenchymatous jaundice typically includes elements of obstruction. In posthepatic (obstructive) jaundice, such as that caused by Cancer of the HEAD of the pancreas, increased hemolysis is inevitable as a consequence of tumor intoxication and, consequently, results in elevated levels of both Direct and Indirect bilirubin in the blood.

Thus, hyperbilirubinemia can result from an excess of either bound or free bilirubin. Measuring their concentrations separately is essential for diagnosing jaundice. If the plasma bilirubin concentration is <100 µmol/L and other liver function tests yield normal results, it can be assumed that the elevation is due to indirect bilirubin. To confirm this, a urinalysis can be performed, since direct bilirubin is absent in the urine when plasma indirect bilirubin concentration is elevated.

In the differential diagnosis of jaundice, it is necessary to account for the urobilinogen content in the urine. Normally, about 4 mg of urobilinogens are excreted from the body in the urine per day. If an increased amount of urobilinogen is excreted in the urine, this indicates impaired liver function, such as in hepatic or hemolytic jaundice. The presence in the urine of not only urobilinogens but also direct bilirubin indicates liver damage and impaired bile flow into the intestine.

C. Hereditary Disorders of Bilirubin Metabolism

Several diseases are known in which jaundice is caused by hereditary disorders of bilirubin metabolism.

Approximately 5% of the population is diagnosed with hereditary jaundice caused by Genetic Defects in The Structure of proteins and enzymes responsible for the transport (uptake) of indirect bilirubin into The Liver and its conjugation with glucuronic acid. This pathology is inherited in an autosomal dominant manner. The concentration of indirect bilirubin is elevated in the blood of patients.

There are 2 known types of hereditary jaundice caused by impaired glucuronidation in the liver—The formation of direct bilirubin.

The first type is characterized by the complete absence of UDP-glucuronosyltransferase. The disease is inherited in an autosomal recessive manner. Administration of phenobarbital, an inducer of UDP-glucuronosyltransferase, does not lead to a decrease in bilirubin levels. Children die at an early age due to The Development of bilirubin encephalopathy.

The second type is characterized by reduced activity (deficiency) of UDP-glucuronosyltransferase, and hyperbilirubinemia occurs due to indirect bilirubin. The jaundice responds well to phenobarbital Treatment.

Impaired Active Transport of bilirubin glucuronides formed in liver cells into the bile is characteristic of jaundice inherited in an autosomal dominant manner. It manifests as hyperbilirubinemia due to direct bilirubin and bilirubinuria (direct bilirubin is detected in the urine).

Familial neonatal hyperbilirubinemia is associated with the presence of Competitive Inhibitors of bilirubin conjugation (estrogens, free Fatty acids) in breast milk. During breastfeeding, these bilirubin conjugation inhibitors are found in the infant's blood serum. This type of hyperbilirubinemia has been termed transient. Hyperbilirubinemia disappears when the infant is switched to formula feeding. Refractory hyperbilirubinemia leads to the development of bilirubin encephalopathy and early death.