Introduction

Heme is a porphyrin ring complexed with ferrous iron and protoporphyrin IX. Heme is an
essential prosthetic group in proteins that is necessary as a subcellular compartment to
perform diverse biological functions like hemoglobin and myoglobin.[1] Other enzymes
which use heme as a prosthetic group includes cytochromes of the electron transport chain,
catalase, and nitric oxide synthase. The major tissues for heme synthesis are bone marrow by
erythrocytes and the liver by hepatocytes.

Fundamentals
Heme synthesis occurs in the cytosol and mitochondria; heme acquisition also occurs through
intestinal absorption and intercellular transport.[2] Heme is a component of different
biological structures mainly, hemoglobin, others include myoglobin, cytochromes, catalases,
heme peroxidase, and endothelial nitric oxide synthase. There are different forms
of biological heme. The most common type is heme b, found in hemoglobin leads to a
derivative of other heme groups. Heme a exists in cytochrome a and heme c in cytochrome c;
they are both involved in the process of oxidative phosphorylation.
5'-Aminolevulinic acid synthase (ALA-S) is the regulated enzyme for heme synthesis in the
liver and erythroid cells. There are two forms of ALA Synthase, ALAS1, and ALAS2. All
cells express ALAS1 while only the liver and bone marrow expresses ALAS2. The gene for
ALAS2 is on the X-chromosome.

Cellular
Porphyrin synthesis is the process that produces heme. Heme synthesis occurs partly in the
mitochondria and partly in the cytosol. The biosynthesis involves an eight-step enzymatic
pathway. Heme biosynthesis starts in mitochondria with the condensation of succinyl Co-A
from the citric acid cycle and an amino acid glycine. They combine to produce a key heme
intermediate, 5'-aminolevulinic acid (ALA) in mitochondria catalyzed by the pyridoxal
phosphate-requiring (vitamin B6) enzyme, aminolevulinic acid synthase (ALAS).[3] This
reaction is the rate-limiting step in the pathway.[4]
The ALA molecule formed exit the mitochondria into the cytosol where two molecules of
ALA condense to produce the pyrrole ring compound porphobilinogen (PBG) catalyzed by a
zinc-requiring enzyme, ALA dehydratase enzyme (also called porphobilinogen synthase).
The next step of the pathway involves condensation of four molecules of porphobilinogen,
aligned to form the linear hydroxymethylbilane (HMB), catalyzed by porphobilinogen
deaminase (PBG deaminase) also known as hydroxymethylbilane synthase. 
Closure of the linear HMB forms an asymmetric pyrrole ring D called uroporphyrinogen III,
catalyzed by uroporphyrinogen-III synthase. This step is vital as an incorrect porphyrin ring
formation leads to protoporphyria. The correct porphyrin ring III forms, and then the side
chains of uroporphyrinogen III are modified, catalyzed by uroporphyrinogen decarboxylase
to produce coproporphyrinogen III.
Following its synthesis, coproporphyrinogen III gets transported into mitochondria. The
coproporphyrinogen III then gets decarboxylated by coproporphyrinogen oxidase enzyme to
form the colorless product protoporphyrinogen IX. 
Finally, protoporphyrinogen IX is converted to protoporphyrin IX using protoporphyrinogen
oxidase.  The final reaction involves the insertion of ferrous iron into protoporphyrin IX
catalyzed by the enzyme ferrochelatase leading to the formation of heme.[5]

Molecular
Heme is ferrous protoporphyrin IX- four pyrrole rings linked via methenyl bridges. The side
chains of heme b are methyl, vinyl, methyl, propionyl, and asymmetric ring D: propionyl,
methyl.

Function
Heme has a variety of functions. As a cofactor, it allows for the following[1]:
 Oxygen transport in hemoglobin
 Storage in myoglobin
 A prosthetic group for cytochrome p450 enzymes
 A reservoir of iron
 Electron shuttle of enzymes in the electron transport chain
 Cellular respiration
 Signal transduction-heme regulates the antioxidant response to circadian rhythms,
microRNA processing
 Cellular differentiation and proliferation

Mechanism
Heme synthesis in erythroid cells: heme is synthesized for incorporation into hemoglobin. In
immature erythrocytes (reticulocytes), heme stimulates protein synthesis of the globin chains
and erythropoietin stimulates heme. The kidney releases erythropoietin hormone at low
oxygen levels in tissues and stimulates RBC and hemoglobin synthesis. Accumulation of
heme in erythroid cells is desired as it leads to more globin chain synthesis and required in
erythroblast maturation. When red cells mature both heme and hemoglobin synthesis ceases.
Additionally, control of heme biosynthesis in erythrocytes is controlled by the availability of
intracellular iron.
Heme synthesis in the liver is highly variable and tightly regulated as heme
outside proteins causes damage to hepatocytes at high concentration. In the liver, cytochrome
P450 (CYP 450) requires heme. Liver contains the isoform ALAS1 which is expressed in
most cells. Drugs increase ALAS1 activity as they lead to CYP 450 synthesis which needs
heme. Low intracellular heme concentration stimulates synthesis of ALAS1. Heme synthesis
stops when heme is not incorporated into proteins and when heme and hemin accumulate.
Hemin decreases the synthesis of ALA synthase 1 in three ways: Hemin reduces the synthesis
of ALAS1 mRNA, destabilizes ALAS1 mRNA, and inhibits import of the enzyme ALAS1
from the cytosol into mitochondria.

Pathophysiology
A defect or mutation in 5’- aminolevulinic acid synthase 2 (ALAS2) leads to a disorder called
X-linked sideroblastic anemia. It reduces protoporphyrin production and decreases heme.
However, Iron continues to enter the erythroblast leading to an accumulation in the
mitochondria and therefore a manifestation of the disease.
During the biosynthetic pathway, the linear hydroxymethylbilane can spontaneously form a
“faulty” porphyrin ring when not immediately used as a substrate for uroporphyrinogen
synthesis. If uroporphyrinogen III synthase is deficient, then hydroxymethylbilane
spontaneously closes and forms a different molecule called uroporphyrinogen I.
Uroporphyrinogen leads to the formation of coproporphyrinogen I. This molecule does not
result in the formation of heme. 

Clinical Significance
Heme synthesis is a biochemical pathway which requires a number of steps, substrates, and
enzymes. A deficiency in an enzyme or substrate leads to accumulation of intermediates of
heme synthesis in blood, tissues, and urine leading to a clinically significant outcome of a
group of disorders called porphyrias. Porphyrias are hepatic or erythropoietic. They can
be acute or chronic, lead to neurologic dysfunction, mental disturbance or photosensitivity.
Defects of heme synthesis after formation of hydroxymethylbilane leads to photosensitivity
of patients. Other symptoms include a change in urine color, abdominal pain, abdominal
colic, highly agitated state, tachycardia, respiratory problems, nausea, confusion, weakness of
lower extremities.  
Porphyrias are acute intermittent, congenital erythropoietic porphyria, porphyria cutanea
tarda, hereditary coproporphyria.
Acute intermittent porphyria: This occurs due to a mutation in hydroxymethylbilane synthase,
which leads to an accumulation of ALA and porphobilinogen.  It does not affect
erythroblasts. The disease presents as severe abdominal pain, vomiting, constipation,
abdominal distention, and behavioral changes (irritability, insomnia, emotional lability),
[6] hypertension and tachycardia. Lab results show elevated urinary porphobilinogen and the
urine darkens on exposure to air and sunlight. Patients presenting with acute intermittent
porphyria are not photosensitive.
Porphyria cutanea tarda: This variant is the most common type of porphyria. It is due to a
deficiency or decreased activity in uroporphyrinogen decarboxylase (UROD). It can be
acquired or hereditary (autosomal dominant). Uroporphyrin accumulates in the urine.
Symptoms include photosensitivity leading to blisters developing in sun-exposed areas and
hyperpigmentation, and hepatic injury. Treatment includes avoidance of sunlight,
hydroxychloroquine, and phlebotomy.
Erythropoietic porphyria: This occurs due to a deficiency in ferrochelatase, the enzyme
responsible for the final formation of heme in the biosynthesis pathway by combining
protoporphyrin IX and ferrous iron. Deficiency leads to an accumulation of protoporphyrin
IX in erythrocytes. Symptoms include painful photosensitivity- swelling burning and itching
in sun-exposed areas; sometimes hepatic dysfunction.
Lead Poisoning: Lead interacts with zinc cofactors for ALA dehydratase and ferrochelatase
leading to inhibition of these two enzymes in the biochemical biosynthetic pathway of heme.
This inhibition leads to mostly ALA and some protoporphyrin IX accumulating in urine.
Symptoms include abdominal pain, vomiting, fatigue irritability and developmental disability
in children.
 Regulation of Heme Biosynthesis
Although heme is synthesized in virtually all tissues, the principal sites of synthesis are
erythroid cells (≈85%) and hepatocytes (accounting for nearly all the rest of heme
synthesis). The differences in these two tissues and their needs for heme result in quite
different mechanisms for regulation of heme biosynthesis.
In hepatocytes, heme is required for incorporation into the cytochromes, in particular,
the P450 class of cytochromes (CYP) that are important for xenobiotic detoxification. In
addition, numerous cytochromes of the oxidative-phosphorylation pathway contain heme.
The rate-limiting step in hepatic heme biosynthesis occurs at the ALA synthase catalyzed
step, which is the committed step in heme synthesis. Heme itself functions as a co-repressor
in the inhibition of ALA synthase gene expression. The Fe3+ oxidation product of heme is
termed hemin. Heme itself, and hemin acts as a feed-back inhibitors on ALA synthase.
Hemin also inhibits transport of ALA synthase from the cytosol (its site of synthesis) into
the mitochondria (its site of action). Because certain pharmaceutical drugs are metabolized
by the hepatic CYP system, which requires heme, increased utilization of heme occurs upon
administration of these drugs. Of particular significance are the barbiturates. Use of
barbiturates should NEVER be prescribed for the pain associated with certain types of
porphyrias. This is because the administration of barbiturates leads to their degradation by
CYP enzymes in the liver, resulting in a reduction in overall heme levels as the heme needs
to be incorporated into the CYP for their function. This results in de-repression of ALA
synthase with the result being an exacerbation of the symptoms of the porphyria due to
increased ALA synthesis and subsequent heme biosynthesis products upstream of the
defective enzyme.
In erythroid cells all of the heme is synthesized for incorporation into hemoglobin and
occurs only upon differentiation when synthesis of hemoglobin proceeds. When red cells
mature both heme and hemoglobin synthesis ceases. The heme (and hemoglobin) must,
therefore, survive for the life of the erythrocyte (normally this is 120 days). In reticulocytes
(immature erythrocytes) heme stimulates protein synthesis. The mechanism of this mode of
heme-mediate regulation of protein synthesis is described in the Protein Synthesis page.
Additionally, control of heme biosynthesis in erythrocytes occurs at numerous sites other
than at the level of ALA synthase. Control has been shown to be exerted on ferrochelatase,
the enzyme responsible for iron insertion into protoporphyrin IX, and on porphobilinogen
deaminase.
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Heme Metabolism and Disposition of Bilirubin

The largest repository of heme in the human body is in red blood cells, which have a life
span of about 120 days. There is thus a turnover of about 6 g/day of hemoglobin, which
presents 2 problems. First, the porphyrin ring is hydrophobic and must be solubilized to be
excreted. Second, iron must be conserved for new heme synthesis.
Normally, senescent red blood cells and heme from other sources are engulfed by cells
of the reticuloendothelial system (phagocytic macrophages primarily of the spleen but also
of the liver, lymph, and bone marrow). The globin is recycled or converted into amino
acids, which in turn are recycled or catabolized as required. Heme is oxidized, with the
heme ring being opened by the endoplasmic reticulum enzyme, heme oxygenase. The
oxidation step requires heme as a substrate, and any hemin (Fe3+) is reduced to heme (Fe2+)
prior to oxidation by heme oxygenase. The heme oxygenase-mediated oxidation occurs on
a specific carbon producing the linear tetrapyrrole biliverdin, ferric iron (Fe3+), and carbon
monoxide (CO). This is the only reaction in the body that is known to produce CO. Most of
the CO is excreted through the lungs, with the result that the CO content of expired air is a
direct measure of the activity of heme oxygenase in an individual. Humans harbor two
distinct heme oxygenase genes identified as HMOX1 and HMOX2. The heme oxygenase
enzyme encoded by the HMOX1 gene is the rate-limiting enzyme of heme catabolism. Both
HMOX1 and HMOX2 genes are constitutively expressed, however, the activity of the
HMOX1 encoded enzyme is inducible by heme, heavy metals, and conditions of stress such
as hypoxia. The HMOX1 gene is located on chromosome 22q12.3 and is composed of 5
exons encoding a protein of 288 amino acids. The HMOX2 gene is located on chromosome
16p13.3 and is composed of 12 exons that generatre nine alternatively spliced mRNAs that
collectively encode three isoforms identified as isoform a (370 amino acids), isoform b (316
amino acids), and isoform c (287 amino acids).
In the next reaction a second bridging methylene (between rings III and IV) is reduced
by biliverdin reductase, producing bilirubin. There are two biliverdin reductase genes in
humans identified as BLVRA and BLVRB. The enzyme encoded by the BLVRA gene is
principally responsible for the catabolism of biliverdin. The enzyme encoded by the
BLVRB gene catalyzes the reduction of not only biliverdin but also a variety of flavins,
such as riboflavin, FAD or FMN, and methemoglobin. The enzyme encoded by the BLVRB
gene is also called NADPH-dependent flavin reductase. The BLVRA gene is located on
chromosome 7p13 and is composed of 11 exons generate two alternatively spliced mRNAs,
both of which encode the same 296 amino acid precursor protein. The BLVRB gene is
located on chromosome 19q13.2 and is composed of 5 exons that encode a protein of 206
amino acids.
Bilirubin is significantly less extensively conjugated than biliverdin causing a change in
the color of the molecule from blue-green (biliverdin) to yellow-red (bilirubin). The latter
catabolic changes in the structure of tetrapyrroles are responsible for the progressive
changes in color of a hematoma, or bruise, in which the damaged tissue changes its color
from an initial dark blue to a red-yellow and finally to a yellow color before all the pigment
is transported out of the affected tissue. Peripherally arising bilirubin is transported to the
liver in association with albumin, where the remaining catabolic reactions take place.
 Pathway for the degradation of heme to bilirubin. The ring of heme is opened through
the action of heme oxygenase which also results in the relase of the iron as the ferric form
(Fe3+) and also releases carbon monoxide, CO. The product of the heme oxygenase reaction
is biluverdin. Biliverdin is converted to bilirubin via the action of biliverdin reductase. The
various substituents on the pentameric rings of biliverdin and bilirubins are M: methyl, P:
propyl, V: vinyl.

In hepatocytes, bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT: a member of the

large UDP glucuronosyltransferase family of enzymes) adds two equivalents of glucuronic
acid to bilirubin to produce the more water soluble, bilirubin diglucuronide derivative. The
increased water solubility of the tetrapyrrole facilitates its excretion with the remainder of
the bile as the bile pigments. The bilirubin UDP-glucuronosyltransferase gene (UGT1A)
which encodes the enzyme that carries out this reaction is located on chromosome 2q37.1.
Several UGT1A enzymes, including bilirubin-UGT (identified as UGT1A1), are encoded
by the UGT1A gene complex. The 5' region of the UGT1A complex contains 13 tandemly
arrayed first exons, including 4 pseudo exons. These tandemly arrayed exons are identified
as 1A1, 1A2, 1A3, etc. Exons 2, 3, 4, and 5 are located in the UGT1A 3' region. All UGT
isoforms contain the same C-terminal domain encoded by exons 2 through 5.  Each first
exon has its own promoter element. The 9 viable first exons are independently spliced to
the common exons 2 through 5 to generate 9 UGT1A transcripts with unique 5' ends and
identical 3' ends. The N-terminal region encoded by each unique first exon determines
acceptor substrate specificity, while the 246-amino acid C-terminal region encoded by the 4
common exons specifies interactions with the common donor substrate, UDP-glucuronic
acid. The bilirubin-UGT isoform (UGT1A1) consists of 533 amino acids.

Bilirubin diglucuronide

In newborn infants, or in individuals with abnormally high red cell lysis, or liver damage
with obstruction of the bile duct, the bilirubin and its precursors accumulate in the
circulation; the result is hyperbilirubinemia, the cause of the abnormal yellowish
pigmentation of the eyes and tissues known as jaundice. All newborn infants undergo
turnover of the red blood cells that contain fetal hemoglobin (HbF) so that new red blood
cells containing adult hemoglobin (HbA) can be produced. In some cases the activation of
the UGT1A gene at birth is insufficient to handle all the red cell turnover resulting in
neonatal jaundice, apparent around day 2 or 3. If the blood levels of bilirubin do not decline
in a short period of time these infants will need to be treated by phototherapy. The blue-
green wavelength light (460-490 nm) used in biliblankets is sufficient to induce breakdown
of bilirubin in the skin so that it can be cleared from the blood. In rare cases the
phototherapy does not work fast enough and in these cases it is appropriate to treat the
infants with phenobarbital which enhances the induction of the UGT1A gene.
In normal individuals, intestinal bilirubin is acted on by bacteria to produce the final
porphyrin products, urobilinogens and stercobilins, that are found in the feces. The
stercobilins oxidize to brownish pigments which impart the brown to brown-black color to
normal feces. Indeed, the color of the stool can be quite diagnostic since chalky clay
colored feces are indicative of a defect in the hepato-biliary circulation, such as in bile
obstruction. Some of the urobilinogen produced by intestinal bacteria is reabsorbed from
the gut and enter the circulation. These urobilinogens are converted to the urobilins which
are then excreted in the urine. Oxidation of the urobilins imparts the yellowish coloration to
urine.

Clinical Aspect of Heme Metabolism and Bilirubin

Clinical problems associated with heme metabolism are of two types. Disorders that
arise from defects in the enzymes of heme biosynthesis are termed the porphyrias (see
Table below and the Porphyrias page) and cause elevations in the serum and urine content
of intermediates in heme synthesis. Inherited disorders in bilirubin metabolism lead
to hyperbilirubinemia (see the Bilirubinemias page).
Hyperbilirubinemias
Bilirubin levels are measured in the serum by an assay utilizing Ehrlich diazo reagent
and results in the formation of an azobilirubin product. Conjugated bilirubin does not
require addition of alcohol to promote the azotization reaction and thus, this is referred to as
measurement of direct bilirubin. The reaction with unconjugated bilirubin requires the
addition of alcohol and thus is referred to as the measurement of indirect bilirubin. Normal
bilirubin measurements are 0.3–1.2md/dL for total (indirect + direct). Direct type bilirubin
does not exist in the plasma, however, a small portion of indirect type bilirubin may present
as direct reacting type and thus the serum measurement may show a direct bilirubin but this
is never above 0.3mg/dL in a normal individual.
Excess circulation and accumulation of bilirubin (hyperbilirubinemia) results in a
yellow-orange discoloration of the tissues and is most easily visible as icteric (yellowish)
discoloration in the sclera of the eyes. Bilirubin toxicity (bilirubin encephalopathy) can be
life threatening in neonates. Bilirubin encephalopathy is characterized by yellow
discoloration of the basal ganglia in babies with intense jaundice and was first described
over a century ago and the term "kernicterus" was coined to describe these physical
changes. Any increase in plasma bilirubin above 20mg/dL is considered dangerous in
neonates. However, individual differences in bilirubin sensitivity can result in kernicterus at
lower bilirubin levels. Kernicterus occurs in infants with severe unconjugated
hyperbilirubinemia and in young adults with high serum levels of unconjugated bilirubin.
The latter is the result of inherited deficiencies in the enzyme responsible for bilirubin
conjugation to glucuronic acid, bilirubin UDP glucuronosyltransferase (bilirubin-UGT).
Bilirubin has been shown to inhibit DNA synthesis, uncouple oxidative phosphorylation,
and inhibit ATPase activity in brain mitochondria. Bilirubin also inhibits a variety of
different classes of enzymes including dehydrogenases, electron transport proteins,
hydrolyases, and enzymes of RNA synthesis, protein synthesis and carbohydrate
metabolism. All of these toxic effects of bilirubin are reversed by binding to albumin. In
fact, albumin plays a vital role in the disposition of bilirubin in the body by keeping the
compound in solution and transporting it from its sites of production (primarily bone
marrow and spleen) to its site of excretion which is the liver.
Several inherited disorders in bilirubin metabolism have been identified. Gilbert
syndrome and the Crigler-Najjar syndromes result from predominantly unconjugated
hyperbilirubinemia. Dubin-Johnson syndrome and Rotor syndrome result from conjugated
hyperbilirubinemia. Once conjugated to glucuronate, bilirubin is water soluble, therefore,
conjugated hyperbilirubinemias are less severe in their symptomology than are the
unconjugated hyperbilirubinemias.
Porphyrias
The porphyrias are both inherited and acquired disorders in heme synthesis. These
disorders are classified as either erythroid or hepatic, depending upon the principal site of
expression of the enzyme defect. Eight different porphyrias have been classified
encompassing defects in each of the enzymes of heme synthesis. Defects in the function of
hepatic uroporhyrinogen decarboxylase (UROD) result in type I porphyria cutanea tarda
(PCT I), whereas mutations in the UROD gene result in type II PCT (PCT II). PCT is the
most commonly occurring type of porphyria. It should be noted that no porphyria has been
identified resulting from defects in the house-keeping form of ALAS (ALAS1). The most
commonly occurring hepatic porphyria is acute intermittent porphyria, AIP, which is
caused by a defect in porphobilinogen deaminase, (PBG deaminase). This enzyme is also
called hydroxymethylbilane synthase (official gene symbol: HMBS) or also but rarely,
uroporphyrinogen I synthase.
All of the porphyrias lead to excretion of heme biosynthetic byproducts that turn the
urine red and when deposited in the teeth turn them reddish brown. Accumulation of these
byproducts in the skin renders it extremely sensitive to sunlight causing ulceration and
disfiguring scars. Increased hair growth (hypertrichosis) is also a symptom of the
porphyrias leading to appearance of fine hairs over the entire face and on the extremities.
This latter symptom lends to the description of "werewolf syndrome" in many porphyria
patients.
In many cases the treatment protocols for the intermittent attacks of the various
porphyrias, in particular in the case of acute intermittent porphyria, include the use of
hemin or hematin and glucose supplementation. Hemin is a form of iron protophorphyrin
IX in which the associated iron has an additional chloride ligand. Hematin is similar except
that instead of a chloride ion there is an hydroxide ion liganded to the iron. The rationale for
the use of these agents is that they act as analogs of heme and strongly inhibit the activity of
ALAS resulting in reductions in the heme biosynthetic intermediates that precipitate the
porphyria attack. The use of glucose to treat porphyrias was a serendipitous observation but
for a long time the mechanism by which glucose infusion alleviated the symptoms of a
porphyria attack were not understood. Quite often there is an association between fasting,
low serum glucose, and the precipitation of an acute porphyria attack which suggested the
utility of glucose infusion. The molecular mechanism was ascertained when it was found
the the transcription factor, PGC-1α, is activated in the liver in respone to hypoglycemia.
Indeed, activation of PGC-1α is required to initiate hepatic gluconeogenesis via the
activation of several genes in this metabolic pathway. In addiiton to gluconeogenic genes,
the ALAS1 gene is activated in the liver via PGC-1α. Thus, hypoglycemia leads to
increased ALAS1 activity and results in accumulation of heme intermediates that result in
the precipitation of the attack.
Protoporphyrin IX