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Porphyrins

Porphyrins (Porphins) are cyclic tetrapyrol compounds formed by the linkage
of four pyrrole rings through methenyl bridges ((

)).

the reduced

porphyrins

(

Porphyrinogens) the linkage of four pyrrole rings (tetrapyrol)

through methylene bridges

((

))

   The  characteristic  property  of  porphyrins  is  the  formation  of  complexes  with
the metal ion bound to nitrogen atoms of the pyrrole rings.
e.g. Heme (iron porphyrin).
Proteins  which  contain  heme  ((hemoproteins))  are  widely  distributed  e.g.
Hemoglobin, Myoglobin, Cytochromes, Catalase & Tryptophan pyrrolase.
Natural  porphyrins  have  substituent  side  chains  on  the  eight  hydrogen  atoms
numbered on the pyrrole rings.
These side chains are:
1-Methyl-group (M)…    ((

))

2-Acetate-group (A)… ((

COOH

))

3-Propionate-group (P)… ((

COOH

))

4-Vinyl-group (V)… ((

))

Porphyrins  with  asymmetric  arrangement  of  the  side  chains  are  classified  as
type III porphyrins while those with symmetric arrangement of the side chains
are classified as type I porphyrins.
Only  types  I  &  III  are  present  in  nature  &  type  III  series  is  more  important
because it includes heme.

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Heme Biosynthesis

Heme biosynthesis occurs through the following steps:

1-The starting reaction is the condensation between succinyl-CoA ((derived from
citric  acid  cycle  in  the  mitochondria))  &  glycine  forming  δ-aminolevulinate
(ALA),  this  reaction  is  a  rate  limiting  reaction  in  the  hepatic  heme  synthesis,  it
occurs  in  mitochondria  &  is  catalyzed  by  ALA  synthase(Aminolevulinate
synthase) enzyme in the presence of pyridoxal phosphate as a cofactor.
2-In  the  cytoplasm  condensation  reaction  between  two  molecules  of  ALA  is
catalyzed by ALA dehydratase enzyme to form two molecules of water & one
molecule of porphobilinogen (PBG) which is a precursor of pyrrole.
ALA dehydratase is inhibited by lead as occurred in lead poisoning.
3-Condensation  of  four  molecules  of  PBG  with  loss  of  four  molecules  of
ammonia  lead  to  formation  of  linear  tetrapyrrole  called  hydroxymethylbilane
(HMB),  this  reaction  is  catalyzed  by  uroporphyrinogen  I  synthase  (PBG
deaminase,  HMB  synthase)

enzyme.

HMB cyclizes spontaneously to form

uroporphyrinogen I or:
4- HMB converted to uroporphyrinogen III by the action of  uroporphyrinogen
III  synthase  enzyme,  under  normal  conditions  the  uroporphyrinogen  formed  is
almost exclusively the III isomer.
Both  of  these  uroporphyrinogens  (I  &  III)  contain  A  &  P  side  chains  &  are
colorless  compounds,  however,  they  readily  reoxidized  to  their  respective
colored porphyrins (as in all porphyrinogens) by light with loss of six hydrogen
atoms.
5-Uroporphyrinogen  I  &  III  are  converted  into  coproporphyrinogen  I  &  III  by
decarboxylation  (loss  of  four  CO

) of all of the acetate (A) groups to be

converted into methyl (M) groups, this reaction is catalyzed by
uroporphyrinogen decarboxylase enzyme.

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6-Coproporphyrinogen  III  then  enters  the  mitochondria  to  be  converted  into
protoporphyrinogen III by oxidative decarboxylation of two (P) side chains into
(V)  side  chains,  this  reaction  is  catalyzed  by  coproporphyrinogen  oxidase
enzyme,  this  enzyme  acts  only  on  coproporphyrinogen  III,  therefore,
coproporphyrinogen I is not further progressed.
7-Protoporphyrinogen  III  is  oxidized  into  Protoporphyrin  III  ((loss  of  six
hydrogen  atoms),  this  reaction  is  catalyzed  by  protoporphyrinogen  oxidase
enzyme & it occurs in the mitochondria, however, this conversion can be induced
by light in vitro.
8-The final step is the formation of heme by the incorporation of ferrous iron into
protoporphyrin  III  in  a  reaction  catalyzed  by  ferrochelatase  (heme  synthase)
enzyme, this reaction occurs in the mitochondria.
Heme synthesis occurs in most human cells with the exception of  mature RBC
because it does not contain mitochondria, however, about 85% of heme synthesis
occurs  in  erythroid  precursors  cells  in  the  bone  marrow  to  form  hemoglobin  &
the  majority  of  the  remainder  in  the  hepatocytes  to  form  other  hemoproteins
mainly cytochrome P450.

ALA Synthase {ALAS}

Two main forms:
1-Hepatic((ALAS  1))..  It  is  a  regulatory  (rate  limiting)  enzyme  in  the  hepatic
heme synthesis, heme acts as a negative regulator for this enzyme.
  Many  drugs  are  porphyrogenic  as  barbiturates  which  can  result  in  a  marked
increase of ALAS 1 activity by utilizing cytochrome P450 which in turn reduces
the intracellular heme concentration lead to the activation of ALAS 1.
Other factors can lead to  marked decrease of ALAS 1 activity as:
  A-Excess glucose which cause reduction of fat burn in which toxins are stored,
therefore,  excess  glucose  reduces  toxin  release  so  that  the  consumption  of
cytochrome P450 is reduced.
  B-  Hematin  (oxidized  form  of  heme)  leads  to  increase  of  intracellular  heme
concentration result in marked decrease of ALAS 1 activity.
2-Erythroid((ALAS 2)).. Unlike ((ALAS 1)), it is not affected by drugs & does
not undergoes feedback regulation by heme.

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Properties of Porphyrins

1-The various porphyrinogens are colorless, where as the various porphyrins are
colored because it contains double bonds joining pyrrole rings which is absent in
porphyrinogens, therefore, all porphyrins ((in 5% HCl)) but not porphyrinogens
have a significant sharp absorbance band near 400 nm wavelength, this band is
called Soret band ((discovered by the French physicist; Charles Soret))

2-Porphyrins  when  illuminated  by  ultraviolet  light,  they  emit  a  strong  red
fluorescence  because  it  contains  double  bonds  joining  pyrrole  rings  while
porphyrinogens (because of absence of double bonds joining pyrrole rings)  they
have no fluorescence.
3-Coproporphyrins,  uroporphyrins  &  protoporphyrins  are  of  clinical  importance
because  they  are  excreted  in  increased  amounts  in  the  porphyrias,  these
compounds  when  present  in  feces  and/or  urine;  can  be  extracted  &  quantified
using spectrophotometric methods.
Generally  uroporphyrins  are  present  in  urine;  coproporphyrins  are  present  in
urine & feces while protoporphyrins are present in feces.
4-ALA & PBG can measured in urine by colorimetric method.

Porphyrias

  Porphyrias are group of disorders due to abnormalities of the heme biosynthesis
pathway, they can be genetic or acquired.
  They  are  not  prevalent;  however,  their  importance  lies  in  the  differential
diagnosis of certain clinical presentations as abdominal pain, neuropsychiatric &
dermatological findings.
Seven types of genetic porphyrias were found according to enzyme involved:
Generally  porphyria  is  inherited  as  autosomal  dominant  except  congenital
erythropoietic porphyria is inherited as autosomal recessive.

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Clinical Features of Porphyria

The  clinical  features  of  porphyria  can  result  from  a  deficiency  of  metabolic
products  beyond  the  enzymatic  block  and/or  from  accumulation  of  metabolites
before the block, the main clinical features are some or all of the followings:
1- Abdominal pain.
2-  Neuropsychiatric presentations.
2- Photosensitivity: When  porphyrinogens are accumulated in the skin, it forms
oxygen free radicals lead to photosensitivity.

Classifications of Porphyrias

*I-According to organs that are mostly affected:
A-Erythropoietic porphyrias: heme synthesis in the bone marrow was affected
as in congenital erythropoietic porphyria & protoporphyria.
B-Hepatic  porphyrias:  heme  synthesis  in  the  liver  was  affected  as  in  ALA
dehydratase  deficient  porphyria,  acute  intermittent  porphyria,  porphyria  cutanea
tarda, hereditary coproporphyria & variegates porphyria.
*II-According to clinical presentations:
A-Acute porphyrias: Patient has abdominal pain & neuropsychiatric symptoms.
B-Cutaneous porphyrias: Patient has photosensitivity.
C-Latent porphyrias: Patient is relatively symptoms-free between the attacks.
*III-According to cause:
A-Genetic  porphyrias:  Inherited  as  autosomal  dominant  except  congenital
erythropoietic porphyria which inherited as autosomal recessive.
B-Acquired porphyrias: as in lead poisoning can affect ALA dehydratase.

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Diagnosis of Porphyrias

1-Clinical & family history.
2-Physical examination.
3-Laboratory tests include:
  A-Measure enzyme activity in blood.
  B-Urine test for uroporphyrins & coproporphyrins.
  C-Stool test for protoporphyrins.
  D-Urine test for ALA & PBG.

Treatment of Porphyrias

1-Gene therapy.
2-Avoid porphyrogenic drugs as barbiturates.
3- Administration of excess glucose & hematin that cause block of ALAS1.
4-Administration  of  β-carotene  which  is  antioxidant  to  reduce  oxygen  free
radicals formation, therefore, reduces photosensitivity.
5-Use  of  sunscreen  that  filter  out  visible  light  preventing  the  exposure  of  light
with wave length 400 nm.
6-Sever pain is treated by analgesia.

Heme Catabolism

   Normally,  senescent  red  blood  cells  &  heme  from  other  sources  are  engulfed
by the cells of the reticuloendothelial system.
-The globins is recycled or converted into amino acids.
-Heme is converted into biliverdin  ((green pigment)) with the release of ferric
iron & CO ((only reaction in the body that is known to produce CO)).
  Finally  by  the  action  of  biliverdin  reductase  biliverdin  was  converted  into
bilirubin ((yellow pigment)) which transported to liver for further metabolism.
The  daily  bilirubin  formation  in  human  adult  is  about  250-350  mg,  derived
mainly from hemoglobin.

Bilirubin

Transport of Bilirubin

   Bilirubin is mildly soluble in aqueous media (as blood), however, its solubility
is increased by binding to serum albumin.
Each molecule of albumin has one high-affinity site & one low-affinity site for
bilirubin binding. In 100 mL of blood, approximately 20-25 mg of bilirubin can
be  tightly  bound  to  albumin  at  its  high-affinity  site,  bilirubin  in  excess  of  this
quantity  can  be  bound  only  loosely  on  low-affinity  site  &  thus  can  easily  be
detached & diffuse into tissues specially CNS causing kernicterus.

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A number of drugs such as antibiotics compete with bilirubin for the high-affinity
binding site on albumin. Thus, these compounds can displace bilirubin from
albumin & so it has significant clinical effects.

Metabolism of Bilirubin

The metabolism of bilirubin occurs through the following sequential steps:
(1) Uptake of Bilirubin.
(2) Conjugation of Bilirubin.
(3) Secretion of Bilirubin into Bile.

(1) Uptake of Bilirubin

In the liver, bilirubin is removed from albumin & taken up by hepatocytes.

(2) Conjugation of Bilirubin

Bilirubin is nonpolar & would persist in liver cells if not rendered water-
soluble. Hepatocytes convert bilirubin to a polar form, which is readily excreted
in the bile, by adding glucuronic acid molecules to it, this process is called
conjugation

that

catalyzed

specific

enzyme

called

UDP-

glucuronosyltransferase (uridine diphosphate-glucuronosyltransferase) or
called

Bilirubin-UGT

(bilirubin-uridine

diphosphate

glucuronosyltransferase),  this  enzyme  transfer  glucuronate  from  UDP-
glucuronic  acid  to  bilirubin  forming  bilirubin  monoglucuronide  which  is  an
intermediate & is subsequently converted to the bilirubin diglucuronide.
   UDP-glucuronosyltransferase  enzyme  activity  can  be  induced  by  a  number  of
clinically useful drugs as phenobarbital.

(3) Secretion of Bilirubin into Bile

  Secretion  of  conjugated  bilirubin  into  the  bile  is  an  active  process  &  it  is
probably the rate-limiting for hepatic bilirubin metabolism.
    The  hepatic  transport  of  conjugated  bilirubin  into  the  bile  is  induced  by  the
same drugs that are capable of inducing the conjugation of bilirubin.

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As  the  conjugated  bilirubin  reaches  the  terminal  ileum  &  large  intestine,  it
converted  by  intestinal  bacterial  flora  to  a  group  of  colorless  tetrapyrrolic
compounds called urobilinogen.
  In  terminal  ileum  &  large  intestine  a  small  fraction  of  urobilinogen  is
reabsorbed  &  reexcreted  by  liver  to  constitute  the  enterohepatic  urobilinogen
cycle while only very trace amount of urobilinogen appears in urine.

Normally, most of the colorless urobilinogen in the colon (in GIT urobilinogen

is called stercobilinogen) are oxidized to a colored compound known as urobilin
(in GIT urobilin is called stercobilin) & are excreted in the feces giving the
brown color of stool.

Reference Ranges of Serum Bilirubin

In adult, the reference ranges of serum bilirubin are:
Total Bilirubin = 0.3-1 mg/dL (5.1-17 µmol/L)

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Unconjugated (Indirect) Bilirubin = 0.2-0.7 mg/dL (3.4 -12 µmol/L)
Conjugated (Direct) Bilirubin = 0.1-0.3 mg/dL (1.7 - 5.1 µmol/L)
However, in newborn, the reference ranges is usually higher than adult.

Hyperbilirubinemia

   Hyperbilirubinemia exists when serum total bilirubin concentration exceeds the
upper  limit  of  its  reference  range  [1  mg/dL  (17µmol/L)]  &  when  it  reaches  a
certain  concentration  (approximately  2.5-3  mg/dL);  it  diffuses  into  the  tissues,
which then become yellow. This condition is called jaundice or icterus.
Hyperbilirubinemia may be due to:-
1-  Overproduction  of  bilirubin  above  the  capacity  of  the  normal  liver  to
metabolize it (Pre-hepatic hyperbilirubinemia) as in hemolysis.
2- Failure of a damaged liver to conjugate bilirubin produced in normal amounts
(Hepatic hyperbilirubinemia).
3- Obstruction of the excretory ducts of the liver that preventing the excretion of
bilirubin (Post-hepatic hyperbilirubinemia).
Depending  on  the  type  of  bilirubin  present  in  plasma  whether  unconjugated
(indirect) and/or conjugated (direct), hyperbilirubinemia may be classified as:
1-  Unconjugated  Hyperbilirubinemia  (Retention  Hyperbilirubinemia):

Due

to overproduction of unconjugated bilirubin lead to:
A-  Elevated  level  of  serum  unconjugated  bilirubin  (Indirect  bilirubin)  above  its
upper reference limit (in adult; 0.7 mg/dL).
B-  Since  unconjugated  bilirubin  is  hydrophobic,  it  can  cross  the  blood-brain
barrier into CNS (especially when serum unconjugated bilirubin level reach 20-
25 mg/dL) causing encephalopathy, this condition called kernicterus.
C-  Since  unconjugated  bilirubin  is  hydrophobic,  it  does  not  appear  in  urine.
Accordingly, acholuric jaundice (absence of bilirubin in the urine) occurs.
D-  The  increased  production  of  bilirubin  leads  to  increased  production  of
urobilinogen which appears in the urine in large amounts (excess urobilinoginuria)
while normally there are very traces amount of urobilinogen in the urine.
2-Conjugated Hyperbilirubinemia (Regurgitation Hyperbilirubinemia): Due
to reflux of conjugated bilirubin into the bloodstream lead to:
A- Elevated level of serum conjugated bilirubin (Direct bilirubin) above its upper
reference limit (in adult; 0.3 mg/dL).
B-  Since  conjugated  bilirubin  is  hydrophilic,  it  cannot  cross  the  blood-brain
barrier so that there is no development of kernicterus.
C- Since conjugated bilirubin is hydrophilic & cannot be excreted (due to biliary
obstruction). It thus regurgitates from the liver & appears in urine. Accordingly,
choluric jaundice (presence of bilirubin in the urine) occurs, clinically the color
of choluric urine is described as a tea-color.
D- Since bilirubin has no access to the intestine (due to biliary obstruction) where
it can be converted to urobilinogen, therefore, no urobilinogen (stercobilinogen)

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is found in urine & feces, therefore, no urobilin (stercobilin) in the stool, the stool
is clinically described as pale-stool.
3- Unconjugated & conjugated hyperbilirubinemia: Due to defect in both
conjugation & secretion of bilirubin as occur mainly in hepatitis.

Examples of Unconjugated Hyperbilirubinemia

1)- Neonatal (Physiologic) Jaundice.
2)- Hemolytic Anemias.
3) - Type I Crigler-Najjar Syndrome.
4)- Type II Crigler-Najjar Syndrome.
5)-Gilbert Syndrome.
6)-Toxic Hyperbilirubinemia.

Examples of Conjugated Hyperbilirubinemia:

1)-Obstruction of Biliary Tree (Cholestatic Jaundice).
2)-Dubin-Johnson Syndrome.
3)-Rotor Syndrome.

Neonatal (Physiologic) Jaundice

  This  transient  condition  is  the  most  common  cause  of  unconjugated
hyperbilirubinemia  in  neonate.  It  results  from  an  accelerated  hemolysis  around
the time  of  birth & an immature hepatic system for  bilirubin  metabolism. Since
the increased amount of bilirubin is unconjugated, it's capable of penetrating the
blood-brain  barrier  when  its  concentration  in  plasma  exceeds  that  which  can  be
tightly bound by albumin (20–25 mg/dL) causing kernicterus.
Treatment
1- Phenobarbital administration because it induce bilirubin-metabolizing system.
2-  Exposure  to  blue  light  (phototherapy)  promotes  hepatic  excretion  of
unconjugated bilirubin.
3- Exchange blood transfusion when plasma bilirubin exceeds that which can be
tightly bound by albumin (20–25 mg/dL) to avoid kernicterus.