Pakistan Society Of Chemical Pathologists

Distance Learning Programme In Chemical Pathology

(DLP-2)

Lesson No 12
Porphyrias- “Made Easy”
By

Brig Aamir Ijaz

MCPS, FCPS, FRCP (Edin)
 Haem in the Body

• Heme is mostly used for its incorporation into haemoglobin and

role in red blood cells.
• But is also needed for cytochrome P450 function.
• Other uses of Haem include:
• Catalase
• Myoglobin
• Nitric oxide synthase
 Introduction
• Porphyrias is a set of diseases that result
from enzyme deficiencies in the heme
synthesis pathway.
• Each disease is associated with a deficiency
in one of the seven enzymes in the pathway
Seven Enzymes – Seven Disorders
 Free Radicals –
The real Culprits
 The respective enzymatic deficiencies lead to the accumulation of porphyrins
and porphyrin precursors that ultimately produce free radicals.
 For acute hepatic porphyrias, delta-aminolaevulinic acid (ALA) and
porphobilinogen (PBG) accumulate and produce free radicals via
autoxidation.
 For erythropoietic porphyrias, uroporphyrins, coproporphyrins, and
protoporphyrins accumulate and produce free radicals via the absorption of
visible light.
 The generated free radicals participate in oxidative stress reactions, such as
lipid oxidation and protein crosslinking, that lead to membrane and
mitochondrial damage, ultimately promoting cell death.
 Loss of negative feedback of heme leads to further accumulation of
porphyrins.
 Genetics
Autosomal Dominant
• AIP
• HCP
• VP
• EPP

Autosomal recessive
• ADP
• CEP

Acquired
• PCT
 Environmental Factors
• Due to incomplete penetrance, inheritance of an enzyme
deficiency of an autosomal dominant porphyria does not
necessarily lead to clinical symptoms.
• Symptoms of acute porphyrias tend to come in the form
of "attacks" which may be induced by other genetic
factors or environmental factors, such as agents that
promote porphyrin and porphyrin precursor synthesis
and/or agents that induce cytochrome P450s.
 Part I
Short Answer Questions:
Q. 1: Porphyria's are usually classified as ‘Acute and Non-acute’,
‘With or Without cutaneous lesions’, pattern of inheritance,
‘congenital or acquired’. Please write name(s) of porphyrias
falling in following five categories. Please write only the names of
the disorders and NOT the names of effected enzymes (one mark
each):
a. Acute (neurovisceral) without any cutaneous lesions
b. Acute (neurovisceral) with blistering cutaneous lesions
c. Non-acute (non-neurovisceral) with blistering cutaneous lesions
d. Acute photosensitivity and non-blistering lesions
e. Acquired

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 Classification of Porphyrias
a. Acute (neurovisceral) without any cutaneous lesions
• Acute intermittent porphyria (AIP)
• ALA dehydratase deficiency porphyria (ALADP)
b. Acute (neurovisceral) with blistering cutaneous lesions
• Variegate porphyria (VP)
• Hereditary coproporphyria (HCP)
c. Non-acute (non-neurovisceral) with blistering cutaneous lesions
• Porphyria cutanea tarda (PCT)
• Hepatic erythropoietic porphyria (HEP)
• Congenital erythropoietic porphyria (CEP)
d. Acute photosensitivity and non-blistering lesions
• Erythropoietic porphyria (EPP)
e. Acquired
• Porphyria cutanea tarda (PCT) 10
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Types of Porphyrias
Slides courtesy of Dr Shahnaz Noor, QAMC BWP
 Types of Porphyrias
Cutaneous
• Bullous lesions
 PCT & HEP
 CEP
• Non-bullous Lesions
 EPP
Acute Presentation (Hepatic Porphyrias):
 AIP
 ADP

Mixed Disorders (Acute and Cutaneous manifestations)

 VP
 HCP
 Symptoms of Cutaneous Forms
 Occur most commonly with exposure to sunlight
 Mainly skin symptoms that occur
 Due to excess porphyrins that accumulate in surface of
skin
• Symptoms:
• Fluid filled blisters
• Changes in pigmentation
• Breakdown (necrosis) of the skin
when exposed to sunlight
• Overall skin can become scarred,
brown, blotchy and fragile
Q. 2: Porphyria's cannot be diagnosed without intelligent use of laboratory investigations. Please
answer following questions regarding appropriate use of laboratory tests helpful in various clinical
situations (one mark each):
a. A 25 years female complains of abdominal pain for the last 24 hours. Her blood counts
(CBC) and abdominal ultrasound are normal. She has been referred to you for the exclusion of a
porphyria. Name TWO laboratory tests which will be most helpful in reaching the diagnosis of a
porphyria and how will you interpret these tests?
b. What patient safety issues can arise if a false negative or false positive test result is given
in the female patient mentioned in 2 (a) above.
C. A 30 year old male has bullous skin lesions which have erupted gradually over a period of
one week. His biochemical findings were:
ALT: 64 U/L
Ferritin: 330 ng/ml
HFE gene mutation: Positive
Which porphyria comes to your mind in this patient and how will you rule out
/diagnose this porphyria by using appropriate laboratory test(s)?

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Q. 2: a.A 25 years female complains of abdominal pain for the last
24 hours. Her blood counts (CBC) and abdominal ultrasound are
normal. She has been referred to you for the exclusion of a
porphyria. Name TWO laboratory tests which will be most helpful
in reaching the diagnosis of a porphyria and how will you interpret
these tests?
Urinary porphobilinogen (PBG) measurement
o High levels are suggestive of acute porphyria (AIP, VP,
HCP)
Urinary δ aminolaevulinic acid (ALA) measurement (and repeat
PBG) on same urine sample
o In symptomatic patient with normal PBG, raised ALA
levels are seen in ALADP, a very rare form of acute porphyrias
and lead toxicity
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Q. 2: b. What patient safety issues can arise if a false negative
or false positive test result is given in the female patient
mentioned in 2 (a) above.

Consequences of false positive result

• Delay in the life saving treatment appropriate for
patient’s actual diagnosis
• Analgesic misuse and dependency
Consequences of false negative result
• Administration of porphyrinogenic drugs that will
aggravate the attack and may increase fatality
• Unnecessary surgery e.g. laparotomy for acute
abdomen not only have its own risks but also deteriorate the
acute porphyria attack 17
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Q. 2: c.A 30 year old male has bullous skin lesions which have
erupted gradually over a period of one week. His biochemical
findings were:
• ALT: 64 U/L
• Ferritin: 330 ng/ml
• HFE gene mutation: Positive
Which porphyria comes to your mind in this
patient and how will you rule out /diagnose this porphyria by
using appropriate laboratory test(s)?

See answers on next slides

Porphyria Cutanea Tarda because of following
points
o Nonacute porphyria
o Bullous skin lesions
o Male predominance
o Raised ALT provides evidence of
hepatocellular damage
o HFE gene mutation has strong positive
association with PCT
Laboratory investigations
o Urine porphyrins – markedly increased
- Chromatographic separation based on carboxyl number reveals
predominance of 8- and 7-carboxyl porphyrin fractions, with lesser amounts
of 6-, 5-, and 4-carboxyl porphyrins, reflecting uroporphyrinogen
decarboxylase (UROD) defect.
o Serum or plasma porphyrins- also show increased polycarboxylated
porphyrins
o Fecal porphyrins – raised. Fractionation reveals complex pattern
with increased isocoproporphyrin
o Urine ALA and PBG – normal which exclude all acute porphyrias
o Erythrocyte porphyrins – within reference range
o Plasma fluorescence peaks at 620 nm
o UROD enzyme activity in red blood cells- markedly reduced and is
diagnostic of PCT
Q. 3: Some tests of porphyrias are essential in any lab for
the diagnosis of these disorders. Please answer following
questions regarding porphyria related tests (one mark
each):
a. How do you test Porphobilinogen in urine and
what precautions you will adopt in collection of urine
sample?
b. Write TWO lines about any ONE method used for
analysis of porphyrins in plasma.

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 Q 3a.Porphobilinogen tests in urine

Qualitative or semi quantitative method

screening method
formation of red colored PBG-Ehrlich compound
on addition of Ehrlich reagent to urine that can be
separated by solvent extraction
rapid, cheap and still widely used but has poor
sensitivity and specificity
 Most important interference: Urobilinogen
 Q 3a.Porphobilinogen tests in urine
(Cont)
Quantitative method
 Preferred method for measurement of PBG in urine
 Spectrophotometric quantification of the red product formed by PBG
reaction with 4-dimethylaminobenzaldehyde in acid (Ehrlich’s reagent)
at 550 nm after removal of urobilinogen and other interfering substances
by anion exchange chromatography. Results should be expressed as
µmol/mmol creatinine.
HPLC
Tandem mass spectrometry
• Sensitive and accurate but time consuming
 Q 3a.Porphobilinogen tests in urine
(Cont)

Specimen collection for urinary porphobilinogen

• Best specimen is freshly voided spot urine preferably early
morning, 10-20ml without any preservative
• Must be collected in colored bottle and kept well protected
from light
• If any delay in transport or analysis and after analysis,
specimen must be kept at 4oC for 48 h and at -20oC for 1
month.
b. Write TWO lines about any ONE method used for
analysis of porphyrins in plasma.

Fluorescence emission spectroscopy

• It utilizes the property of porphyrins to fluoresce at
neutral pH between 610-640 nm depending upon their
structure and protein complex
• Simple method that can differentiate among
various cutaneous porphyrias

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 Part II
MCQs (One Best Type)
Q. 4 Heme is an important prosthetic group in many
important substances of the body. Which of the
following tissues / organs are the most active site of
heme synthesis:

a. Bone marrow
b. Kidneys
c. Liver
d. Lungs
e. Muscles

a. Bone marrow
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Biosynthesis of Heme
Slides courtesy of Dr Anwar Magsi, PNS SHIFA Karachi
 Biosynthesis of Heme

• Synthesized in every human

cell
• Liver (15%):
• Bone Marrow (80%)
Q 5. Two different delta aminolaevulinic acid
synthase (ALAS) enzyme systems have been
found. Only ONE of the following features is
common in ALAS1 and ALAS2 enzymes :
a. Down-regulation by heme
b. First step in the synthesis of heme
c. Liver is the major location
d. Mutations have been described in the genes coding
these enzymes
e. Up-regulation by iron
b. First step in the synthesis of heme30
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 Two Types of ALAS

ALAS-1
• Present in liver and all other tissues for
production of haem used for non-haemoglobin
substances e.g. Cytochrome P450
ALAS-2
• Present in erythrocytes for production of
Haemoglobin
Q 6. The most common human porphyria is:

a. Acute intermittent porphyria

b. ALA dehydratase porphyria
c. Congenital erythropoietic porphyria
d. Porphyria cutanea tarda
e. Hereditary coproporphyria

.
d. Porphyria cutanea tarda 32
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 Two Most Important Disorders

• Acute intermittent porphyria (AIP) ---

Most common acute
• Porphyria Cutanea Tarda (PCT)-------
The commonest
Acute Intermittent Porphyria (AIP)
 Acute intermittent porphyria (AIP)
 2nd most common form of porphyria
 Caused by deficiency of PGB deaminase
 Metabolite porphobilinogen accumulates in cytoplasm
 Symptoms:
• Localized abdominal pain
• Urinary symptoms
• Peripheral neuropathy
• raised concentration of urinary porphyrins
 Treatment
• Haematin, Heme arginate
 Do not cure but reduces symptoms
 Inhibit ALA synthase which occurs at the beginning of heme biosynthesis
 A Supplementary Question
The commonest presenting clinical feature of Acute Intermittent Porphyria is:

Best answer:
a. Abdominal pain
b. Dark urine
c. Muscular weakness
d. Severe constipation
e. Tachycardia

a.Abdominal pain
 Symptoms of Acute Forms
 Originate mainly in nervous system
 Symptoms last around 1-2 weeks
 Possible mechanisms include damage by free radicals, direct neurotoxicity of ALA, and
the deficiency in nervous tissue

Symptoms:
 Severe abdominal pain
 Muscle weakness and pain, tingling, or numbness and possibly paralysis
 Pain in arms, legs, back
 Constipation
 Vomiting
 Diarrhea
 Insomnia
 Seizures and Confusion
 Anxiety and paranoia
 Fever
Q 7: Poisoning of which of the following metals
resembles acute porphyria due to accumulation of ALA:

a. Arsenic
b. Copper
c. Iron
d. Lead
e. Zinc
d. Lead 38
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 Lead Toxicity and Porphyrias

• Lead exposure causes increased ALA

due to replacement of Zn by Lead in the
enzyme.
• The condition resembles ALADP
• Also called ‘plumbporphyria’
Q 8: A 31 year female doctor has recently been diagnosed as a
patient of Acute Intermittent Porphyria. She shows you a list of
factors she has downloaded from internet that causes
precipitation of an acute attack. The list is given below, which of
these factors she has wrongly noted:

a. Ethanol
b. High carbohydrate diet
c. Non-steroidal anti-inflammatory drugs
d. Oral contraceptive pills
e. Smoking

b. High carbohydrate diet 40

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 Treatment for Acute Forms
Carbohydrate such as glucose
To help limit the synthesis of porphyrins

Phlebotomy
To reduce excessive iron stores which improves heme synthesis

Sedatives to help with anxiety

Pain medications such as opiates
Haem arginine
Haem inhibit ALA synthase and the accumulation of toxic precursors
Q 9 : In the 1950s, in eastern Turkey an outbreak of
Porphyria Cutanea Tarda (PCT) occurred in thousands
of adults and children after consumption of stored wheat
to fight famine. This outbreak of PCT was a result of:

a. Long exposure to sun during queues for wheat

b. Periods of prolonged starvation due to famine
c. Poor hygiene of the dwellers
d. Treatment of wheat with a fungicide
e. Tuberculosis due to malnutrition

d. Treatment of wheat with a

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Q 10: PCT has been found to be associated with which
of the following viral infections:

a. Cytomegalovirus
b. Ebola virus
c. Epstein Barr virus
d. Hepatitis C virus
e. Human papilloma virus

d. Hepatitis C virus 43
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Q 11:An 18 months infant has been referred to you for the investigation of porphyria. His mother describes that
whenever she takes his son in the sun, his face and back of hands immediately get red and the child starts crying
with pain. On examination you find no lesions on the face or hands, but his Serum AST levels are 2 folds
increased.
His laboratory investigations related to porphyrias carried out in your lab shows following results:

• Urine Porphyrin 25.0 nmol/mmol creat (0 - 35 )

• Urine PBG 0.8 µmol/mmol creat (0 - 1.5 )
• Urine ALA 1.2 µmol /mmol creat (0 - 3.8 )
• Urine Creatinine 4.2 mmol/L
• Urinary porphyrin fractions: Not increased
• Plasma fluorescence peak at 630 nm
• Erythrocyte total protoporphyrins: markedly increased
• Erythrocyte metal-free protoporphyria:
Markedly increased (92% of total Erythrocytic protoporphyrins)
• Erythrocyte Zinc Protoporphyrins:
Marginally increased (8% of total Erythrocytic protoporphyrins)
• Gene Mutation Studies: Ferrochelatase gene mutation detected
What is the most probable diagnosis in this patient?
a. Erythropoietic protoporphyria
b. Hepatoerythropoietic porphyria
c. Lead poisoning
d. Porphyria cutanea tarda
e. X-linked protoporphyria

a. Erythropoietic protoporphyria44
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Erythropoietic Protoporphyria (EPP)
 Erythropoietic Protoporphyria

Caused by deficiency of Ferrochelatase

Autosomal dominant
Photosensitivity- can be managed by limiting exposure
neuropathic pain and neuropathy. His urine tests showed following results:

• Total Porphyrin 64.0 nmol/mmol creat (0 - 35 )

• Porphobilinogen 0.8 µmol/mmol creat (0 - 1.5 )

• Delta-Aminolaevulinic Acid 7.2 µmol /mmol creat (0 - 3.8 )

• Creatinine 3.9 mmol/L

• Plasma fluorescence peak at 620 nm
What is the most probable diagnosis in this patient?

a. Acute intermittent porphyria

b. Aminolaevulinic acid dehydratase porphyria
c. Congenital erythropoietic porphyria
d. Erythropoietic protoporphyria
e. Hepatoerythropoietic porphyria

b. Aminolaevulinic acid dehydratase 47

porphyria 04/28/2024
ALA Dehydratase Deficiency Porphyria (ALADP)
Q 13: A 3 month infant presents with blisters on the skin and photosensitivity. Mother also tells about
reddish-brown coloration of the diapers of the infant. He also has hemolytic anaemia and splenomegaly
His laboratory investigations showed following results:

• Urine Total Porphyrin 71.0 nmol/mmol creat (0 - 35 )

• Urine PBG 0.68 µmol/mmol creat (0 - 1.5 )
• Urine ALA 1.9 µmol /mmol creat (0 - 3.8 )
• Urine Creatinine 3.1 mmol/L
• Plasma fluorescence peak at 618 nm
• Urinary porphyrin fractions:
o Uroporphyrin I : Increased
o Coproporphyrins I: Increased
• Erythrocyte total protoporphyrins: markedly increased
• Erythrocyte metal-free protoporphyria:
Markedly increased (22% of total Erythrocytic protoporphyrins)
• Erythrocyte Zinc Protoporphyrins:
Marginally increased (68% of total Erythrocytic protoporphyrins)
• Gene Mutation Studies: UROS gene mutation detected
What is the most probable diagnosis in this patient?
a. Congenital erythropoietic porphyria
b. Hepatoerythropoietic porphyria
c. Hereditary coproporphyria
d. Porphyria cutanea tarda
e. Variegate porphyria
a. Congenital erythropoietic 49
porphyria 04/28/2024
Congenital Erythropoietic Porphyria (CEP)
Congenital erythropoietic porphyria (CEP)

• Deficiency of Uroporphyrinogen III synthase

• Rare autosomal recessive (1 in 1,000,000)
• Severe photosensitivity
Other Porphyrias
Hereditary Coproporphyria (HCP)
 HCP
• Deficiency of Coproporphyrinogen III Oxidase
• Autosomal dominant
• No cure exists
Variegate Porphyria (VP)
 VP
• Deficiency in protoporphyrinogen IX-oxidase
• Autosomal dominant
Inside Erythrocytes
(Important for Haematologists, too !!!)
Sideroblastic Anaemias and
Erythropoietic Porphyrias

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Algorithms of Porphyrias
 63
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 64
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 65
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 66
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• ALA-1 is inhibited by haematin not two
• ALA-2--def—X-linked erythropoietic protoporphyria—other name---
sideroblastic anemia
• ALA-1– is not associated with any disease
• All pt come-----dermatology only AIP --from surgical or medical
• Sample requirement
• ALA---urine---acidic ph req
• PBG----alkaine 8-9
• PCT---spordic type1-(acquired –liver-only have positive UROD def)and
type2-familial(UROD def in all tissue)
• Long term complication of acute porphyria---CRF. Hepatcellular ca, HTN
• Main role of heme in liver-----cytochrome p450
• AIP---ALL TEST NORMAL AND PAIN RELIEVED WITH SWEET---
DIAGNOSTIC
• CEP----Congenital ,child had teeth gold and brownish
• Initial part of manegement----Family screening of metabolite,enzyme
level ,DNA analysis, gene tracking
• Differential----Hereditary tyrosinemia,lead poisoning,heaptobiliary
disorder, pseudoporphyria, hemological, GIT 67
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 72
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Thank You and Best Of Luck