CHAPTER 33: HEME DEGRADATION

Chapter 33: Heme Degradation

Hemoglobin (Hb) is catabolized in reticuloendothelial (RE) cells (mononuclear phagocytes) of the spleen and bone marrow, where the protein portion, globin, is cleaved off, hydrolyzed, and exported as amino acids for reutilization by the organism, while the heme portion is digested. Iron (Fe++) is exported (bound to transferrin in plasma) toward bone marrow (for new erythropoiesis), and other tissues (e.g., liver) for storage. Heme is oxidized to biliverdin (a green pigment found, for instance, in high concentration in the bile of snakes and birds), whereby the porphyrin ring is opened up and small amounts of carbon monoxide (CO) are formed. Carbon monoxide, which is highly toxic, normally competes with O2 for Hb binding, and it is slowly expired through the lungs. Biliverdin is next reduced to bilirubin by RE cells, which then diffuses into blood where it is bound to albumin and transported to the liver for ultimate removal from blood, conjugation with a glucuronide, and excretion into bile.

The average production of bilirubin from heme in adult mammals is about 3-5 mg/kg BW/day. Under normal circumstances, 75-85% of total bilirubin production occurs in the RE system from degradation of Hb heme derived from senescent erythrocytes. Although all mammalian cells contain heme in the form of hemoproteins, with the exception of the liver the tissue concentrations of these hemoproteins are so low, or their turnover rates so slow (e.g., myoglobin), that this contribution to bilirubin production is normally considered insignificant.

Studies using radiolabeled heme precursors in primates (glycine-14C and D-ALA-3 H) demonstrated the existence of two early-labeled plasma bilirubin peaks, and one late-labeled peak. The first peak occurring 1-6 hours post injection is derived from rapidly turning over substances such as hepatic hemoproteins (cytochromes, catalase, and tryptophan pyrrolase), free hepatic heme, and hepatic porphyrins (uroporphyrin I and III, coproporphyrin I and III, etc.) involved in heme biosynthesis. The second peak occurring at 1-3 days is bilirubin derived from erythrocytic bilirubin produced by ineffective or increased erythropoiesis, or intramedullary hemolysis. Some degree of ineffective erythropoiesis is normal accounting for this fraction. Peaks one and two are termed "shunt bilirubin", since they represent bilirubin which does not come from normally senescent erythrocytes. Disorders of peak two bilirubin are found with either thalassemia or pernicious anemia (e.g., folate or vitamin B12 deficiency). Peak three bilirubin usually originates from normal senescent hemolysis. In many hemolytic anemias, however, jaundice (i.e., bilirubin accumulation in tissues) is largely due to extramedullary hemolysis (peak three). In addition, extramedullary hemolysis causes bone marrow to produce erythrocytes at an increased rate, therefore an even greater amount of ineffective erythropoiesis or intramedullary hemolysis will be occurring, and peak two bilirubin will also be increased.

Hepatic Bilirubin Uptake, Conjugation, and Excretion: The liver takes up bilirubin from sinusoidal blood by way of a specific transport system, moves it to the smooth endoplasmic reticulum, conjugates it, and excretes it into bile (causing bile's orange-green color). Transfer across the hepatocyte plasma membrane is bidirectional, and some investigators believe that as much as 30% of bilirubin initially taken up by liver cells may normally reflux back into plasma. Although hereditary defects in the hepatic uptake mechanism for bilirubin occur, a common cause of unconjugated hyperbilirubinemia in animals (due to the reduced ability of the liver to remove bilirubin from the circulation), occurs with starvation. This effect is seen most often with equine species.

Studies have shown that bilirubin is conjugated in the liver by esterification with glucuronides in most mammalian species. In the horse, dog, cat, mouse, and rabbit, glucose and xylose are also conjugated to bilirubin. In pony bile, for example, bilirubin diglucuronide, bilirubin diglucoside, bilirubin glucuronide-glucoside, bilirubin monoglucuronide, bilirubin monoglucoside, and bilirubin monoxyloside conjugates are present. Hepatic bilirubin mono- and diglucuronide formation is catalyzed by UDPglucuronosyltransferase (UGT).

If hemolysis is excessive in dogs, free hemoglobin may sometimes be extracted by the kidney tubules, where small amounts can be converted to bilirubin, conjugated, and excreted into urine. Although this has not been reported in other animal species, the observation that the canine kidney can produce bilirubin from heme, conjugate it and excrete it into urine may explain the presence of bilirubinuria during periods when the plasma conjugated bilirubin concentration is minimal.

Active secretion of conjugated bilirubin into bile occurs against a large concentration gradient. As conjugated bilirubin reaches the terminal ileum and large intestine, some deconjugation by bacterial enzymes (glucuronidases) occurs, and the pigment is subsequently reduced by fecal flora to a group of colorless tetrapyrrolic compounds known as urobilinogens. Urobilinogen can be further reduced to stercobilinogen which, upon oxidation yields stercobilin (which imparts a dark color to feces. Only about 15% of urobilinogen formed in the intestine is normally reabsorbed, and the liver normally extracts urobilinogen efficiently from portal blood. Urobilinogen is excreted into bile unchanged, returning to the intestine (thereby completing an enterohepatic urobilinogen cycle). Since the liver extracts most of the urobilinogen from portal blood, normally there are mere traces which pass on into the peripheral circulation. Although urobilinogen is 80% bound to plasma protein, what little escapes the liver quickly reaches the kidneys, where it can undergo glomerular filtration. Urobilinogen excretion into urine correlates well with urinary pH, with higher values being recorded in alkaline than in acidic urine. The colorless urobilinogen is oxidized by light to the highly colored urobilin, which can impart color to urine.

Characterization of Plasma Bilirubin: Assay of plasma bilirubin can be diagnostically useful, depending upon the total amount present, its source, and its ratio of unconjugated: conjugated fractions. According to the most commonly used Van den Burgh reaction, bilirubin is coupled with diazotized sulfanilic acid to form azobilirubin. The color of this derivative is pH dependent, becoming blue under alkaline conditions. The color, thus, is proportional to the concentration. When the reaction occurs in the presence of methanol, both the unconjugated (UCB) and conjugated (CB) bilirubin pigments react, therefore the concentration is termed total bilirubin. When water is used in place of methanol, only the water-soluble conjugated pigment reacts, the concentration then is termed direct-reacting bilirubin. The unconjugated bilirubin thus can only be resolved as the difference between the two, and is referred to as the indirect fraction.

Under normal conditions, most of the bilirubin in plasma is in the unconjugated form (bound to albumin), for biliary excretion of conjugated bilirubin is the preferred route. Therefore, elevations in plasma conjugated bilirubin would be anticipated during pathophysiologic processes associated with biliary obstruction, or in other conditions where liver cells are unable to transport conjugated bilirubin across canalicular membranes into bile, therefore regurgitating it into plasma. It should be noted in this regard that canalicular excretion is usually rate-limiting for hepatic bilirubin transport. Therefore, in situations where bilirubin production is increased (e.g., hemolysis), the normal liver continues to conjugate it at a faster rate than it can be excreted into bile; therefore, the residual conjugated bilirubin may be refluxed back into the circulation, adding a conjugated component to an existing unconjugated hyperbilirubinemia.

Unconjugated bilirubin is relatively insoluble in water, urine and bile, but it is lipophilic and thus has a high affinity for brain tissue. Unconjugated bilirubin is also tightly bound to plasma albumin, and therefore is rarely found in urine. Conversely, CB is more hydrophilic, it is loosely bound to albumin in the circulation, and therefore is more likely to be filtered by the kidneys and excreted into urine. Conjugated bilirubin is also less likely to cross the blood-brain-barrier.

Bilirubin encephalopathy (kernicterus) is an acquired condition caused by extreme unconjugated hyperbilirubinemia which exceeds the capacity of plasma albumin to bind bilirubin, and thus keep it from crossing the blood-brain barrier. Hemolytic diseases or conditions where the liver is deficient in conjugating bilirubin are best associated with kernicterus. Since unconjugated bilirubin is lipid soluble, it penetrates neuronal and glial membranes easily and subsequently disrupts oxidative phosphorylation. Patients surviving kernicterus may exhibit a range of neurologic symptoms, including spasticity, muscular rigidity, ataxia and mental retardation.

SUMMARY

Chapter 33 discusses the process of heme degradation in the body. Hemoglobin is broken down in the spleen and bone marrow, with the globin portion being hydrolyzed and the heme portion being oxidized to biliverdin. Iron is exported for reuse, while biliverdin is reduced to bilirubin and transported to the liver for excretion. The liver takes up bilirubin, conjugates it with glucuronides, and excretes it into bile. Bilirubin is then converted to urobilinogens in the intestines, which are further reduced to stercobilin and excreted in feces. Some urobilinogen is reabsorbed and excreted in urine. Plasma bilirubin can be measured to diagnose certain conditions, with elevations in conjugated bilirubin indicating biliary obstruction or liver dysfunction. Unconjugated bilirubin is insoluble in water and has a high affinity for brain tissue, and extreme hyperbilirubinemia can lead to bilirubin encephalopathy (kernicterus) with neurological symptoms.

OUTLINE

  • Hemoglobin (Hb) is catabolized in reticuloendothelial (RE) cells of the spleen and bone marrow
  • Globin portion of Hb is cleaved off, hydrolyzed, and exported as amino acids for reutilization
  • Heme portion of Hb is oxidized to biliverdin, releasing small amounts of carbon monoxide (CO)
  • Biliverdin is reduced to bilirubin by RE cells, which then diffuses into blood and binds to albumin
  • Bilirubin is transported to the liver for conjugation with glucuronide and excretion into bile
  • Average production of bilirubin from heme in adult mammals is about 3-5 mg/kg BW/day
  • 75-85% of total bilirubin production occurs in the RE system from degradation of Hb heme derived from senescent erythrocytes
  • Studies using radiolabeled heme precursors demonstrate the existence of two early-labeled plasma bilirubin peaks and one late-labeled peak
  • Hepatic bilirubin uptake, conjugation, and excretion occurs in the liver
  • Bilirubin is conjugated with glucuronides in most mammalian species, but other conjugates may be present in certain animals
  • Excessive hemolysis in dogs can lead to bilirubin conversion, conjugation, and excretion in urine
  • Conjugated bilirubin is actively secreted into bile and undergoes deconjugation and reduction in the intestine, forming urobilinogens and stercobilin
  • Urobilinogen is excreted into bile and can be reabsorbed or excreted in urine
  • Plasma bilirubin can be assayed to determine total bilirubin, direct-reacting bilirubin, and indirect bilirubin fractions
  • Elevations in plasma conjugated bilirubin indicate biliary obstruction or impaired hepatic transport
  • Unconjugated bilirubin is relatively insoluble and has a high affinity for brain tissue, leading to bilirubin encephalopathy (kernicterus) in extreme cases of hyperbilirubinemia
  • Kernicterus is associated with hemolytic diseases or liver deficiency in bilirubin conjugation

QUESTIONS

Q: What happens to hemoglobin during heme degradation?

A: Hemoglobin is catabolized in reticuloendothelial (RE) cells, where the protein portion, globin, is cleaved off and exported as amino acids, while the heme portion is digested.

Q: What happens to iron during heme degradation?

A: Iron is exported and bound to transferrin in plasma, and is transported to the bone marrow for new erythropoiesis, and other tissues for storage.

Q: What is the fate of heme during heme degradation?

A: Heme is oxidized to biliverdin, whereby the porphyrin ring is opened up and small amounts of carbon monoxide are formed.

Q: How is bilirubin formed during heme degradation?

A: Biliverdin is reduced to bilirubin by reticuloendothelial (RE) cells, and then diffuses into blood where it is bound to albumin and transported to the liver for removal from blood, conjugation with a glucuronide, and excretion into bile.

Q: What are the three peaks of plasma bilirubin production?

A: The first peak is derived from hepatic hemoproteins and porphyrins involved in heme biosynthesis, the second peak is derived from erythrocytic bilirubin produced by ineffective or increased erythropoiesis, and the third peak is derived from normal senescent hemolysis.

Q: How is bilirubin conjugated in the liver?

A: Bilirubin is conjugated with glucuronides in the liver by UDPglucuronosyltransferase (UGT).

Q: What happens to bilirubin in the intestine?

A: Conjugated bilirubin is deconjugated by bacterial enzymes and reduced to urobilinogens, which can be further reduced to stercobilinogen and stercobilin, giving feces its dark color.

Q: How is bilirubin excreted in urine?

A: Urobilinogen, which is oxidized to urobilin, can be excreted in urine, with higher values in alkaline urine.

Q: What is the difference between unconjugated and conjugated bilirubin

A: Unconjugated bilirubin is bound to albumin and is relatively insoluble, while?

Mind Map: Chapter 33: Heme Degradation

Central Idea: Heme Degradation and Bilirubin Metabolism

Main Branches:
  1. Heme Degradation
    • Hemoglobin (Hb) catabolism in RE cells
    • Cleavage of globin and export as amino acids
    • Digestion of heme
    • Export of iron (Fe++) for erythropoiesis and storage
  2. Bilirubin Metabolism
    • Oxidation of heme to biliverdin
    • Reduction of biliverdin to bilirubin
    • Binding of bilirubin to albumin and transport to liver
    • Conjugation of bilirubin with glucuronide in liver
    • Excretion of conjugated bilirubin into bile
Sub-Branches:
  1. Heme Degradation
    • Iron export and reutilization
    • Formation of biliverdin and carbon monoxide
    • Reduction of biliverdin to bilirubin
    • Transport of bilirubin to liver
  2. Bilirubin Metabolism
    • Uptake of bilirubin by liver cells
    • Conjugation of bilirubin with glucuronides
    • Excretion of conjugated bilirubin into bile
    • Deconjugation and reduction of bilirubin in intestine
    • Formation of urobilinogens and stercobilinogen
    • Reabsorption and excretion of urobilinogen
    • Coloration of urine by urobilin
  3. Plasma Bilirubin
    • Assay of plasma bilirubin
    • Total bilirubin vs. direct-reacting bilirubin
    • Unconjugated vs. conjugated bilirubin
    • Elevations in plasma conjugated bilirubin
    • Unconjugated bilirubin and its affinity for brain tissue
  4. Bilirubin Encephalopathy (Kernicterus)
    • Extreme unconjugated hyperbilirubinemia
    • Capacity of plasma albumin to bind bilirubin
    • Hemolytic diseases and liver deficiency
    • Neurologic symptoms and long-term effects

Study Plan: Chapter 33: Heme Degradation

Day 1:

  • Read and understand the overall process of heme degradation.
  • Focus on the role of reticuloendothelial (RE) cells in the catabolism of hemoglobin.
  • Learn about the cleavage of globin and its export as amino acids.
  • Study the export of iron (Fe++) and its binding to transferrin for erythropoiesis and storage.
  • Understand the oxidation of heme to biliverdin and the formation of carbon monoxide (CO).
  • Take note of the reduction of biliverdin to bilirubin by RE cells.

Day 2:

  • Review the average production of bilirubin from heme in adult mammals.
  • Understand the contribution of senescent erythrocytes to bilirubin production.
  • Learn about the significance of hepatic hemoproteins and heme biosynthesis in bilirubin production.
  • Study the concept of "shunt bilirubin" and its association with thalassemia and pernicious anemia.
  • Explore the role of extramedullary hemolysis in bilirubin accumulation and increased ineffective erythropoiesis.

Day 3:

  • Focus on hepatic bilirubin uptake, conjugation, and excretion.
  • Understand the specific transport system for bilirubin uptake by liver cells.
  • Learn about the conjugation of bilirubin with glucuronides and other conjugates in different mammalian species.
  • Study the hepatic bilirubin mono- and diglucuronide formation catalyzed by UDPglucuronosyltransferase (UGT).
  • Take note of the extraction of free hemoglobin by kidney tubules and its conversion to bilirubin for excretion in urine.

Day 4:

  • Review the active secretion of conjugated bilirubin into bile against a concentration gradient.
  • Understand the deconjugation of bilirubin by bacterial enzymes in the terminal ileum and large intestine.
  • Study the reduction of bilirubin to urobilinogens by fecal flora and the formation of stercobilin.
  • Learn about the enterohepatic urobilinogen cycle and the reabsorption of urobilinogen by the liver.
  • Explore the excretion of urobilin