PORPHYRIAS AND THE 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 Empire!

INTRODUCTION:

Not a ‘vampire’s’ disease

Some symptoms of porphyrias have lead people to believe that these diseases provide some basis for vampire legends:

· Extreme sensitivity to sunlight · Anemia

This idea has been discarded both for scientific reasons:

· Porphyrias do not cause a craving for blood.· Drinking blood would not help a victim of

porphyria.

COORDINATED REGULATION OF HEME AND GLOBIN SYNTHESIS:

↓Heme:inhibits activity of pre-existing -ALA synthase

Diminishes the transport of -ALA synthase from cytoplasm to mitochondria after synthesis of the enzyme.

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.

Definition :

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.

Neuropsychiatric:1.Acute intermittant porphyrias(AIP)2.ALA dehydratase porphyria(ADP)

Cutaneous(photosensitivity):1.congenital erythropoietic porphyria(CEP)2.porphyria cutanea tarda(PCT)3.Erythropoietic porphyria(EPP)

Mixed:1.Hereditary copro-porphyria(HCP)2.Variegate porphyria(VP)

Classification Based On Predominant Clinical Manifestations

Hepatic: 1.AIP 2.ADP 3.HCP 4.VP Erythrocytic: 1.CEP 2.EPP Both: 1.PCT

Classification Based On Site Of Expression Of Disease :

Acute:1.AIP2.ADP3.HCP4.VP Nonacute(cutaneous):1.PCT2.EPP3.CEP

Classification Based On Mode Of Presentation Of Disease

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.

HEPATIC PORPHYRIAS

clinical presentation depends on the amount of residual ALA dehydratase activity.

commonly affects- male adolescents

symptoms resembling those of AIP-abdominal pain and neuropathy.

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.

Clinical Features

Diagnosis :

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

Intravenous hemin

Liver transplantation.

TREATMENT OF ADP

Acute Intermittent Porphyria (AIP)

AD diseases resulting from the half-normal level of HMB synthase activity.

Disease is widespread

Clinical expression is highly variable

Activation of the disease is often related to environmental or hormonal factors

Common precipitating factors include:

1.Endogenous and exogenous steroids

2.Porphyrinogenic drugs

3.Alcohol ingestion

4.Low-calorie diets, usually instituted for weight loss

Attacks can be prevented by avoiding known precipitating factors.

CAUSATIVE 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 puberty.

Symptoms are more common in women, suggesting a role for estrogens or progestins.

Premenstrual attacks are probably due to endogenousprogesterone.

Exacerbated by exogenous steroids, including OCP preparations containing progestins

pregnancy ,usually well tolerated,suggesting -beneficial metabolic changes - ameliorates effects of high levels of progesterone.

Fasting• 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

CLINICAL FEATURES

Neurovisceral symptoms rarely occur before puberty

Abdominal pain, the most common symptom, is usually steady andpoorly 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

Mental symptoms

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.

COMPLICATIONS:

Sudden death-results from sympathetic overactivity and cardiac arrhythmia.

Treatment of seizures is difficult because most antiseizure drugs can exacerbate AIP

When an attack resolves-

Abdominal pain may disappear within hours

paresis begins to improve within days and may continue to improve over several years

Diagnosis

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 hemin administration.

Urine During Attack Of AIP

No history of symptoms -have normal urinary excretion of ALAand PBG.

1.Detection of the family’s HMBS mutation will diagnose asymptomatic family members.

2.Prognosis of individuals with HMBS mutations is generally favorable

Fecal porphyrins are usually normal or minimallyincreased 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 separately.

HCP is less common than AIP and VP.

Clinical Features

HCP is influenced by the same factors that cause attacks in AIP.

The disease is latent before puberty, and symptoms,which are virtually identical to those of AIP, are more common inwomen.

HCP is generally less severe than AIP.

Blistering skin lesions are identical to PCT and VP and begin in childhood in rare homozygous cases.

Diagnosis :

COPRO III is markedly ↑ in the urine and feces in symptomatic patients.

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 lesions.

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 lesions.

Treatment

.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 the disorder.

Clinical Features

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

Neurologic symptoms

Developmental disturbances, including growth retardation, in infancy or childhood;

Diagnosis ↑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 homozygous porphyrias.

TREATMENT -Variegate Porphyria

Acute attacks are treated as in AIP, and hemin should be startedearly in most cases.

Other than avoiding sun exposure, β-Carotene for treating the skin lesions

phlebotomy,and chloroquine are not helpful.

Porphyria Cutanea Tarda

PCT, the most common of the porphyrias, can be

[A]sporadic(type 1)

[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

symptomatic PCT.

Causative factors:

Inherited UROD mutations in type 2 PCT

hepatitis C

HIV

Excess alcohol

Elevated iron levels

Estrogens

Hemochromatosis gene (HFE) mutations C282Y and H63D.

After exposure to halogenated aromatic hydrocarbons.

Blistering skin lesions appears most commonlyon the backs of the hands -major clinical feature .

These rupture and crust over, leaving areas of atrophy and scarring.

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.

Clinical Features

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.

ERYTHROPOIETIC PORPHYRIAS

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 hemosiderosis.

PS – hypochromic, microcytic anemiastriking anisopoikilocytosis and polychromasialeukocytes 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 fecalporphyrins are normal.

Definitive diagnosis requires the demonstration of mutations in the erythroid ALAS2 gene

Diagnosis

Severe anemia may respond to pyridoxine supplementation.

This cofactor is essential for ALA synthase activity, and mutationsin the pyridoxine binding site of the enzyme have been found in association with XLSA.

TREATMENT -X-Linked Sideroblastic Anemia

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 splenomegaly.

odontoporphyria

Porphyrins are deposited in teeth and in bones; teeth are brownish and fluoresce on exposure to long-wave ultraviolet light.

Skin thickening, focal hypo- and hyperpigmentation, and hypertrichosis of the face and extremities are characteristic

Diagnosis

URO and COPRO (mostly type I isomers) accumulate in

1.Bone marrow2.Erythrocytes3.Plasma4.Urine 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

β-Carotene

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 lymphocytesand 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 feces.

Protoporphyrin transported to the vessels in the skin -nonblistering photosensitivity.

Clinical Features

Skin photosensitivity, usually begins in childhood –

Asso.with substantial ↑ in erythrocyte protoporphyrin

occurs only in patients with ferrochelatase activities below ~35% of normal.

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

Diagnosis 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 lead poisoningIron deficiencyvarious 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.

Prognosis, complications and Treatment

Avoiding sunlight exposure and wearing clothing designed toprovide protection

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 improvement.

Treatment:

Splenectomy – In hemolysis & significant splenomegaly.

Plasmapheresis and intravenous hemin

Liver transplantation-in severe liver complications.

Bone marrow transplantation.

Tests For Porphyrinogen:1.Ehlrichs Aldehyde Test2.Watson Schwartz Test3.Hoesch’s Test

Tests For Total Porphrins In Urine: Spectrophotometry-porphyrins have intense absorbance peak at 400nm.

Tests for total porphyrin in feces:- Spectrophotometric estimation of acidic extract of fecal sample(5-10gm

wet wt).

Tests for porphyrins in erythrocytes and plasma(EDTA-blood sample)-1.Visual examination of porphyrin fluroscenceand solvent fractionation2.spectrophotometry.

LAB DIAGNOSIS

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 1 week.

Urine may darken if the patient has porphyria, especially if left at room temperature.

Laboratory test for porphoblinogen in urine

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.

.

Ehrlich’s aldehyde test

• 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 urobilinogen

• Both urobillinogen and porphobilinogen produce similar reactions

Procedure

In the presence of

1.UTI-Nitrites oxidize urobilinogen to urobilin.

2.Antibiotic therapy (gut bacteria which produces urobilinogen are destroyed).

False negative test:

The Ehrlich's aldehyde reaction and Watson–Schwartz tests are based on solubility differences between urobilinogen and porphobilinogen.

Urobilinogen can be extracted by chloroform and/or butanol, where as porphobilinogen will remain in an aqueous phase.

Watson–Schwartz Test

Procedure

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 urobilinogen concentration.

Interpretation

Hoesch's Test.

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.

Watson–Schwartz test

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 porphyria.

False-positive reaction:

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 with HCl.

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 standard.

Fluorescence Screening Procedure For Porphyrin

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..

Principle: 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 ethyl acetate

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.

Procedure

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 blue.

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.

Results:

Precautions for Quantitative estimation

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 acidic

Quantitated by eluting from different columns and reacting with Ehrlich's reagent.

Micellar electrokinetic capillary chromatographic method -allows separation of ALA and porphobilinogen

Coproporphyrin and uroporphyrin can be separated by thin-layer chromatography

-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.

Acute abdominal pain(ACUTE PORPHYRIAS)

Test for urinary porphobilinogen

positive Negative

AIP,VP,HCP Exclusion of porphyria

Measure fecal COPROIII&PROTOIX

Negative protoIX COPROIII

AIP VP HCP

Cutaneous photosensitivity(CUTANEOUS PORPHYRIAS)

Free erythrocyte protoporphyrin

Positive Negative

EPP urinary/fecal total porphyrin positive

Urine-UROI,COPROI Feces-COPROI

CEP

Urine-UROIFeces-ISOCOPRO

PCT

TYPE OF PORPHYRIAS

URINE FAECES

1.AIP PBG,COPROIII

2.VP PBG,COPROIII PROTOIX

3.HCP PBG,COPROIII COPROIII

Diagnostic Pattern Of Conc. Of Heme Precursors In Acute Porphyrias

Diagnostic Pattern Of Conc.Of Heme Precursors In cutaneous Porphyrias TYPE OF PORPHYRIA

URINE FAECES ERYTHROCYTES

1.CEP UROI,COPROI COPROI

2.PCT UROPORPHYRIN ISOCOPRO

3.EPP

PROTOPORPHYRIN