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chapter 29
Metabolism of Iron and Heme
per volume of plasma and minimizes diffusion of bilirubin
into extrahepatic tissues, thereby preventing bilirubin tox-
icity. Because of formation of this complex, bilirubin
does not normally appear in urine. Urinary bilirubin is
almost invariably conjugated bilirubin (see below) and
signifies the presence of a pathological process. An al-
bumin molecule binds two molecules of bilirubin at one
high-affinity site and at one to three secondary sites. Biliru-
bin conjugated with glucuronic acid also binds to albumin
but with much lower affinity. Another form of bilirubin
(probably conjugated), very tightly (probably covalently)
bound to albumin, has been described. The mechanism of
its formation is not known, although blockage of biliary
flow associated with an intact hepatic conjugating system
releases a chemically reactive form of bilirubin into the
circulation.
If the capacity of albumin to bind bilirubin is exceeded
because of increased amounts of unconjugated bilirubin
or decreased concentration of albumin, bilirubin readily
enters extrahepatic tissues. In neonates, this can cause
kernicterus,
a serious condition associated with perma-
nent neurological damage (see below). Bilirubin can be
displaced from binding to albumin by sulfonamides, sali-
cylates (notably aspirin), and Cholangiographie contrast
media. Use of these substances in jaundiced newborn
infants increases the risk of occurrence of kernicterus.
Medium-chain fatty acids increase and short-chain fatty
acids decrease bilirubin binding to albumin. Binding, at
least to the primary site, is independent of pH. Esti-
mation of reserve bilirubin binding capacity has been
used to evaluate the risk of bilirubin toxicity in icteric
patients.
Hepatic Uptake, Conjugation, and
Secretion of Bilirubin
Hepatocytes take up bilirubin from the sinusoidal plasma
and excrete it after conjugation with glucuronic acid across
the canalicular membrane into the bile. The entry and exit
steps and the transport of bilirubin within the cell are not
completely understood. The following is a plausible inter-
pretation of the available data.
Since binding of bilirubin to albumin is usually re-
versible, a small amount of free bilirubin is present in
plasma in equilibrium with albumin-bound bilirubin. It is
probably this free bilirubin that is taken up at a rate deter-
mined by its plasma concentration. As this free bilirubin
concentration decreases, more bilirubin is released from
albumin and becomes available for uptake. Alternatively,
the albumin-bilirubin complex may bind to specific hepa-
tocyte plasma membrane receptors, and thereby bilirubin
is released to enter the cell. Both models are consistent
with the finding that albumin does not accompany biliru-
bin into the hepatocyte.
The entry step seems to be carrier-mediated, is sat-
urable, is reversible, and is competitively inhibited by sul-
fobromophthalein, indocyanine green, cholecystographic
agents, and several drugs. Bile salts do not compete with
bilirubin for hepatic uptake.
After it enters hepatocytes, bilirubin is transported to
the smooth endoplasmic reticulum for glucuronidation
bound to a protein. Two cytosolic proteins, Z
protein
(fatty acid-binding protein) and
ligandin
(Y protein), bind
bilirubin and other organic anions. Ligandin, which con-
stitutes about 2-5% of the total soluble protein in rat
and human liver, has lower capacity but higher affin-
ity for bilirubin than Z protein. Ligandin (M.W. 47,000)
has two subunits, A and B, which appear to be iden-
tical except for a 30-amino-acid extension at the car-
boxyl terminus of the B subunit. Bilirubin is bound
entirely to the A subunit (two molecules per A sub-
unit). Ligandin also has glutathione S-transferase, glu-
tathione peroxidase, and ketosteroid isomerase activities,
which depend on both subunits. Glutathione S-transferases
catalyze detoxification reactions for a number of sub-
stances. Binding of bilirubin and other organic an-
ions to ligandin occurs at sites unrelated to its enzyme
activities.
Under normal conditions, ligandin is probably the prin-
cipal hepatic bilirubin-binding protein and may serve in-
tracellularly the same protective and transport functions
as albumin in plasma. It may also help limit reflux of
bilirubin into plasma, since its affinity for bilirubin is at
least five times greater than that of albumin. Z protein
(M.W. 11,000) becomes important at high plasma biliru-
bin concentrations. The concentration of ligandin in the
liver does not reach adult levels until several weeks after
birth, whereas neonatal and adult levels of Z protein are
the same. This lack of ligandin, together with low hep-
atic glucuronyltransferase activity, is the probable cause
of transient, “physiological,” nonhemolytic,
neonatal
jaundice.
Glucuronidation of bilirubin in the endoplasmic retic-
ulum by UDP-glucuronyltransferase produces an ester
between the
1
-hydroxyl group of glucuronic acid and
the carboxyl group of a propionic acid side chain of
bilirubin (Figure 29-13). In bile, about 85% of biliru-
bin is in the diglucuronide form and the remainder is in
the monoglucuronide form. Glucuronidation increases the
water solubility of several lipophilic substances. There ap-
pear to be many UDP-glucuronyltransferases in the endo-
plasmic reticulum, which differ in substrate specificity.
(Biosynthesis of UDP-glucuronic acid was described in
Chapter 15.)
