BCH 301/381: SPECIAL TOPICS IN MEDICAL.

/FUNCTIONAL BIOCHEMISTRY

PORPHYRINS AND PORPHYRIA

In addition to their role as the building blocks of proteins, amino acids are precursors of many
specialized biomolecules such as porphyrins. Porphyrins are components of hemeproteins of
animals and invertebrates. Heme is metalloporphyrin. It contains metal iron in the centre of
porphyrin ring. Hence heme proteins are referred as metalloporphyrinoproteins.

Occurrence in nature: Porphyrins readily bind metal ions, usually ferrous (Fe 2+) or ferric (Fe3+)
iron. The most prevalent metalloporphyrin in humans is heme, which consists of one Fe 2+
coordinated in the center of the tetrapyrrole ring of protoporphyrin IX. Heme is the prosthetic
group for hemoglobin, myoglobin, the cytochromes, the cytochrome P450 (CYP)
monooxygenase system, catalase, nitric oxide synthase, and peroxidase. These hemeproteins are
rapidly synthesized and degraded. E.g is synthesis and degradation of erythrocytes on daily basis.

Medical and Biological Importance

1. Porphyrins are present in biological fluids like blood, bile, urine and feces of animals and
invertebrates. They are also found in plants and bacteria.

2. Hemeproteins like hemoglobin and myoglobin are involved in O 2 transport in animals and
vertebrates.

3. In invertebrates erythrocruorins which are also hemeproteins are responsible for the O 2
transport.

4. Hemeproteins like cytochromes and cytochrome oxidase are components of respiratory chain
and involved in electron transport.

5. Cytochrome P450 which is involved in detoxification of drugs is a hemeprotein.

6. Some hemeproteins are involved in metabolism. For example tryptophan dioxygenase an

enzyme of tryptophan catabolism is a metelloprophyrinoprotein and cyclooxygenase an enzyme
of prostaglandin synthesis is a hemeprotein.

7. Hemeproteins like catalase and peroxidase are involved in the removal of H2O2.

8. In plants porphyrins are components of chlorophyll and phycobilins.

PORPHYRIN METABOLISM

Heme is synthesized by most of the cells except mature erythrocytes. However bonemarrow

and liver are chief organs involved in heme production. Bonemarrow produces about 80%
and rest is produced by liver.

Glycine as the precursor of porphyrin: The biosynthesis of porphyrins, for which glycine is a
major precursor, is of the central importance because of the porphyrin nucleus in heme proteins
such as hemoglobin and the cytochromes. In higher eukaryotes, glycine reacts with succinyl-
CoA in the first step to yield α-amino- β-ketoadipate which is then decarboxylated to δ-
aminolevulinate. In plants, algae, and most bacteria, δ-aminolevulinate is formed from
glutamate.

In all organisms, two molecules of δ-aminolevulinate condense to form porphobilinogen and,

through a series of complex enzymatic reactions, four molecules of porphobilinogen come
together to form protoporphyrin. The iron atom is incorporated after the protoporphyrin has been
assembled, in a step catalyzed by ferrochelatase. Porphyrin biosynthesis is regulated in higher
eukaryotes by heme, which serves as a feedback inhibitor of early steps in the synthetic pathway.
Genetic defects in the biosynthesis of porphyrins can lead to the accumulation of pathway
intermediates, causing a variety of human diseases known collectively as porphyrias. One of
such porphyria is the intermittent porphyria. Intermittent porphyria occurs by the inhibition of
formation of pre- uroporphyrinogen from porphobilinogen leading to accumulation of δ-
aminolevulinate and porphobilinogen in the liver.

Reaction steps of porphyrin biosynthesis: Synthesis of the tetrapyrrole ring (heme) starts in the
mitochondria then progresses in the cytoplasm and end up in the mitochondria. The reaction
steps in the biosynthentic pathways are as follows:

1. Formation of δ-aminolevulinic acid: All the carbon and nitrogen atoms of the porphyrin
molecule are provided by glycine (a nonessential amino acid) and succinyl coenzyme A (a citric
acid cycle intermediate) that condense to form δ-aminolevulinic acid (ALA) in a reaction
catalyzed by ALA synthase ([ALAS]. This reaction requires pyridoxal phosphate (PLP) as a
coenzyme and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are
two isoforms of ALAS, 1 and 2, each produced by different genes and controlled by different
mechanisms. ALAS1 is found in all tissues, whereas ALAS2 is erythroid specific. Loss of
function mutations in ALAS2 result in X-linked sideroblastic anemia- i.e absent of fe 2+ at the
centre of heme]

2. Formation of porphobilinogen: The condensation of two molecules of ALA to form

porphobilinogen by zinc-containing ALA dehydratase (porphobilinogen synthase) is extremely
sensitive to inhibition by heavy metal ions (for example, lead) that replace the zinc. This
inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning.

3. Formation of uroporphyrinogen: The condensation of four porphobilinogens produces the

linear tetrapyrrole hydroxymethylbilane, which is cyclized and isomerized by uroporphyrinogen
III synthase to produce the asymmetric uroporphyrinogen III. This cyclic tetrapyrrole undergoes
decarboxylation of its acetate groups, generating coproporphyrinogen III. These reactions occur
in the cytosol.

4. Formation of heme: Coproporphyrinogen III enters the mitochondrion, and two propionate
side chains are decarboxylated by coproporphyrinogen III oxidase to vinyl groups generating
protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as
Fe2+) into protoporphyrin IX can occur spontaneously, but the rate is enhanced by ferrochelatase,
an enzyme that, like ALA dehydratase, is inhibited by lead.
Fig.1: Pathways of Porphyrin biosynthesis.

Cooperativity interaction of hemoglobin protein: Iron atom is coordinately linked with 5

nitrogen atoms (4 nitrogen of pyrrole rings of protoporphyrin and 1 st nitrogen atom of a histidine
residue of globin). The remaining valency of iron atom is satisfied with water or oxygen atom.
The iron’s sixth site is coordinatedwith oxygen in oxyhemoglobin and with H 2O in
deoxyhemoglobin.
Fig. 1. In the heme molecule, iron atom is coordinately linked with nitrogen atoms

Hemoglobin can exist in two different states (conformations), known as the

(a) T form
(b) R form.

The T form (for tense) and has a much lower O2 affinity than the R form (for relaxed). Binding
of O2 to one of the subunits of the T form leads to a local conformational change that weakens
the association between the subunits. Increasing O2 partial pressure thus means that more and
more molecules convert to the higher affinity R form. This cooperative interaction between the
subunits increases the O2 affinity of Hemoglobin with increasing O2 concentrations.

DISORDERS OF HEME SYNTHESIS (PORPHYRIA)

Porphyrias are diseases of heme biosynthesis characterized by increased excretion of porphyrins

or porphyrin precursors(e.g Delta amino levulinic acid(ALA) + Porphobilinogen (PBG)) in urine
and feces. Accumulation of porphyrins or porphyrin precursors in plasma and tissues also occurs.
They may be inherited (autosomal) and acquired. (Greek ‘porphyria' means purple).

There are several types of inherited porphyrias and they are classified into 2 groups based on
organ or cells that are affected.

a. Hepatic porphyrias

b. Erythropoietic porphyrias

This classification is based on the major site, where the enzyme deficiency is manifested. The
clinical manifestations vary. Porphyrias in general, are not associated with anemia. Depending
on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the
liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals
with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and
neuropsychiatric signs, whereas those with enzyme defects leading to the accumulation of
tetrapyrrole intermediates show photosensitivity (that is, their skin itches and burns [pruritus]
when exposed to visible light). [Note: Photosenstivity is a result of the oxidation of colorless
porphyrinogens to colored porphyrins, which are photosensitizing molecules thought to
participate in the formation of superoxide radicals from oxygen].

a.Chronic hepatic porphyria: Porphyria cutanea tarda, the most common porphyria, is a
chronic disease of the liver. The disease is associated with a deficiency in uroporphyrinogen
decarboxylase,(or coproporphyrinogen III synthase) but clinical expression of the enzyme
deficiency is influenced by various factors, such as hepatic iron overload, exposure to sunlight,
alcohol ingestion, estrogen therapy, and the presence of hepatitis B or C or HIV infections.
Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads
to cutaneous symptoms (cutanea tarda) as well as urine that is red to brown in natural light and
pink to red in fluorescent light.

b. Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase deficiency

porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are
characterized by acute attacks of gastrointestinal (GI), neuropsychiatric, and motor symptoms
that may be accompanied by photosensitivity. Symptoms of the acute hepatic porphyrias are
often precipitated by use of drugs, such as barbiturates and ethanol, which induce the synthesis
of the heme-containing CYP microsomal drug oxidation system. This further decreases the
amount of available heme, which, in turn, promotes increased synthesis of ALAS1.

c. Erythropoietic porphyrias: The chronic erythropoietic porphyrias (congenital erythropoietic

porphyria and erythropoietic protoporphyria) are characterized by skin rashes and blisters that
appear in early childhood.
Acute Intermittent Porphyria (AIP): It is inherited as an autosomal dominant trait. PBG-
deaminase (uroporphyrinogen-I-synthase) is deficient. As the name indicates, the symptoms
appear intermittently and they are quite vague. Hence it is at times called the "little imitator".
This leads to a secondary increase in activity of ALA synthase, since the end-product inhibition
is not effective. The levels of ALA and PBG are elevated in blood and urine. As they are
colorless compounds, urine is colorless when voided, but the color is increased on standing due
to photo-oxidation of PBG to porphobilin. Hence urine samples for PBG estimation should be
freshly collected and transported in dark bottles. Women have less severe manifestations before
menarche and after menopause. Thus, the female sex hormones have a stimulatory effect on
ALA synthase.. An attack is precipitated by starvation and symptoms are alleviated by a high
carbohydrate diet. Drugs like barbiturates, which are known to induce ALA synthase, can
precipitate an attack. Most commonly, patients present with acute abdominal pain. Another
group may have neurological manifestations like sensory and motor disturbances, confusion and
agitation. Some patients may present with psychiatric problems and may be treated accordingly.

ALA synthase (ALAS) deficiency: It is the key enzyme of the pathway. It is inhibited by heme
in non-erythroid cells. Defective gene leads to Pyridoxine responsive sideroblastic anemia.
Administration of porpyrinogenic drugs (barbiturates, ethanol, anticonvulsants) depletes heme;
so feedback inhibition on ALAS is removed. This in turn leads to excessive production of heme
intermediaries causing neurological porphyrias. Definitive diagnosis is by demonstration of
mutation in erythroid ALA synthase.

ALA dehydratase (Porphobilinogen synthase) deficiency: This condition is very rare.

Features are similar to AIP (abdominal pain and neuropathy). Differential diagnosis are lead
poisoning (inhibit ALA dehydratase) and tyrosinemia Type I (accumulation of succinyl acetone
which is structurally similar to ALA). Characteristic features are increased plasma ALA,
increased urine ALA and coproporphyrin III, decreased ALAD in erythrocytes. Prenatal
diagnosis is by ALAD activity in cultured chorionic villi.

Congenital Erythropoietic Porphyria: It is inherited as an autosomal recessive trait. Normally

the type III isomer is produced in larger amounts, but in this condition, type I isomer is formed
considerably. They are converted to porphyrins type I. This would lead to ineffective feedback
inhibition which further increases the rate of formation of type I porphyrins. Their level in blood
increases leading to photosensitivity. Their excretion in urine makes the urine dark red in color
(port wine appearance). The major manifestations relate to the skin due to the photo-
sensitization by the presence of porphyrins in the capillaries. Reactive oxygen species (free
radicals) are the cause for cell destruction. Repeated attacks of dermatitis and scarring lead to a
typical facial deformity often referred to as 'monkey face'. Repeated ulceration and scarring may
cause mutilation of nose, ear and cartilage. This may mimic leprosy. When UV light is reflected
on to teeth a red fluorescence is seen; this is called erythrodontia.

Ferrochelatase deficiency (Erythropoietic protoporphyria): It is characterized by increased

free protoporphyrin in RBC and decreased ferrochelatase activity in cultured lymphocytes. RBCs
exhibit red fluorescence at 620 nm. Accumulation of porphyrin precursors, ALA and
porphobilinogen leads to neurovisceral manifestations. Accumulated uroporphyrin and
coproporphyrin cause delayed bullous lesions. On the other hand, being more lipophilic,
protoporphyrin associates with cell membranes and causes burning sensation and inflammatory
reaction in skin exposed to sun. Hence biochemical diagnosis of porphyria will be considered
based on the major clinical features, namely: (a) neurovisceral and (b) cutaneous
manifestations.

Acquired Porphyrias: Porphyria can result from lead poisoning. Most of the paints contain
lead more than the permitted levels. Children suck painted toys; and they get the poison. Causes
of acquired porphyria such as ethanol. The toxic effect of lead is due to inhibition of ferro
chelatase. So, there is decreased level of heme with consequent increased activity of ALA
synthase. A characteristic difference from congenital porphyrias is that there is associated
anemia in the acquired variety.
Diagnosis of Porphyrias: To demonstrate porphyrins, UV fluorescence is the best technique.
The presence of porphyrin precursor in urine is detected by Ehrlich's reagent. When urine is
observed under ultraviolet light; porphyrins if present, will emit strong red fluorescence.

Fig 2: Enzyme deficiencies in porphyrias (identifies the defective enzyme at each step)

DEGRADATION OF HEME

After approximately 3 - 4 months in the circulation, red blood cells are taken up and degraded by
the reticuloendothelial system, particularly in the liver and spleen. Approximately 85% of heme
destined for degradation comes from senescent RBCs. The remainder is from the degradation of
heme proteins other than hemoglobin. The end-products of heme catabolism are bile pigments.
Bile pigments are Bilirubin and Biliverdin. They are the breakdown products of heme; they are
useless excretory products.
1. Formation of biliverdin: The first step in the degradation of heme is catalyzed by the
microsomal heme oxygenase system of the reticuloendothelial cells. In the presence of
nicotinamide adenine dinucleotide phosphate (NADPH + H) and O 2, the enzyme catalyzes three
successive oxygenations that result in opening of the porphyrin ring (converting cyclic heme to
linear biliverdin), production of carbon monoxide (CO), and release of Fe 2+. The CO has biologic
function, acting as:

(a) Signaling molecule and (b) Anti-inflammatory.

Biliverdin, a green pigment, is reduced, forming the red-orange bilirubin. Bilirubin and its
derivatives are collectively termed bile pigments (Biliverdin and Bilirubin).

Fig3; Heme degradation

2. Uptake of bilirubin by the liver: Bilirubin is only slightly soluble in plasma and, therefore, is
transported to the liver by binding noncovalently to albumin. Certain anionic drugs, such as
salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter
the central nervous system (CNS). This causes the potential for neural damage in infants.
Bilirubin dissociates from the carrier albumin molecule; enters a hepatocyte via facilitated
diffusion; and binds to intracellular proteins, particularly the protein ligandin.

3. Formation of bilirubin diglucuronide: In the hepatocyte, the solubility of bilirubin is

increased by the addition of two molecules of glucuronic acid, producing bilirubin diglucuronide.
This process is referred to as conjugation. The reaction is catalyzed by microsomal bilirubin
UDP-glucuronosyltransferase (bilirubin UGT) using uridine diphosphate (UDP)-glucuronic acid
as the glucuronate donor.
Fig.4: Production of Bilirubin diglucuronide.

4. Secretion of bilirubin into bile: Bilirubin diglucuronide (conjugated bilirubin [CB]) is

actively transported against a concentration gradient into the bile canaliculi and then into the
bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease.
[Note: A rare deficiency in the protein required for transport of CB out of the liver results in
Dubin-Johnson syndrome.] Unconjugated bilirubin (UCB) is normally not secreted into bile.

5. Formation of urobilins in the intestine: Bilirubin diglucuronide is hydrolyzed and reduced

by bacteria in the gut to yield urobilinogen, a colorless compound. Most of the urobilinogen is
oxidized by intestinal bacteria to stercobilin, which gives feces the characteristic brown color.
However, some of the urobilinogen is reabsorbed from the gut and enters the portal blood. A
portion of this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is
taken up by the liver and then resecreted into the bile. The remainder of the urobilinogen is
transported by the blood to the kidney, where it is converted to yellow urobilin and excreted,
giving urine its characteristic color.

BILE ACIDS AND SALTS

Bile Acids /Salts: Studies on the chemistry of compounds present in the bile had begun in the
early nineteenth century. Remarkable experiments performed by Thenard and Berzelius led to
the identification of choleic acid and bilin. Bile salts are the sodium salts of bile acids
(glycocholate and taurocholate). They are produced from cholesterol; they help in the absorption
of fat. Both bile pigments and bile salts are present in the bile.
Fig.1: Bile acids are formed from cholesterol. (By decarboxylation and hydroxylation)

Bile salts are steroidal detergents, which together with lipids/fats/cholesterol form mixed
micelles in the intestine to enable fat digestion and absorption through the intestinal wall. They
are biosynthesized from cholesterol in the liver and stored in the gall bladder. Bile is secreted
through the bile duct into the intestine when food passes from the stomach to the duodenum.
Most of the bile salts secreted into the upper region of the small intestine are absorbed along with
dietary lipids at the lower end of the small intestine. They are separated from the dietary lipid
and returned (more than 85%) to the liver for re-circulation. This movement of bile salts is
termed as enterohepatic circulation.

The most abundant bile salts in humans are cholate, chenodeoxycholate and deoxycholate, and
they are normally conjugated with either glycine (75%) or taurine (25%). Conjugation increases
the aqueous solubility of bile salts under physiological conditions. All primary bile acids appear
to have three features in common: (i) they are the major end-products of cholesterol metabolism,
(ii) they are secreted into the bile largely in a conjugated form, and (iii) these conjugates are
membrane-impermeable, water-soluble, amphiphilic molecules having a powerful ability to
transform lamellar arrays of lipids into mixed micelles.

Physiological functions of Bile acids

1. Solubilization and transport of lipids: Bile acids emulsify dietary fat droplets through the
formation of mixed micelle. This significantly increases the surface area of fat, making it
available for digestion by lipases, which otherwise cannot access the interior of lipid droplets.
Bile acids are lipid carriers and are able to solubilize many lipids by forming mixed micelles
with fatty acids, cholesterol and monoglycerides. These micelles are responsible for the
solubilization and absorption of fat-soluble vitamins such as vitamin E.

2. Bile salt-activated lipase: The intestinally located pancreatic enzyme, bile salt-activated
lipase (BAL), possesses unique activities for digesting different types of lipids. The reaction
scheme for the conversion of trioleoylglyceride to glycerol catalysed by BAL can be described
by consecutive first-order reactions comprising three pseudo-first-order rate constants (k1, k2 and
k3: Scheme 2). BALs differ from other lipases in their requirement of bile salts for activity.

3. Cholesterol homeostasis: The hepatic synthesis of bile acids accounts for the majority of
cholesterol breakdown in the body. In humans, every day, 500 mg of cholesterol is converted to
bile acids. This route for the elimination of excess cholesterol is probably important in all
animals. It has recently been discovered that bile acids can also act as hormones, which bind to
nuclear receptors and subsequently modulate the expression of proteins involved in cholesterol
homeostasis.

4. It has also been found that bile acids have pharmacological application because of the
antibacterial and antifungal properties of bile acid derivatives.