Physiology and pathology

1-Bilirubin production.
2-Transport in blood.
3-Hepatocellular uptake.
4-Intracellular transport in hepatocytes.
5-Conjugation with glucuronic acid.
6-Secretion into bile ducts.
7- Intestinal metabolism.
8- Renal excretion of bilirubin
9- Renal excretion oh urobilinogen

1-BILIRUBIN PRODUCTION
Bilirubin is the terminal product of heme metabolism. Heme is present in hemoglobin and in other
oxidative compounds such as hepatic mitochondrial and microsomal cytochromes (P-450). Thus plasma
bilirubin is part erythropoietic and part non-erythropoietic. Approximately, 85 % erythropoietic and 15%
non-erythropoietic.
The erythropoietic fraction originates from two sources: the circulating normal aging red cells and the
immature defective red cells of the bone marrow. The daily production of bilirubin is 250 to 350 mg.
Shunt bilirubin is called that portion that does not originate from senescent circulating red cells but
originates from immature and defective red cells (7%) and from non- hemoglobin heme compounds,
particularly from hepatic cytochromes and from myoglobin. These two fractions were discovered by
labeling hemoglobin with a radioactive glycin, and observing that one fraction (78 %) of bilirubin is
excreted in the feces in 120 days and another fraction is excreted in 10 days or less. The first was called
late labeled bilirubin, the second was called early labeled bilirubin or shunt bilirubin. Shunt bilirubin
may be markedly elevated in certain pathologic states: sideroblastic anemia, megaloblastic anemia,
erythroleukemia, lead poisoning and a congenital disorder called "idiopathic dyserythropoietic
jaundice". The patients affected by this condition do not have hemolysis. They have hyperbilirubinemia
and jaundice. The hyprbilirubinemia is due to shunt bilirubin.
Bilirubin from erythropoietic heme is produced by monocytic macrophages, reticulo-endothelium, in every
organ but especially in the spleen, liver and bone marrow in order of importance.. The bilirubin from non-
erythropoietic hepatic heme is produced in the hepatocytes.
The tetrapyrrolic ring of heme is broken by an oxygenase at the alpha bridge, the bond between the
two carbons opposite to the gamma bridge which is between the two carbons carrying the two propionic
acids. The tetrapyrrolic molecule from a ring is transformed into a tetrapyrrolic chain without iron.
HEME + Heme oxygenase = OXY- HEME ( closed tetrapyrrolic ring with iron)
OXY- HEME + heme reductase = BILIVERDIN (open tetrapyrrolic ring without iron)
BILIVERDIN + biliverdin reductase = BILIRUBIN (unconjugated)
Pathology of bilirubin production
Hyprbilirubinimia with jaundice occurs in increased destruction of red blood cells namely:
hemolysis. It occurs in 1)congenital disorders of red cells (sickle cells, thalassemia, spherocytosis), 2)
immune hemolysis (erythroblastosis fetalis, 3) acquired diseases of red cells (dyserythropoiesis), etc.

In the adult, even a marked hemolysis does not produce significant increase of serum bilirubin if the
hepatic bilirubin clearance is normal. In the newborn, however, a marked hemolysis will be catastrophic.
At levels of 20mg/dl of serum bilirubin the infant will be deeply jaundiced and will develop kernicterus
(Nuclear jaundice: a grave form of yellow staining and degeneration of intracranial gray matter especially
of lenticular nucleus, ammon,s horn and subthalamic area).
Phototherapy is used for treatment of hyerbilirubinemia in neonates.
Bilirubin is a photoreceptor. The blue light transforms bilirubin into colorless products of oxidation which
are excreted in the urine.
Synthetic porphyrins containing tin or zinc instead of iron cause decrease of bilirubin formation by
competing for the heme oxygenase activity of macrophages. These compounds have been used in the
treatment of hyperbilirubinemia in animals and humans (e.g. Gilberts syndrome) with limited success.

2-BILIRUBIN TRANSPORT IN BLOOD

Bilirubin is toxic to tissues, therefore, it is transported in the blood bound to albumin. Only a minute
amount of free form is present in the blood.
Pathology of bilirubin transport in blood.
If the free fraction increases, bilirubin will invade and damage the tissues. It will cross the blood -brain
barrier and cause kernicterus in the neonate. Free plasma bilirubin can increase in the fallowing
pathologic conditions:
-1- overproduction.
-2- defective conjugation in the hepatocyte.
-3- presence of substances interfering with bilirubin-albumin binding: sulphonamides , long-chain fatty
acids from breast milk, salycilates, contrast media, etc. These agents compete for albumin binding sites.

3-HEPATOCELLULAR UPTAKE OF BILIRUBIN.

Bilirubin is taken up by hepatocytes at their sinusoidal surface. The albumin-bilirubin bond is broken.
Albumin remains in the plasma. The free molecule of bilirubin enters the hepatocyte.This uptake is very
rapid.

Pathology of bilirubin uptake by hepatocytes.

The impairment of uptake will result in unconjugated hyperbilirubinemia.
Occurrence:
1) Male fern oil jaundice. This oil was used to treat tape worm. (Aspidium).
3) Jegzichte sheep.

4-INTRACELLULAR TRANSPORT OF BILIRUBIN IN HEPATOCYTES.

In the hepatocye bilirubin is bound to cytoplasmic proteins: ligandins and Z protein. Ligandins are a
group of enzymes that represent 2% of cytosolic proteins. Z proteins bind fatty acids. The primary function
of these proteins is that of avoiding the reflux of free bilirubin into the blood. Apparently, the time lapse
between uptake of bilirubin and cojugation is relatively long.
Pathology of intracellular transport.
No hperbilirubinemia and jaundice is known due to deficiency of ligandins.

5-CONJUGATION WITH GLUCURONIC ACID

One way for cells to neutralize unwanted compounds is to conjugate them with a modified sugar, a
glycosyl. The sugars used for this reaction are xylose, glucose or glucuronic acid. Glucose is normally
present in the cell sap, xylose and glucuronic acid are formed from glucose by UDP-glucose
dehydrogenase. Xylosidation is predominant in plants, glucosidation in bacteria and glucuronidation
in mammals. Unconjugated bilirubinin is lipophilic. Its conjugation with glucuronic acid renders it
hydrophilic, thus, it can be eliminated in the bile. Many other agents are eliminated by conjugation with
glucuronic acid: steroids, thyroid hormone, catecholamines, estradiol, testosterone, bile acids, phenols,
morphine, which can be conjugated by other cells besides hepatocytes.
The glucuronidation of bile proceeds in two steps: first glucuronic aid (GA) is synthesized from cytosolic
glucose that is complexed with uridinediphosphate (UDP) ad forms udpglucuronic acid (UDPGA). From
this compound, the glucuronic acid is transferred to blirubin. The first reaction is catalyzed by a UP-
glucose dehydrogenate, the second reaction is catalyzed by bilirubin- DUGAN- transferees that is
synthesized by microsomes. Any deficiency of these two enzymes will result in defective conjugation and
elimination of bilirubin. On the other end, administration of microsomal enzyme inducers such as
phenobarbital, glutethimide and antipyrine favor bilirubin conjugation and elimination by increasing
blirubin transferase activity. Conjugation occurs in the endoplasmic reticulum and consists of forming an
ester between glucuronic acid and one or both propionic side-chains of bilirubin. The result will be
formation of bilirubin mono and di-glucuronides. In general, about 80% of the di and less than 20% of
the mono are formed. Human bile cotains also small amounts of unconjugated bilirubin. In summary:

GLUCOSE + UDP-Glucose-dehydrogenase = UDP-GLUCURONIC ACID (UDPGA)

UDPGA + BILIRUBIN + Glucuronyl transferase = BILIRUBIN MONO &
DI- GLUCURONIDES.

Pathology of bilirubin conjugation

GILBERTSYNDROME
Is due to a very mild deficiency of glucuronyl transferase.It is a very frequent disorder. It affects 5 to 7% of
the general population. More common in males. It consists of mild fluctuating jaundice due to non-
hemolytic unconjugated hyperbilirubinemia in the range of 5 to 7mg/dl or rarely higher. The liver is
morphologically normal. State of health and life-span are normal. Hemolysis, low caloric diet, nicotinic
acid will increase the jaundice. A lipid diet will decrease the jaundice. Phenobarbital and other enzyme
inducing agents are beneficial. Some individuals with this syndrome beside a defect of bilirubin
CRYGLER-NAJJAR SYNDROME, TYPE I
Is due to a severe deficiency of glucuronyl tranferase. Deep jaundice develops tat birth, High serom
unconjugated hyprbilirubinemia, >20 mg/dl., not responding to phenobarbital. Absent formation of
diglucuronides. Death usually in the first year or two with kernicterus. Phototherapy, plasmaferesis and
albumin exchange are beneficial. Liver transplantation may be life-saving. The liver is histologically
normal. A similar condition exists in Gann rat. Fortunately this syndrome is rare. Only 100 or more cases
have been described. It is apparently a hereditary autosomal recessive trait.
CRYGLER- NAJJAR SYNDROME TYPE II
Is due to a moderate deficiency of glucuronyl transferase. Milder unconjugated hyperbilirubinemia
responding to enzyme inducing agents: phenobarbital, gltethimide, phenazone, chlorpromazine. Both,
mono and di-glucuronides are formed. Patients develop normally but some may suffer bilirubin
encephalopathy, kernicterus. They will have unremitting jaundice for the whole life. It is a familial disorder.
The mode of genetic transmission is not clear.Thi defect of conjugation may have an associated defect of
bilirubin uptake by hepatocytes.
PYSILOLOGICAL JAUNDICE OF THE NEWBORN.
It is due to a very transient insufficiency of glucuronyl transferase. During the first few days of life there is
an overproduction of bilirubin and an underdeveloped mechanism of the liver to dispose of bilirubin.
Together with deficient conjugation, bilirubin production, blood transport, hepatic uptake and secretion are
all deficient. Sometimes extrahepatic factors exist to aggravate the situation: infections, drugs competing
for binding sites of bilirubin and above all, breast feeding. The long chains of fatty acids of the breast milk
interfere with bilirubin-albumin binding sites.

6- BILE SECRETION FROM HEPATOCYTES

The liver is an endocrine and an exocrine gland. It secretes synthesized products internally into the blood
through the sinusoidal surface such as blood proteins, coagulation factors etc. and secretes external into
the biliary tract and the intestine bile and many other substances, the terminal products of detoxifying
function. The mechanism of this external secretion is the least clear in the physiology of the liver. It seems
that many cellular organelles are involved in this process: vesicles, Golgi complexes, lysosomes, plasma
membranes, mitochondria, cytoskeleton, plasma membranes, canalicular villi. Are however clear the
consequences of the malfunction of this apparatus especially in the secretion bile which will result in
conjugated hyperbilirubinemia.

Pathology of bile secretion

DUBIN-JOHNSON SYNDROME.
The syndrome consists of chronic benign jaundice due to conjugated hyperbilirubinemia without pruritus
or elevation of serum alkaline phosphatase nor histological evidence of cholestasis. The hepatocytes
contain an abundance of coarse dark-brown pigment similar to melanin . The liver is black but normal.
Serum bilirubin ranges between 2 and 20mg/dl, 60% conjugated. Jaundice appears in the first 3 decades
of life and is intermittent. Sometimes the onset is acute, simulating a hepatitis. The prognosis is excellent.
The disease is inherited as autosomal recessive trait. The diagnosis is made by needle biopsy. Corriedale
sheep have similar black liver disease.
Click on the pictures to enlarge

Fig.11-1-1 Dubin-Johnson Syndrome.

The liver is brown-black because of the large amount of brownish coarse pigment stored in the
hepatocytes. Typically there is no intrahepatic cholestasis in this condition. The pigment predominates in
the centrolobular zone.
 Fig.11-1-2 Dubin-Johnson Syndrome.
There could be a moderate portal fibrosis in older patients. The pigment is stored in lysosomes like
lipofuscin. Bile canaliculi do not contain bile. According to studies conducted in Corriedale sheep, the
pigment contains a melanin-like component and and its formation is attributable to a defect of excretion
of epinephrine metabolites.
ROTOR SYNDROME.
This is a condition similar to Dubin-Johnson. There is intermittent jaundice with conjugated
hyperbilirubinemia, similar clinical course, excellent prognosis but no pigment in the liver tissue.
BENIGN RECURRENT INTRAHEPATIC CHOLESTASIS.
A syndrome characterized by recurrent attacks of rather severe jaundice. The attacks start usually before
puberty but they may start later. They are preceded by 2-4 weeks of pruritus malaise, anorexia followed
by
increasing jaundice without pain or fever and lasting an average of 2-3 months during each attack. It may
last from tow weeks to two years.
Nausea, vomiting , abdominal pain and skin rash occur in some cases. An affected individual may have
up to 30 attacks during his life. Biochemically these patients have elevated serum bilirubin, 10 to 20
mg/dl, mostly conjugated, elevated alkaline phosphatase and bile acids. Alpa-glutamyl transferase (GGT)
is elevated. Serum bile acids are elevated 2-30 folds. Transaminases are occasionally markedly elevated.
These abnormalities and the clinical symptoms disappear completely in disease-free intervals. In the
cholestatic phase there is acinar zone 3 cholestasis with bile plugs and mononuclear cell infiltration in the
cholestatic area. In some cases there may be mild hepatocytic damage and portal mononuclear infiltrate.
These changes do not produce any fibrosis or cirrhosis. Liver biopsies taken during clear intervals were
normal. The disorder is rather rare and appears to be familial with autosomal recessive character.
FAMILIAL RECURRENT INTRAHEPATIC CHOLESTASIS OF PREGNANCY.
This disorder is clinically and biochemically similar to benign intrahepatic cholestasis. It occurs in the third
trimester of pregnancy when the estrogen level is the highest and disappears postpartum. The affected
subjects appear to belong to families with benign intrahepatic cholestasis trait. Gonadal steroid appear to
ply a determining role in the cause of this syndrome. Histology of the liver shows centrolobular cholestasis
similar to benign intrahepatic cholestasis. It is most frequent in Scandinavia (1/100), Bolivia and Chile
(1/10).The disorder is safe for the mother but not for the fetus who will suffer premature births and
stillbirths due to placental infarcts. The mothers have higher incidence of gallstones. Sometimes the
disorder manifests itself only with presence of pruritus without jaundice. (Pruritus gravidarum). The
patients are not severely ill as in fatty liver of pregnancy, hepatitis, obstructive jaundice.
DRUG- INDUCED INTRAHEPATIC CHOLESTASIS.
Many drugs produce cholestasis. The first cases reported were due to chlopromazine and synthetic
steroids now out of market (Nilavar). Synthetic oral contraceptives are high in the list. They appeared to
act on sensitivity base and affect only sensitive individuals. Many appear to impair the secretory function
of the hepatocytes. And the list is increasing with the advent of new drugs. The liver in these cases may
show
marked and fatal necrosis.

POST-OPERATIVE INTRAHEPATIC CHOLESTASIS

It is attributable to the combined effect of bilirubin overload deriving from blood transfusions and to defect
of hepatocytic secretory function. Usually the jaundice appears in 1-2 postoperative days and disappears
in one or two weeks, Hyperbilirubinemia is predominantly conjugated with rater normal alkaline
phosphatase and transaminases.
BACTERIAL INFECTIONS
It is a form of intrahepatic cholestasis. The hyperbilirubinemia is conjugated in all cases. Elevation of
serum alkaline phosphatase in some cases. Hepatic histology without much hepatocellular damage.
Three types of morphological changes have been described:
1-canalicular cholestasis , the most common, mostly pericentral without hepatocellular damage.
2-ductular cholestasis, characterized by the presence of big bile thrombi in bile ductules and canals of
Hering at the periphery of portal fields. No bile plugs in interlobular bile ducts.
3-Toxic shock syndrome due to infection with staphylococcus aureus producing Toxic Shock Syndrome
Toxin-1 (TSST-1). This toxin was produced by this organism growing in polyacrylate tampons in
menstruating women. The liver suffers inflammation of intrahepatic bile ducts and canaliculi.
with rupture of bile ducts and microvesicular steatosis. There is inflammatory reaction in portal fields with
neutrophils, eosinophils lymphocytes and monocytes. There is centrolobular cholestasis in 50% of cases.

7- INTESTINAL METABOLISM OF BILIRUBIN

Bilirubin in the intestine is reduced to urobilins according to the following cascade:
BILIRUBIN GLUCURONIDE + bacterial or intestinal beta-glucuronidase = FREE BILIRUBIN
FREE BILIRUBIN + bacterial dehydrogenase = UROBILINOGEN (colorless)
UROBILINOGEN + dehydrogenase = UROBILIN (orange-yellow).
The bulk of bilirubin, urobilinogen and urobilin is excreted in the feces. Small amounts of bilirubin and
urobilinogen are reabsorbed by the intestine and return to the liver. The bilirubin is recunjugated in the
liver and re-excreted in the feces. The reabsorbed urobilinogen is excreted in the urine, about 4 mg/ day
and 0,1 to 1 mg in a random urine sample.

Pathology of biliary excretion into the intestine

COMPLETE BILIARY OBSTRUCTION.
The bile does not reach the intestine therefore the feces are acholic. There is conjugated
hyperbilirubinemia and bilirubinuria. Urobilinogen is not formed in the intestine and there is no
urobilinogen in the urine. because since the bile does not reach the intestine, urolinogen is not formed.
PARTIAL BILIARY OBSTRUCTION.
Less bile reaches the intestine. Urobilinogen is formed but in smaller amounts. There is less conjugated
hyperbilirubinemia, absent bilirubinuria and small amounts of urobilinogen in the urine.
HEMOLYSIS.
Hemolysis causes unconjugated hyperbilirubinemia. There is no bilirubinuria because unconjugated
bilirubin is not hydrophilic and cannot be excreted in the urine. There is increased urobilinogen in the
urine because more bilrubin reaches the intestine and more urobilinogen is formed an reabsorbed.

8- RENAL EXCRETION OF BILIRUBIN

Only conjugated bilirubin (the direct fraction) is excreted in the urine when its level in the plasma is
increased above normal. It not present in the urine of normal subjects and it is not eliminated in the urine
in cases of unconjugated (the indirect fraction) hyperbilirubinemia, such as in cases of hemolysis.
Only the small fraction of non-protein bound bilirubin in the plasma passes in the urine. Some drugs and
bile salts which compete for protein binding (salicylates, sofosoxazole) increase The theshold of
elimination depends on the degree of protein binding which varies and its quantity in the urine does not
have clinical relevance.
Conjugated bilirubin can be demonstrated in the proximal renal tubules.

9-RENAL EXCRETION OF UROBILINOGEN

Urobilinogen is formed by bacteria in the small intestine and in the colon.
It is then reabsorbed by the small intestine and the colon and re-xcreted by the by the liver into the
intestine almost entirely. A very small amount is therefore excreted into the urine: 0-4 mg/day. This
amount will increase when more urobilinogen is formed or when the liver is sick and unable to re-excrete
it. This amount will decrease when its formation in the intestine is decreased such as in the case of
complete bile duct obstruction when the bile cannot flow to the intestine where urobilinogen is formed by
the specific bacteria. The urobilinogen formed by bacteria in the small intestine is re-absorbed better than
that formed in the colon.