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molecular studies of ADP patients - nine point mutations,two splice-site mutations, and a two-base deletion in the ALAD gene .
Prenatal diagnosis -determination of ALA dehydratase activity and/or gene mutations in cultured chorionic villi or amniocytes.
In patients with no history of acute symptoms,porphyrin precursor excretion is usually normal, suggesting that half normal hepatic HMB synthase activity is sufficient and that hepatic ALA synthase activity is not increased. under conditions where heme synthesis is increased in the liver, half-normal HMB synthase activity may become limiting, and ALA, PBG, and other heme pathway intermediates may accumulate and be excreted in the urine.
(clonazepam may be safer than phenytoin or barbiturates).
Patients with HMBS mutations in the initiation of translation codon in exon 1 and in the intron 15′-splice donor site have normal enzyme levels in erythrocytes and deficient activity only in nonerythroid tissues. This occurs because the erythroid and housekeeping forms of HMB synthase are encoded by a single gene, which has two promoters. Thus, the enzyme assay may not be diagnostic, and genetic testing should be used to confirm the diagnosis. More than 390 HMBS mutations have been identified in AIP, including missense, nonsense, and splicing mutations and insertions and deletions, with most mutations found in only one or a few familiESThe prenatal diagnosis of a fetus at risk can be made with cultured amniotic cells or chorionic villi. However, this is seldom done, because the prognosis of
The primary source of excess protoporphyrin is the bone marrow reticulocytes. Erythrocyte protoporphyrin is free (not complexed with zinc) and is mostly bound to hemoglobin. In plasma, protoporphyrin is bound to albumin. Hemolysis and anemia are usually absent or mild.
Fast atom bombardment mass spectrometry and laser desorption/ionization
Time-of-flight mass spectrometry .
Porphyrias and lab diagnosis
GUIDED BY -DR.KAMAL MALUKANI
PRESENTED BY- DR.GARGI TIGNATH
Cruelly referred to as a Vampire’s disease. Thought to be a
cause of the madness of King George III.
Can be caused by lead poisoning: The fall of the Roman
Some symptoms of porphyrias have lead
people to believe that these diseases provide
some basis for vampire legends:
Extreme sensitivity to sunlight
This idea has been discarded both for scientific
Porphyrias do not cause a craving for blood.
Drinking blood would not help a victim of
COORDINATED REGULATION OF HEME
AND GLOBIN SYNTHESIS:
inhibits activity of pre-existing -ALA synthase
Diminishes the transport of -ALA synthase from
cytoplasm to mitochondria after synthesis of the
represses the production of -ALA synthase by
regulating gene transcription.
stimulates globin synthesis to ensure that levels of
free heme remain low in concentration.
Inhibition of the synthase and stimulation of globin
synthesis are the most important aspects in
balancing hemoglobin production.
The term porphyria in greek meaning purple discolouration of
some body fluids during attack.
The porphyrias are a group of diseases resulting from defects
in the synthesis of heme.
Inherited enzyme deficiencies in which the enzyme substrate
is usually excreted in excess in urine and/or feces.
During acute attacks, high levels of porphobilinogen are
excreted, but between attacks levels of porphobilinogen may
be increased or normal.
Heme is mainly required in bone marrow(for hemeglobin synthesis)
and in liver (for cytocrome synthesis).
On the basis of that porphyrias are divided into Erythropoietic
porphyrias and Hepatic porphyrias.
Hepatic porphyrias mainly affects nervous system,while
erythropoietic porphyrias primarily affects-skin.
ALA DEHYDRATASE-DEFICIENT PORPHYRIA (ADP)
Rare, autosomal recessive .
Caused by -severe deficiency of ALA dehydratase activity
Age- children or young adults
specific gene mutations ,affected homozygotes have <10% of
normal ALA dehydratase activity in erythrocytes.
As there are multiple causes for deficient ALA dehydratase activity, It
is important to confirm the diagnosis of ADP by mutation analysis.
clinical presentation depends on the amount of residual ALA
commonly affects- male adolescents
symptoms resembling those of AIP-abdominal pain and
Infants–presents with more severe disease, including failure
to thrive beginning at birth.
Earlier age of onset & more severe manifestations -reflects
significant deficiency of ALA dehydratase activity.
significantly elevated levels of
-plasma and urinary ALA
-urinary coproporphyrin (COPRO) III.
ALAD activities in erythrocytes were <10% of normal.
Differential diagnosis –
1.Hereditary tyrosinemia type1
(fumarylacetoacetase deficiency-succinylacetone-(which accumulates
in hereditary tyrosinemia is structurally similar to ALA)
2. Lead poisoning
lead inhibits ALA dehydratase- ↑ urinary excretion of ALA and
COPRO III-cause manifestations resembling acute porphyrias.
Heterozygotes are clinically asymptomatic , do not excrete
increased levels of ALA can be detected by-
Intermediate levels of erythrocyte ALA dehydratase activity or
specific mutation in the ALAD gene.
Acute attacks is similar to that of AIP
severely affected infants- supported by hyperalimentation.
periodic blood transfusions
Acute Intermittent Porphyria (AIP)
AD diseases resulting from the half-normal level of HMB synthase
Disease is widespread
Clinical expression is highly variable
Activation of the disease is often related to environmental or hormonal
Common precipitating factors include:
1.Endogenous and exogenous steroids
4.Low-calorie diets, usually instituted for weight loss
Attacks can be prevented by avoiding known precipitating factors.
Induction of the rate-limiting hepatic enzyme ALA synthase in
heterozygotes with half-normal HMB synthase activity
AIP almost always latent before puberty –suggests adult levels of
steroid hormones are important for clinical expression.
Heterozygous for HMBS mutations, causes for attacks of AIP prior to
Symptoms are more common in women, suggesting a role for estrogens
Premenstrual attacks are probably due to endogenous
Exacerbated by exogenous steroids, including OCP preparations
pregnancy ,usually well tolerated,suggesting -beneficial metabolic
changes - ameliorates effects of high levels of progesterone.
• peroxisome proliferator-activated
receptor γ coactivator 1α (PGC-1α).
• Increased ALAS1 transcription,
• Increased heme biosynthesis-
• Increased Porphyrins synthesis
Important link between nutritional status and the attacks in acute porphyrias.
Increased carbohydrate intake may ameliorate attacks
Neurovisceral symptoms rarely occur before puberty
Abdominal pain, the most common symptom, is usually steady and
poorly localized but may be cramping.
Ileus , abdominal distention & decreased bowel sounds are common.
Fever, and leukocytosis are usually absent or mild (symptoms are
neurologic rather than inflammatory).
Nausea; vomiting; constipation
Tachycardia; hypertension – sympathetic overactivity
Pain in the limbs, head,neck or chest
Muscle weakness – proximal muscles
Sensory loss- axonal degeneration
Dysuria and urinary incontinence
Progression to respiratory and bulbar paralysis and death occurs
especially when the diagnosis and treatment are delayed.
Sudden death-results from sympathetic overactivity and cardiac
Treatment of seizures is difficult because most antiseizure drugs can
When an attack resolves-
Abdominal pain may disappear within hours
paresis begins to improve within days and may continue to improve over
Urine and Plasma ALA and PBG levels - are substantially ↑during acute
attacks, become normal only after prolonged latency.
[ Normal urinary PBG excretion-0–4 mg/24 h;50-150 μmol/24 hrs]
[ Normal urinary ALA excretion -1–7 mg/24 h ;8–53 μmol/24 h].
Diagnosis of an acute attack in a patient with biochemically proven AIP
is based primarily on clinical features.
Excretion of ALA and PBG decreases over a few days after intravenous
No history of symptoms -have normal urinary excretion of ALA
1.Detection of the family’s HMBS mutation will diagnose
asymptomatic family members.
2.Prognosis of individuals with HMBS mutations is generally
Fecal porphyrins are usually normal or minimally
increased in AIP, in contrast to HCP and VP.
HEREDITARY COPROPORPHYRIA (HCP)
AD,due to half-normal activity of COPRO oxidase.
Disease presents with acute attacks, as in AIP.
Cutaneous photosensitivity also may occur but much less commonly
than in VP.
Acute attacks and cutaneous photosensitivity may occur together or
HCP is less common than AIP and VP.
HCP is influenced by the same factors that cause attacks in
The disease is latent before puberty, and symptoms,which are
virtually identical to those of AIP, are more common in
HCP is generally less severe than AIP.
Blistering skin lesions are identical to PCT and VP and begin in
childhood in rare homozygous cases.
COPRO III is markedly ↑ in the urine and feces in symptomatic
Often persists, especially in feces, when there are no symptoms.
Urinary ALA and PBG levels ↑ during acute attacks, but may revert to
normal when symptoms resolve.
Plasma porphyrinsare usually N or only slightly ↑- in cases with skin
Diagnosis of HCP -confirmed by ↑ fecal porphyrins (consisting almost
entirely of COPRO III,distinguishes it from other porphyrias).
Neurologic symptoms are treated as in AIP .
Phlebotomy and chloroquine are not effective for the cutaneous
VARIEGATE PORPHYRIA (VP)
AD, due to deficient activity of PROTO oxidase, seventh enzyme in the
heme biosynthetic pathway.
Presents with neurologic symptoms, photosensitivity, or both.
VP is particularly common in South Africa, 3 of every 1000 whites have
skin photosensitivity, acute neurovisceral crises, or both.
Acute attacks identical to those in AIP , precipitated by same
factors as AIP
Blistering skin lesions similar to PCT, but more difficult to treat ,
usually of longer duration.
Homozygous VP :
Associated with photosensitivity
Developmental disturbances, including growth retardation, in
infancy or childhood;
↑Urinary ALA and PBG during acute attacks, but return to normal more
quickly than in AIP.
↑ fecal protoporphyrin and COPRO III
↑ urinary COPRO III are more persistent.
Plasma porphyrin levels also ↑,particularly with cutaneous lesions.
VP can be distinguished rapidly from all other porphyrias by examining the
fluorescence emission spectrum of porphyrins in plasma –
VP has a unique fluorescence peak at neutral pH.
Increased erythrocyte levels of zinc protoporphyrin, characteristic finding in all
TREATMENT -Variegate Porphyria
Acute attacks are treated as in AIP, and hemin should be started
early in most cases.
Other than avoiding sun exposure, β-Carotene for treating the skin
phlebotomy,and chloroquine are not helpful.
PCT, the most common of the porphyrias, can be
[B]familial (type 2)
[C] Also develop after exposure to
Hepatic URO decarboxylase is deficient in all types of PCT.
Generation of an URO decarboxylase inhibitor in the liver, which
forms uroporphomethene in the presence of iron and under
conditions of oxidative stress.
Majority of PCTpatients (~80%) have no UROD mutations
sporadic(type 1) disease.
Heterozygous for UROD mutations have familial (type 2)
Inheritance of UROD mutation from one parent results in- half N
enzyme activity in liver and all other tissues.
-A significant predisposing factor, but insufficient to cause
Inherited UROD mutations in type 2 PCT
Elevated iron levels
Hemochromatosis gene (HFE) mutations C282Y
After exposure to halogenated aromatic hydrocarbons.
Blistering skin lesions appears most commonly
on the backs of the hands -major clinical feature .
These rupture and crust over, leaving areas of atrophy and
Lesions may also occur on the forearms, face, legs, and feet.
Skin friability and small white papules termed milia are common,
especially on the backs of the hands and fingers.
Hypertrichosis and hyperpigmentation,especially of the face.
skin over sun-exposed areas becomes severely thickened,with
scarring and calcification that resembles systemic sclerosis.
Neurologic features are absent.
TREATMENT :Porphyria Cutanea Tarda
Alcohol, estrogens, iron supplements, and if possible, any
drugs that exacerbates the disease should be discontinued.
Repeated phlebotomy, to reduce hepatic iron.
A unit (450 mL) of blood can be removed every 1–2 weeks,to
gradually reduce excess hepatic iron until the serum ferritin level
reaches the lower limits of normal.
In the erythropoietic porphyrias, excess porphyrins from bone marrow
erythrocyte precursors are transported via the plasma to the skin and
lead to cutaneous photosensitivity.
X-linked Sideroblastic Anemia (XLSA)
Deficient activity of the erythroid form of ALA synthase
Associated with ineffective erythropoiesis,weakness, and pallor.
Clinical Features :
Typically, males with XLSA develop refractory hemolytic anemia,
pallor, and weakness during infancy.
Secondary hypersplenism, become iron overloaded, and can develop
hypochromic, microcytic anemia
striking anisopoikilocytosis and polychromasia
leukocytes and platelets appear normal.
Hb, MCV and MCHC- reduced
Bone marrow examination
Hypercellularity with a left shift and megaloblastic erythropoiesis
with an abnormal maturation.
Prussian blue-staining sideroblasts are observed.
Levels of urinary porphyrin precursors and of both urinary and fecal
porphyrins are normal.
Definitive diagnosis requires the demonstration of mutations in the
erythroid ALAS2 gene
Severe anemia may respond to pyridoxine supplementation.
This cofactor is essential for ALA synthase activity, and mutations
in the pyridoxine binding site of the enzyme have been found in
association with XLSA.
Congenital Erythropoietic Porphyria (CEP)
Also known as Günther’s disease, AR disorder.
It is due to the markedly deficient, but not absent, activity of URO
synthase-results in accumulation of URO I and COPRO I isomers.
CEP is associated with hemolytic anemia and cutaneous lesions.
Clinical Features :
Severe cutaneous photosensitivity typically begins in early infancy.
skin over light-exposed areas is friable and bullae and vesicles -
prone to rupture & infection.
Secondary infections of cutaneous lesions - lead to disfigurement
of the face & hands.
Hemolysis due to marked ↑ in erythrocyte porphyrins leads to
Porphyrins are deposited in teeth and in bones; teeth are
brownish and fluoresce on exposure to long-wave ultraviolet
Skin thickening, focal hypo- and hyperpigmentation, and hypertrichosis of the
face and extremities are characteristic
URO and COPRO (mostly type I isomers) accumulate in
5. Feces( predominant porphyrin in feces is COPRO I).
Diagnosis of CEP can be confirmed by
1.Demonstration of markedly deficient URO synthase activity
2 .Identification of specific mutations in the UROS gene.
Severe cases often require transfusions for anemia.
Chronic transfusions - to suppress erythropoiesis -effective in
reducing porphyrin production
Splenectomy -reduces hemolysis & ↓ transfusion requirements.
Protection from sunlight & minor skin trauma
prompt treatment of complicating bacterial infections.
TREATMENT :Congenital Erythropoietic Porphyria
ERYTHROPOIETIC PROTOPORPHYRIA (EPP)
Deficient activity of ferrochelatase (FECH), the last enzyme in the
heme biosynthetic pathway.
Most common erythropoietic porphyria in children after PCT
second most common porphyria in adults
FECH activities as low as 15–25% of normal in lymphocytes
and cultured fibroblasts.
Protoporphyrin accumulates in bone marrow reticulocytes and then
appears in plasma, is taken up in the liver, and is excreted in bile and
Protoporphyrin transported to the vessels in the skin -nonblistering
Skin photosensitivity, usually begins in childhood –
Asso.with substantial ↑ in erythrocyte protoporphyrin
occurs only in patients with ferrochelatase activities below ~35% of
consists of pain,redness and itching-within minutes of sunlight
exposure resemble angioedema
Vesicular lesions are uncommon.
Chronic skin changes include lichenification,leathery pseudovesicles, labial
grooving, and nail changes
substantial increase in erythrocyte protoporphyrin,-which is
predominantly free and not complexed with zinc, is hallmark of EPP.
Protoporphyrin levels ↑ in bone marrow, plasma, bile, and feces.
Erythrocyte protoporphyrin concentrations increases in other conditions
various hemolytic disorders
All homozygous forms of porphyrias
sometimes even in acute porphyrias
In all these conditions, in contrast to EPP, It is complexed with zinc.
Urinary levels of porphyrins and porphyrin precursors are normal.
↓Ferrochelatase activity in cultured lymphocytes or fibroblasts
DNA diagnosis by mutation analysis - to detect FECH mutation.
In a suspected EPP, confirm the diagnosis by assay that
distinguishes free and zinc-complexed protoporphyrin.
Erythrocytes in EPP also exhibit red fluorescence under a
fluorescence microscopy at 620 nm.
20% of EPP patients - minor abnormalities of liver function
About 5% of patients -accumulation of protoporphyrins – chronic liver
disease -progress to liver failure -death.
Protoporphyrin is insoluble, excess amounts form crystalline
structures in liver cells - ↓hepatic bile flow- bile duct epithelium ,
damaged by toxic bile, leads to biliary fibrosis.
Avoiding sunlight exposure and wearing clothing designed to
Oral β-carotene (120–180 mg/dL).
Afamelanotide,an α-melanocyte-stimulating hormone (MSH).
Cholestyramine and other porphyrin absorbents
-like activated charcoal -interrupts enterohepatic circulation of
protoporphyrin and promote its fecal excretion, leading to some
Splenectomy – In hemolysis & significant splenomegaly.
Plasmapheresis and intravenous hemin
Liver transplantation-in severe liver complications.
Bone marrow transplantation.
Tests For Porphyrinogen:
1.Ehlrichs Aldehyde Test
2.Watson Schwartz Test
Tests For Total Porphrins In Urine:
Spectrophotometry-porphyrins have intense absorbance peak at
Tests for total porphyrin in feces:
- Spectrophotometric estimation of acidic extract of fecal sample(5-10gm
Tests for porphyrins in erythrocytes and plasma(EDTA-blood sample)-
1.Visual examination of porphyrin fluroscenceand solvent fractionation
Urine specimens for urobilinogen or porphobilinogen must be
fresh. (10-20 ml).
If the testing will be delayed-
1.pH should be adjusted to near neutral (pH 7)
2. specimen stored in a refrigerator, where it is stable for about
Urine may darken if the patient has porphyria, especially if
left at room temperature.
Principle: Ehrlich’s reagent(p-dimethylamino benzeldehyde) reacts
with urobilinogen in urine to produce a pink colour.
Intensity of colour depend on amount urobilinogen present.
• 5ml of fresh urine in a test tube+
• 0.5ml of ehrlich’s aldehyde reagent (20ml HCL+ 80 ml distilled
water+ 2gm paradimethylaminobenzaldehyde).
• Allow to stand at room temperature for five minutes..
• Development of pink colour indicates normal amount of
• Both urobillinogen and porphobilinogen produce similar
In the presence of
1.UTI-Nitrites oxidize urobilinogen to urobilin.
2.Antibiotic therapy (gut bacteria which produces urobilinogen are
The Ehrlich's aldehyde reaction and Watson–Schwartz tests
are based on solubility differences between urobilinogen and
Urobilinogen can be extracted by chloroform and/or butanol,
where as porphobilinogen will remain in an aqueous phase.
Permit the phases to separate
Insert a stopper and shake vigorously for 1 minute
Add 5 mL of chloroform
Add 5 mL saturated sodium acetate and mix
Check with pH paper to confirm (pH4-5)
2.5 mL fresh urine
.Add 2.5 mL Ehrlich's reagent and mix
1.Examine the upper (aqueous) phase. If the color is absent, consider
the result of the screening test to be negative and stop.
2. If color is present, separate the upper (aqueous) phase and add 5
mL of butanol.
3. A ‘pink to rose red' color in the lower aqueous layer indicates a
positive result -Suggests a concentration of porphobilinogen that is
several times normal.
4.A color in the upper butanol layer indicates an increase in
Based on the inverse Ehrlich's reaction (i.e., of maintaining an acid
solution by adding a small urine volume to a relatively large reagent
volume), eliminating the problem of urobilinogen reactivity.
The sensitivity is similar to that of the Watson–Schwartz test, but the
reaction is for porphobilinogen.
Detects about 20-100 mg/L of porphobilinogen, and urobilinogen in
amounts up to 200 mg/L does not give a positive result (red color).
A yellow color may be caused by urea.
Detects greater than 6 mg/L and the Hoesch test greater than 11
mg/L of porphobilinogen.
More sensitive than the Hoesch test for porphobilinogen
May yield a positive result between attacks of acute intermittent
Urorosein urinary pigment related to indoleacetic acid –
produces a positive Hoesch test (in response to strong HCl -rose
color may be confused with a positive porphobilinogen result.
false-positive problems may be excluded by testing the specimen
with concentrated HCl (6 mol/L).
Urine from a patient having an acute porphyric attack may be dark
red in color, necessitating a 1:10 dilution with water prior to test.
FALSE POSITIVE RESULTS :
Large doses of methyldopa(Aldomet) .
Indoles in some patients with intestinal ileus.
Drug-phenazopyridine (Pyridium), which becomes orange
A quantitative porphobilinogen test is necessary-
If either the Watson–Schwartz test or the Hoesch test result
is questionable; because of the instability of porphobilinogen.
Alternative urine screening tests for porphobilinogen are:
Micellar electrokinetic capillary chromatographic method ,
A semiquantitative kit -urine is pretreated with ion-exchange resin &
color of the Ehrlich–porphobilinogen adduct is compared to a set of
Uroporphyrin and coproporphyrin can be detected by fluorescence.
An orange-red fluorescence is seen if a positive specimen is
placed near an ultraviolet light source..
In this method, the urine is acidified and the extracted porphyrin
exposed to ultraviolet light.
1.Place 5 mL urine in a stoppered glass centrifuge tube.
2.Add 3 mL of a mixture of one part glacial acetic acid with four parts of
3.Shake and allow to separate. Centrifuging will accelerate the separation.
4.Using a Wood's lamp, observe the upper layer for fluorescence.
Inspect the tube in a dark room.
1. With ultraviolet reflected light- lavender to violet color indicates the
presence of porphyrins .
2. Pink to red fluorescence indicates higher levels of porphyrin.
3. Pale blue with no pink color is negative.Normal urine may fluoresce
4. To increase the sensitivity of the test and remove interfering drug
metabolites, transfer the upper layer to a glass tube
5. Acidify with 0.5 mL of 3 M HCl (25 mL concentrated HCl diluted to 100
mL with water).
6. Shake. Porphyrins are extracted into the lower aqueous layer and will
give a red-orange fluorescence.
Urine specimens for quantitative porphobilinogen - collection:
In a dark container containing 5 g sodium carbonate for a 24-hour
to produce urine of neutral pH.
Frozen specimens are fairly stable, ALA is more stable if urine is
Quantitated by eluting from different columns and reacting with
Micellar electrokinetic capillary chromatographic method -allows
separation of ALA and porphobilinogen
Coproporphyrin and uroporphyrin can be separated by thin-layer
-Quantitated using ion-exchange columns.
Fecal porphyrins can be qualitatively estimated using extraction with
ultraviolet (UV) light, or quantitated.
In some porphyrias, erythrocytes may show fluorescence when an
unstained blood smear is examine microscopically.
The nucleated bone marrow erythrocytes give greater fluorescence.
Test for urinary
AIP,VP,HCP Exclusion of porphyria
Measure fecal COPROIII&PROTOIX
Negative protoIX COPROIII