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 O
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
Audio: bilirubin and jaundice
POSSIBLE CAUSES OF HYPERBILIRUBINEMIA
liver prematurity (neonatal jaundice)
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)
Dubin-Johnson syndrome (hereditary, autosomal recessive, benign)
Rotor syndrome (hereditary, benign)
intrahepatic cholestasis, causing bile reabsorption in the blood. May occur because of cholelithiasis, hepatitis, cirrosis, biliary cancer, etc.
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.
An important laboratory feature of neonatal jaundices is that they usually are due to the increase of just one bilirubin component (either conjugated or unconjugated); by contrast adult jaundices are usually of the mixed variety (i.e. with increase of both bilirubin components)
, because the same disease that impairs conjugation (thus increasing unconjugated serum bilirubin) also causes intraepatic cholestasis and reabsorption of conjugated bilir ubin in the blood
Audio: jaundice due to increase of unconjugated bilirubin
Audio: mixed and conjugated bilirubin jaundices
Jaundice is most often an early sign of impaired liver function. Thus it may be associated or precede the impairment of other functions of the liver, and it is important to have a clear idea of the multiple functions played by this organ. This is especially relevant in laboratory medicine because several laboratory parameters may be altered in diseases of this organ.
conjugation and excretion of bilirubin
Production of urea
(approx. 90% of urea production via the urea cycle occurs in the liver; liver failure may be associated to hyperammonemia).
Biosynthesis of cholesterol
; production and elaboration of
. (in chronic liver failure reduction of HDL may be observed)
Production and excretion of bile salts
(cholesterol derivatives required for emulsifying dietary triglycerides; reduced or absent production of bile salts impairs the digestion and absorption of triglycerides and causes steatorrhea; reabsorption of bile salts causes itching).
Production, accumulation and utilization of glycogen
. This liver function is essential for the control of glycemia, and liver failure may cause hypoglycemic crises.
Production of serum albumin
and the majority of serum proteins (liver failure may be a cause of hypoalbuminemia with diffuse peripheral oedemas and ascites).
are also produced by the liver, and liver failure may cause coagulation disorders.
Conjugation and excretion of xenobiotics
(many substances we absorb with our diet cannot be utilized by our metabolism and must be excreted; the kidney excretes water soluble ones, the liver conjugates and excretes in the bile water insoluble ones. Special attention should be given to drugs: liver failure may cause increased persistence of drugs in our blood and may lead to overdosage).
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
: 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).
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.
is increased in many cases of liver disease (e.g. ethilism, drug toxicity, etc.).
Serum albumin concentration
is decreased in advanced states of liver failure (e.g. ethilism, drug toxicity, chronic hepatitis, cirrhosis, etc.).
may be increased because of reduced production of coagulation factors.
Serum ammonia concentration
may be increased in advanced states of liver failure (e.g. ethilism, drug toxicity, chronic hepatitis, cirrhosis, etc.), due to reduced production of urea. The urea concentration (BUN) is not a reliable indicator of liver function because it strongly depends on kidney excretion, that may be decreased; however, BUN may be decreased if liver failure is present and the kidney function is maintained. Ammonia is the principal, but not the only, substance that may cause hepatic encephalopathy, with mental confusion, and possibly coma.
: if an obvious anatomic lesion is suspected (e.g. biliary cancer, cirrosis) imaging methods (MRI, echography) and liver biopsy should be considered.
Laboratory findings for some important diseases of the liver
. Several different and unrelated viruses have orgna specificity for the liver. The infection causes death of liver cells, and inflammation of the organ with exudate and cholestasis. The reduced function of the liver causes increase of unconjugated bilirubin in the serum; the intrahepatic cholestasis causes reabsorption and increase of conjugated bilirubin in the serum. Thus viral hepatitis is associated to
jaundice and mixed hyperbilirubinemia
. often to very significant levels. Conjugated bilirubin appears in the urine, which becomes dark, whereas the feces are pale.
may be due to the reabsorption of bile salts. The death of liver cells is associated to
very significant increases of the liver enzymes
in the serum (sGOT, sGPT, γ-GT, etc.).
If viral hepatitis is suspected, the viral antigen, antiviral antibodies and viral genetic material 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 no 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.
Viral hepatitis D is caused by a defective RNA virus which co-infects hepatocytes with HBV. It is associated to high risk of cirrhosis and liver cancer.
Viral hepatitis E is particular in that it is usuallty transmitted via the oro-fecal route but may also be transmitted by contaminated blood transfusions. It is associated to high risk of cirrhosis and liver cancer.The laboratory may search for:
(usually proteins from the viral capsid);
, and the
viral genetic material
(than may be either RNA or DNA, see below). Given that the viruses capable of causing acute hepatitis are numerous, the laboratory tests should be requested to cover all possibilities, as listed in the table below:
Differential laboratory diagnosis of viral hepatitis
mode of transmission
genetic material to sequence
HV-A (an enteric picornavirus)
single stranded, linear RNA
HB-Ag (Australia antigen)
single stranded, linear RNA
single stranded, circular RNA (defective virus)
oral or blood transfusion
single stranded, linear RNA
P Valenzuela: Hepatitis A, B, C, D and E viruses: structure of their genomes and general properties
P. Farci et al.: Hepatitis D Virus and Hepatocellular Carcinoma
P. Kar and R. Karna: A Review of the Diagnosis and Management of Hepatitis E
Alcoholic liver disease
. Excess alcohol intake has profound damaging effects on the liver, because of several reasons: (i) direct toxicity of alcohol and its metabolytes, notably acetaldehyde; (ii) nutritional imbalances and vitamin deficiency often associated with ethylism. The first lesion observed is accumulation of fat in the parenchyma (steatosis). Chronic liver disease due to ethylism may progress to liver cirrhosis.
is a chronic condition resulting from virtually any disease that causes inflammation of the organ and death of its cells. Chronic viral hepatitis and alcohol abuse are two important possible causes. Essentially, cirrhosis consists in the replacement of dead parenchima with fibrous connective tissue. The liver is usually enlarged and has hard at palpation. Cirrhosis implies progressive liver failure. Moreover the formation of fibrous, scarry tissue causes compression of the intraepatic veins and
. Portal hypertension, associated to hypoalbuminemia is responsible for
, the extravasation of oedematous fluid in the peritoneal cavity.
The laboratory signs of liver cirrhosis are those of progressive liver failure, and more or less follow a predictable course: mixed bilirubin increase is usually the first sign, associated with jaundice. Bile salts are reabsorbed together with conjugated bilirubin and excite the nervous terminations in the skin causing itching. Reduced production of serum proteins (albumin, coagulation factors,etc.) comes next; hyperammonemia with hepatic encephalopathy usually appear only in advanced states of liver failure and have severe prognosis.
Neoplastic liver disease
. The liver may be affected by primitive or metatsatic cancers. Primary liver cancer is uncommon unless chronic viral hepatitis or liver cirrhosis are present. It may be scarcely symptomatic in its early stages, but as it invaeds the organ and impair its functions it produces a clinical an laboratory condition of progressive liver failure. Secondary (metastatic) cancer of the liver is usually due to primitive intestinal cancers (of the stomach, colon or small intestine) whose cells reach the liver via the mesenteric / portal venous system. It causes a progressive liver failure. Secondary (or tertiary) dissemination from the liver is usually to the lungs.
is essential to confirm the diagnosis of liver cancers.
Neonatal jaundice; congenital (inherited) diseases of the liver
. Neonatal jaundice may be due to
, in which case it is only transient and benign, or to
inherited genetic defects
of the liver. Some of these are benign, other have an infavorable prognosis. The most severe inherited disease of heme metabolism is
, due to reduced (type II) or absent (type I) activity of glucuronyl transferase. A severe jaundice due only to unconjugated bilirubin is present at birth or appears immediately thereafter. Accumulation of unconjugated bilirubin in the basal ganglia of the brain (kernicterus)causes mental retardation and utimately death. A milder and benign defect of the same enzyme is present in Gilbert's syndrome. Dubin-Johnson's and Rotor's syndromes are hereditary defects of the secretion of conjugated bilirubin in the bile; they cause neonatal jaundice due to conjugated bilirubin and have mild symptoms because conjugated bilirubin is efficiently excreted by the kidney (producing a dark urine).
1) 25 year old patient complaining of mild jaundice, tiredness, anorexia. Blood test reveals:
white cell count
13 x 10
9 mg/dL (3.2 mMol/L)
5 mg/dL (3 mg/dL conjugated)*
17 s (normal value: 11-14 s)*
alkaline phosphatase (ALP)
380 IU/L *
570 IU/L *
Analysis of the case
: acute liver failure; probably due to acute viral hepatitis. Notice that all liver functions are compromised to some extent (e.g. production of coagulation factors and urea). Very large increase of ALT with moderate increase of ALP. Carry out all virological tests: look for viral antigens, antiviral antibodies, viral genetic material (DNA or RNA).
2) 55 year old patient complaining of jaundice, tiredness, weight loss. Physical examination reveals ascitis. Blood test reveals:
12 g/dL *
3 x 10
mean corpuscular volume
108 fL (normal value 80-100 fL) *
white cell count
10 x 10
6 mg/dL (2.1 mMol/L)
2.5 mg/dL (1.5 mg/dL conjugated)*
total serum proteins
4 g/dL *
alkaline phosphatase (ALP)
350 IU/L *
570 IU/L *
γ-glutamyl transferase (γGT)
850 IU/L *
Analysis of the case
: chronic liver failure probably due to liver cirrhosis. Inquire about possible causes: chronic hepatitis, alcohol abuse, chronic professional intoxication. Liver biopsy indicated.
3) 40 year old patient complaining of fever, malaise, mild jaundice. Refers traveling in tropical countries. Blood test reveals:
10 g/dL *
2 mg/dL (mostly unconjugated)*
alkaline phosphatase (ALP)
γ-glutamyl transferase (γGT)
Analysis of the case
: anemia and increased unconjugated bilirubin, with normal liver enzymes suggests hemolytic disease. In a newborn you would also consider liver prematurity or a genetic defect of glucoronyl transferase, but in this case we have an adult patient. Hemolytic syndromes may be of allergic (e.g. drug allergies) or toxic origin. Since the patient refers traveling to tropical countries, suspect malaria or other parasitic disease causing hemolysis (e.g. Schistosoma). Malaria is diagnosed by microscopic examination of blood smear stained with Giemsa; for schistosomiasis look for parasite eggs in the feces (in the urine for Schistosoma haematobium).
4) 60 year old patient complaining of jaundice, tiredness, weight loss. Blood test reveals:
4.5 x 10
white cell count
6.5 x 10
5 mg/dL (4.5 mg/dL conjugated)*
total serum proteins
4 g/dL *
alkaline phosphatase (ALP)
1010 IU/L *
γ-glutamyl transferase (γGT)
500 IU/L *
Analysis of the case
: Obstructive jaundice, with conjugated hyperbilirubinemia. Some characteristic liver enzymes are normal (ALT), other are increased (ALP, γGT). The enzymatic pattern is suggestive of damage of the bile duct cells (colangiocytes). Suspect cholelythiasis, infection of the gallbladder, colangiocarcinoma, carcinoma of the pancreas obstructing the bile duct. NMR of the abdomen is indicated.
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
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
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).
is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.
Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.
In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R
is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.
What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.
How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.
When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.
Why do we have abnormal electrolytes in hematological neoplasia e.g.
Bellelli: I do not have a good explanation for this effect, which may have
multiple causes. However, you should consider two factors: (i) acute leukemias cause a massive
proliferation of leukocytes (or lymphocytes depending on the cell type affected) with a very
shortened lifetime; thus you observe an excess death rate of the neoplastic cells. The dying
cells release in the bloodstream their content, which has an electrolyte composition different
from that of plasma: the cell cytoplasm is rich in K and poor in Na, thus causing hyperkalemia.
(ii) the kidney may be affected by the accumulation of neoplastic white cells or their lytic products.
Gaussian curve: If it is bimodal is it more likely to be a "certain diagnosis" than if it is
unimodal or does it only show the distinguishment from health?
Bellelli an obviously bimodal Gaussian curve indicates that the disease is clearly
separated from health: usually it is a matter of how precise and clear-cut is the definition of the disease.
For example tuberculosis is the disease caused by M. tuberculosis, thus if the culture of the sputum is
positive for this bacterium you have a "certain" diagnosis (caution: the patient may suffer of two diseases,
e.g. tuberculosis and COPD diagnosis of the first does not exclude the second). However, in order to have
a "certain" diagnosis it is not enough that distribution of the parameter is bimodal, it is also required that the
patient's parameter is out of the range of the healthy condition: this is because a distribution can be
bimodal even though it is composed by two Gaussians that present a large overlap, and the patient's
parameter may fall in the overlapping region. Thus, in order to obtain a "certain" diagnosis you need to
consider not only the distribution of the parameter(s) but also the patient's values and the extent of the
Prof can you please elaborate a bit more on the interhuman variability and its difference
with the interpopulation variability please?
Bellelli: every individual is a unique combination of different alleles of the same genes;
this is the source of interindividual variability. Every population is a group of individuals who intermarry and
share the same gene pool (better: allele pool). Every allele in a population has its own frequency. Two
population may differ because of the diffferent frequencies of the same alleles; in some cases one
population may completely lack some alleles. The number and frequencies of alleles of each gene
determine the variance. If you take two populations and calculate the cumulative interindividual variance
of the population the number you obtain is the sum of two contributions: the interindividual variance within each population, plus the interpopulation variance
between the means of the allele frequencies. For example, there are human population in which the frequency of blood group B is close to 0% and other populati
ons in which it is 30% or more.
Prof can you please explain again the graph you have showed us in class about thromboplastin?
(Y axis=abs X axis= time)
Bellelli: the graph that I crudely sketched in class represented the signal
of the instrument (an absorbance spectrophotometer) used to record the turbidity of the
sample (turbidimetry). The plasma is more or less transparent, before coagulation starts.
When calcium and the tissue factor (or collagen) are added. thrombin is activated and begins
digesting fibrinogen to fibrin; then fibrin aggregates. The macroscopic fibrin aggregates cause
the sample to become turbid, which means it scatters the incident light. The instrument reads
this as a decrease of transmitted light (i.re an increase of the apparent absorbance) and the
time profile of the signal presents an initial lag phase, which is called the protrombin or
thromboplastin time depending on the component which was added to start coagulation
(tissue factor or collagen).
Prof can you please explain the concept you have described in class about
the simultaneous hypercoagulation and hemorrhagic syndrome? How can this occur?
Bellelli: The condition you describe is observed only in the Disseminated
Intravascular Coagulation syndrome. Suppose that the patient experiences an episode of
acute pancreatitis: tripsin and chymotripsin are reabsorbed in the blood and proteolytically
activate coagulation causing an extensive consumption of fibrinogen and other coagulation
factors. Tripsin and chymotripsin also damage the vessel walls and may cause internal
hemorrages, but at that point the consumption of fibrinogen may have been so massive that
not enough is left to form the clot where the vessel has been damaged, causing an internal
hemorrage. Pancreatitis is a very severe, potentially lethal condition, and DIC is only one of
the reasons of its severity.
You said that certain drugs (ethanol, cocaine, cannabis, opiates...) cause a
necessity of higher and higher dosage, for two reasons: the enzyme in the liver is inducible and
the receptors in the brain are expressed less and less. So, first, I am not sure I got it right, and
second I did not understand how expressing less receptors leads to a necessity of higher
Bellelli: You got it correctly, but the detailed mechanism of resistance may
vary among different substances, and not all drugs cause adaptation.
The reason why reducing the number of receptors may require an increased dosage of the drug
is as follows: suppose that a certain cell has 10,000 receptors for a drug. When bound to its
agonist/effector, each receptor produces an intracellular second messenger. Suppose that in
order for the cell to respond 1,000 receptors must be activated. The concentration of the
effector required is thus the concentration that produces 10% saturation. You can easily
calculate that this concentration is approximately 1/10 of the equilibrium dissociation constant
of the receptor-effector complex (its Kd), the law being
Fraction bound = [X] / ([X]+Kd)
where [X] is the concentration of the free drug.
After repeated administration, the subject becomes adapted to the drug, and his/her cells
express less receptors, say 5,000. The cell response will in any case require that 1,000
receptors are bound to the effector and activated, but this now represents 20% of the total
receptors, instead of 10%. The drug concentration required is now 1/4 of the Kd.
Continuing administration of the drug further reduces the cell receptors, but the absolute
number of activated receptors required to start the response is constant; thus the fewer
receptors on the cell membrane, the higher the fraction of activated receptors required.
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