|The majority of bilirubin (80%) is produced from the degradation
of hemoglobin from erythrocytes undergoing normal (removal of aged
or effete cells) or abnormal destruction (i.e. intravascular or
extravascular hemolysis) within mononuclear phagocytes (principally
splenic, hepatic and bone marrow macrophages). A small percentage
(20%) is derived from the catabolism of various hepatic hemoproteins
(myoglobin, cytochrome P450) as well as from the overproduction
of heme from ineffective erythropoiesis in the bone marrow. Within
macrophages, a free heme group (iron + porphyrin ring) is oxidized
by microsomal heme oxygenase into biliverdin and the iron is released
(the iron is then stored as ferritin or released into plasma, where
it is bound to the transport protein, transferrin). Biliverdin reductase
then reduces the green water-soluble biliverdin into unconjugated
bilirubin. Heme oxygenase is also located in renal and hepatic parenchyma,
enabling these tissues to take up heme and convert it to bilirubin.
Birds lack biliverdin reductase, thus they excrete heme breakdown
products as biliverdin rather than bilirubin.
Unconjugated or free bilirubin is then released into plasma where
it binds to albumin. Uptake of unconjugated bilirubin occurs in
the liver and is carrier-mediated. The carrier-mediated uptake is
shared with unconjugated bile acids and dyes such as BSP. Once within
the hepatocyte, unconjugated bilirubin is transported with ligand
(Y protein) or other proteins (e.g. Z protein) and the majority
is conjugated to glucuronic acid by UDP-glucuronyl transferase.
The remainder is conjugated to a variety of neutral glycosides (glucose,
xylose). In the horse, the majority of bilirubin is conjugated to
glucose. Bilirubin must be conjugated before it can be excreted
into bile (conjugation makes bilirubin water soluble). Excretion
into biliary canaliculi is the rate-limiting step of the entire
bilirubin metabolism pathway and occurs via specific transporters,
which are energy (ATP) dependent. Transfer into the canaliculi is
facilitated by bile salt-dependent and bile salt-independent biliary
flow (the latter of which is generated by a basolateral (sinusoidal
or blood-side) Na/K ATPase pump).
Bilirubin is secreted, along with bile salts (and sodium) into the
intestine, where the bile salts form micelles facilitating absorption
of fat. In the intestine, bacterial enzymes deconjugate bilirubin
and degrade it to urobilinogen. Urobilinogen is reaborbed (about
10%) or broken down (90%) into urobilin and stercobilin (both of
which are excreted in the feces). Of the resorbed urobilinogen,
most is taken up by the liver (enterohepatic circulation, i.e. the
urobilinogen is absorbed into the portal vein, taken up by the liver
and re-excreted into bile - this re-excretion into bile is not depicted
in the picture below), whilst the rest bypasses the liver and is
excreted into the urine.
Conjugated bilirubin is not normally found in the urine of domestic
animals, although small to 1+ amounts of conjugated bilirubin may be
seen in concentrated urine from dogs (particularly males), due to the
low canine renal threshold for bilirubin. In all species (but dogs,
in particular), bilirubinuria may precede an increase in serum bilirubin
in cholestatic disorders. Remember, only conjugated bilirubin can be
excreted in urine as it is water soluble.
Circulating bilirubin exists in two main forms as determined by the
Van den Bergh reaction, which differentiates bilirubin into conjugated
(direct) and unconjugated (indirect) forms. There is a third form of
bilirubin, called delta bilirubin (or biliprotein), which is conjugated
bilirubin bound to proteins. Delta bilirubin increases in serum when
hepatic excretion of conjugated bilirubin is impaired (cholestasis)
and the liver retains intact conjugation mechanisms. It has a long half-life
and is not excreted in the urine (as it is protein bound). Delta bilirubin
may be responsible for a persistent bilirubinemia without bilirubinuria
seen in some animals with cholestasis. It does react with the diazo
dyes, similar to direct or conjugated bilirubin.
Although we usually think of bilirubin in terms of its diagnostic utility
(i.e. to support a diagnosis of hemolytic anemia or hepatobiliary disease),
bilirubin is actually an anti-oxidant, which is its main physiological
Causes of increased total bilirubin
Clinical icterus is observed when total bilirubin values exceed 1.5
- Artifact: With some analyzers and reagents, hemolysis and
lipemia (even mild) will cause artifactually high bilirubin values.
The procedures used by the chemistry analyzer at Cornell University
are minimally impacted by hemolysis and lipemia. The analyzer also
gives an estimation of the amount of bilirubin in the sample (free
from hemolysis and lipemia interference) as an icteric index. This
index correlates closely (often to the nearest 1-2 mg/dL) to the total
bilirubin values and can be used to confirm true increases in total
bilirubin. Remember that bilirubin is unstable in light and samples
stored for several days, in the presence of light, may have falsely
reduced bilirubin values.
- Hemolysis: Destruction of red cells, whether through extravascular
or intravascular hemolysis will increase the production of unconjugated
bilirubin because of enhanced hemoglobin metabolism by mononuclear
phagocytes. A healthy liver can handle substantial hemolysis without
allowing an increase in total bilirubin, therefore, hyperbilirubinemia
is usually due to severe, rapid hemolysis. In these cases, the bilirubin
is mostly unconjugated bilirubin and the total bilirubin is usually
< 2 -3 mg/dL. In some cases of hemolytic anemia (perhaps with longer
standing hemolysis), secondary hepatic hypoxia/dysfunction will interfere
with bilirubin excretion into the bile ducts (remember this is the
rate-limiting step of bilirubin metabolism and is ATP-dependent),
resulting in cholestasis. This occurs predominantly in small animal
patients with immune-mediated hemolytic anemia and some foals with
neonatal isoerythrolysis (NI, up to 40-60% of total bilirubin may
be conjugated in foals with NI). Therefore, animals with hemolytic
anemia and bilirubinemia > 2-3 mg/dL often have a cholestatic component
to the icterus, i.e. there are substantial increases in both conjugated
(which can dominate) and unconjugated bilirubin. This reflects both
cholestasis and increased unconjugated bilirubin production from heme
breakdown. Note that icterus in cattle is mostly due to hemolysis
(and is usually unconjugated) and rarely due to liver disease or post-hepatic
bile duct obstruction. You may find schematic illustrations of bilirubin
metabolism in intravascular and
extravascular hemolysis helpful.
- Liver disease: Hepatic disease may cause increases in both
unconjugated and conjugated bilirubin. Increases in bilirubin in dogs
often occurs after elevation of cholestatic enzymes (GGT, ALP) due
to the low renal threshold for bilirubin. In acutely developing icterus,
ALP and GGT levels may be normal because they require time for induction.
In large animals with liver disease, increases in bilirubin are usually
due to unconjugated bilirubin. Only cattle with very severe liver
disease will have increased bilirubin (usually unconjugated).
- Cholestasis: This is defined as decreased bile flow and can
be due to physical obstruction of bile flow or functional defects
in the transporters that deliver bile salts or bilirubin into the
biliary system. Obstructed bile flow can be intrahepatic (e.g. hepatocyte swelling due to hepatic lipidosis in cats) or extrahepatic
(e.g. bile duct obstruction from pancreatic neoplasia, cholelithiasis,
Fasciola hepatica in cattle). Changes in the character of bile (e.g. thick sludged bile in cats with dehydration) can also result in decreased bile flow. Functional defects in bile salt
or bilirubin transporters occur secondary to inflammatory cytokines
(e.g. endotoxemia) and drugs. Defects in these transporters also occur
with physical obstructions to bile flow. Cholestasis will result in
bilirubinemia, with increased direct bilirubin, and bilirubinuria (excess conjugated bilirubin
in blood is excreted into the urine, because it is water soluble). Indirect bilirubin is also usually increased in cholestasis due to the toxic effect of accumulated bile salts on hepatocytes or cholestasis-induced decreases in the hepatic transporters which take up unconjugated bilirubin from blood). Cholestasis frequently (but not always) results in a higher conjugated than unconjugated bilirubin, particularly when there is a physical obstruction to bile flow (e.g. cholelithiasis, biliary mucocele). The exception is the horse, where unconjugated bilirubinemia still dominates in cholestatic conditions, i.e. direct bilirubin rarely exceeds 50% of total bilirubin in horses with cholestasis.
1) Fasting: In horses, fasting will produce
a hyperbilirubinemia due to unconjugated bilirubin. The precise mechanism
for this is
unknown but it is thought to be due to either decreased uptake of
bilirubin (due to competition for uptake with free fatty acids) or
impaired conjugation of bilirubin (due to low glucose within hepatocytes
- remember, in horses most of unconjugated bilirubin is conjugated
to glucose). Increases in bilirubin are noticeable within 12 hours
of fasting and may reach levels as high as 10-12 mg/dL within 2-4
days of anorexia, with clinical icterus. This occurs in the absence
of significant liver disease. Note that horses have higher reference
intervals for bilirubin than donkeys. Mild increases in total bilirubin
(mostly unconjugated) are seen in cows that are sick and/or anorectic.
The total bilirubin usually does not exceed 4 mg/dL in these cows.
The precise mechanism is unknown.
Figure reproduced with permission
from: Hepatic function by Tennant B in "Clinical
Biochemistry of Domestic Animals", 5th edition,
Kaneko et al, eds, pg 344, 1997; Copyright Elsevier.
2) Neonatal: Young animals, especially
foals, often have jaundice (due primarily to unconjugated bilirubin).
This is due to multifactorial causes, including hemolysis of fetal
red blood cells, decreased liver uptake of bilirubin, immaturity of
hepatic conjugation mechanisms and poor albumin binding.
- Inherited: Inherited defects in hepatic uptake, conjugation
and excretion of bilirubin occur in monkeys, sheep, and rats. Southdown
sheep have an inherited defect in bilirubin uptake, resulting in a
fasting hyperbilirubinemia due to unconjugated bilirubin. Corriedale
sheep have Dubin-Johnson syndrome, a fasting hyperbilirubinemia due
to conjugated bilirubin from defective excretion of conjugated bilirubin.
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