MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
Jaundice and the Catabolism of the Heme

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      Some 400 mg of heme are degraded and excreted daily by healthy adults as bilirubin derivatives. Over 70% of the heme derives from the turnover of hemoglobin (i.e. from aged erythrocytes or erythrocyte precursors), the rest from other hemoproteins present in all the body cells.
 
      Free heme is toxic because of its ability to activate O2 to superoxide and peroxide anions. It is transported in the blood plasma bound to hemopexin or albumin and removed by the cells of the reticuloendothelial system, in the liver or elsewhere. The same cells are able to endocytose and degrade aging red cells. Reticuloendothelial cells degrade the heme with the enzyme heme oxygenase, that opens the tetrapirrole ring and releases iron (which is recycled). A product of this reaction is the toxic gas CO; this is the only reaction known to produce CO in our body. The heme derivative thus obtained is called biliverdin and is further processed to bilirubin by the enzyme biliverdin reductase.
 
      Bilirubin is water insoluble and when it is released by the reticoloendothelial cells, is taken up by albumin and carried to the liver (many reticuloendothelial cells do actually reside in the liver). Glucuronyl transferase, an enzyme of the hepatocyte, conjugates one or two molecules of glucuronic acid to bilirubin. The reaction products, bilirubin-glucuronide or bilirubin-diglucuronide, are soluble in water and are excreted via the bile in the faeces.



Audio: the heme degradation pathway
 
      During the intestinal transit, bilirubin is partially metabolyzed by the bacterial flora and converted to other pigments called stercobilinogens and urobilinogens, thay may be partly readsorbed by the gut and excreted in the urine. Bilirubin and the bilinogens are of a brownish colour, and are responsible for the colour of the faeces. If readsorbed and excreted in the urine, they cause the urine to become dark brown.
 
      Bilirubin is normally present in the human blood, in two forms: (i) unconjugated bilirubin is bound to albumin and is present during its transfer from reticuloendothelial cells to the liver. This form of bilirubin is also called indirect (because it requires ti be activated in order to react with the standard diazo reagents) or pre-hepatic. (ii) Conjugated bilirubin is present in the blood because of reabsorption from the smallest biliary vessels. This form is also called direct or post-hepatic.
 
      JAUNDICE (or ICTERUS)
      In the healthy adul the total serum concentration of bilirubin is <1 mg/dL, mostly due to unconjugated bilirubin in transit from the spleen or other reticuloendothelial system sites to the liver. Conjugated bilirubin is <0.3 mg/dL. In the newborn, especially the premature newborn, the serum bilirubin may be significantly higher, up to 5-7 mg/dL.
      Bilirubin concentration may be increased because of several reasons; above 2 mg/dL it causes a yellow staining of the eye sclera, the skin and all visible tissues rich in elastin, to which it binds (e.g. the frenulus of the tongue). Ths condition is called jaundice.
Audio: bilirubin and jaundice


POSSIBLE CAUSES OF HYPERBILIRUBINEMIA
Bilirubin type Possible causes
unconjugated liver prematurity (neonatal jaundice)
hemolysis
Gilbert's syndrome (congenital, benign, due to mildly reduced activity of glycuronyl transferase)
Crigler-Najjar syndrome type I (hereditary, autosomal recessive, lethal, due to complete absence of glycuronyl transferase)
Crigler-Najjar syndrome type II (hereditary, autosomal dominant, due to severely reduced activity of glycuronyl transferase)
Primary shunt hyperbilirubinemia (familial, benign)
conjugated Dubin-Johnson syndrome (hereditary, autosomal recessive, benign)
Rotor syndrome (hereditary, benign)
both intrahepatic cholestasis, causing bile reabsorption in the blood. May occur because of cholelithiasis, hepatitis, cirrosis, biliary cancer, etc.

      Neonatal jaundice is due to the incomplete maturity of the liver at birth and is specially common in premature newborns. It is usually moderate and is due to unconjugated bilrubin, that may attain levels of 2 mg/dL or more. It is treated by UV light (the eyes must be protected), that photochemically degrades bilirubin accumulated in the subcutaneous tissues and favors its renal excretion. In severe cases of congenital jaundice (e.g. Crigler Najjar syndrome) unconjugated bilirubin may accumulate in the brain, mainly in the basal ganglia (kernicterus). This condition causes permanent brain damage and mental retardation, and should be avoided at all costs, whenever possible.

Audio: jaundice due to increase of unconjugated bilirubin

Audio: mixed and conjugated bilirubin jaundices

      LABORATORY EVALUATION OF LIVER FUNCTION
      Jaundice is a common sign that may be caused by a host of diseases (see Table). It is a generic clinical sign, that may accompany a host of hepatic conditions (and some non-hepatic ones); the diagnosing the underlying disease is usually based on a series of clinical tests ordered sequentially to exclude or ascertain the different possibilities. Jaundice occurring in an adult patient is first evaluated by ascertaining two points: which type of bilirubin is increased, and whether the condition is acute or chronic. In the presence of jaundice (even mild jaundice), laboratory signs of liver damage should be systematically looked for.

      Serum bilirubin concentration: in the healthy adult does not usually exceed 1 mg/dL, of which one third is due to conjugated bilirubin and the remaining two thirds to unconjugated bilirubin. Jaundice becomes visible (initially in the sclerae) when bilirubin concentration in the serum exceeds 2 mg/dL. Unconjugated bilirubin is mostly bound to albumin and must be solubilized with organic solvents for the test. The standard test for bilirubin is based upon the reaction of this compound with p-diazobenzenesulfonic acid (van den Bergh reaction); the reaction product is red-colored and can be measured by absorption spectrophotometry. This reaction transfers the diazo-dye to bilirubin:

      Bilirubin conjugated with glicuronid acid reacts with diazo-compounds in water and is thus called "direct" bilirubin; the reaction of unconjugated bilirubin requires that ethanol is added as a co-solvent, thus unconjugated bilirubin is also called "indirect" bilirubin.

      Conjugated bilirubin is water-soluble and is filtered by the kidney; it causes the urine to be yellowish. In case of obstructive jaundice and cholestasis, conjugated bilirubin is reabsorbed in the serum and causes the urine to become dark, whereas the faeces become paler.

Audio: Laboratory methods for the determination of bilirubin

      Aminotransferases: aspartate aminotransferase (AST, GOT) and alanine aminotransferase (ALT, GPT) are intracellular enzymes present in several tissues, but most notably in the hepatocytes. Diseases that cause necrosis of hepatocytes (e.g. viral hepatitis, cirrosis, liver cancer) they are released in the serum. Their serum concentration in the healthy adult is non-zero may because of the physiological turnover of liver cells but should not exceed 40 u/mL; in viral hepatitis may exceed 400 u/mL. Jaundice is often associated to increase of aminotransferases, but the reverse is not true, because of two reasons: (i) liver damage may be sufficient to yield a significant increase in the enzymes, but insufficient to cause a significant increase of bilirubin concentration; and (ii) damage of tissues other than the liver may cause increase of the aminotransferases (e.g. the heart and the central nervous system).
      If viral hepatitis is suspected, the viral antigen, antiviral antibodies and viral DNA may be looked for. Viral hepatitis A is transmitted by contaminated food and water, has short incubation time and is relatively benign; the characteristic viral antigen HAAg is found in the serum, stool and liver tissue. The disease runs an acute course and there is non chronic carrier state; occasionally it may cause epidemic outbreaks in regions with poor sanitation.
      Viral hepatitis B is transmitted by blood transfusions and contact with infected blood, runs an acute course but may chronicize and is associated to chronic hepatitis and liver cancer. Diagnosis is established by the finding of the specific viral antigens HBsAg (Australia antigen), HBcAg, HBeAg and the delta antigen.
      Viral hepatitis C resembles B under many aspects but is due to a different virus. Diagnosis relies on the demonstration of specific antibodies by immunassay, or on the sequencing of viral RNA from the serum samples.

      Alkaline phosphatase is characteristic of the biliary duct cells and is increased in many cases of obstructive jaundice and liver cancer. Other organs that may release this enzyme in the serum are pancreas, lung and bone.

      gamma-Glutamyl transpeptidase is increased in many cases of liver disease (e.g. ethilism, drug toxicity, etc.).

      Other tests: if an obvious anatomic lesion is suspected (e.g. biliary cancer, cirrosis) imaging methods (MRI, echography) and liver biopsy should be considered.

Questions and exercises:
1) Bilirubin concentration in the serum of healthy adults should be:
less than 1 mg/dL, mostly unconjugated
less than 1 mg/dL, mostly conjugated
less than 2 mg/dL, mostly unconjugated
less than 2 mg/dL, mostly conjugated

2) The most common type of jaundice is mixed (i.e. due to both conjugated and unconjugated bilirubin), because lesions which damage the epatocyte may often damage also, or compress, the intraepatic bile ducts. Epatocyte damage causes increase of unconjugated bilirubin; compression of the intraepatic bile ducts causes reabsorption of conjugated bilirubin in the blood. An example of this condition is observed in
Crigler-Najjar type I syndrome
Viral hepatitis
Rotor syndrome
Hemolytic crisis

3) The most relevant enzymes released in the serum as a consequece of diseases causing liver cells death are:
Aminotransferases (ALT, AST), alkaline phosphatase, gamma-glutamyl traspeptidase (gammaGT)
Aminotransferases (ALT, AST), acidic phosphatase, gamma-glutamyl traspeptidase (gammaGT)
Aminotransferases (ALT, AST), alkaline phosphatase, cytochrome-c
Aminotransferases (ALT, AST), lactate dehydrogenase (LDH), creatine kinase (CPK)

4) Jaundice and liver enzymes lead you to suspect that your patient is affected by acute viral hepatitis. Which Laboratory exam(s) would you carry out to confirm your diagnosis:
Antibodies against the different viruses that cause hepatitis
Search of the viral RNA by reverse transcriptase and PCR followed by DNA sequencing
Assay of viral antigen(s)
All the preceding tests

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).



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