Chapter 19: Porphyrins and Hemoglobin - PowerPoint Presentation

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Chapter 19: Porphyrins and Hemoglobin

By Louann W. Lawrence, Larry A. Broussard

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Introduction

Porphyrins, hemoglobin, & myoglobin all contain porphyrin ring: 4 pyrrole groups bonded by methene bridges.

Porphyrins can chelate metals

to form functional groups that participate in oxidative metabolism.

Hemoglobin designed to bind, deliver, and release oxygen.

Porphyrias

:

disturbances heme synthesis

Hemoglobinopathies:

disorders of

qualitative defects

in

hemoglobin

molecule

(example: Sickle Cell Anemia)

Thalassemias:

disorders of

quantitative defects

in production of normal hemoglobin molecules

(examples: alpha and beta thalassemia)

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Introduction

Basic structure of porphyrins

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Introduction

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Porphyrins

Porphyrins

(in the body):

Act as chemical intermediates in synthesis of hemoglobin, myoglobin

, & other respiratory pigments called cytochromes

P

art of peroxidase & catalase enzymes, which contribute to efficiency of internal respiration

Chelate iron to form

heme

Used to diagnose

porphyrias

(result from

heme

disturbances)

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Heme Synthesis

Main synthesis sites are the bone marrow and liver

Synthesis of

heme

:

Delta-

aminolevulinic

acid

Porphobilinogen

Uroporphyrinogen

Coproporphyrinogen

Protoporphyrin

Heme

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Porphyrins

Chemistry of Porphyrins

Porphyrins are organic compounds found

in nature

.

Pigment Chlorophyl is a magnesium porphyrin

Four basic isomers may exist for every porphyrin compound (I-IV) however, only types I & III, which differ in side chain arrangement, occur in nature.

Only Type III isomers form heme

Porphyrins are stable & red-violet to red-brown and fluoresce red when excited by light near 400 nm.

Porphyrinogens:

reduced form of of porphyrins, functional forms that must be used in heme synthesis; unstable, colorless, do not fluoresce

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Porphyrins

3 porphyrin compounds clinically significant in humans:

Protoporphyrin (PROTO) – primarily excreted in feces

Uroporphyrin (URO) – primarily excreted in urine

Coprophorphyrin (COPRO) – can be both

Excess of the 3 in biological fluids is a sign of abnormal heme synthesis.

Again – porphyrinogens are the functional form of the compound that MUST be used in heme synthesis.

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Porphyrins

Porphyrin Synthesis

All cells contain hemoproteins & can synthesize heme, but bone marrow & liver are main sites.

Rate of heme synthesis in cells of liver is achieved largely through regulation of enzyme

-aminolevulinic acid (ALA) synthase.

Increase in heme causes decrease in ALA synthase.

Decrease in heme causes increase in ALA synthase.

Rate of heme synthesis is flexible & can change rapidly in response to many external stimuli.

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Porphyrins

Clinical Significance and Disease Correlation

Porphyrias: inherited or acquired enzyme deficiencies that result in overproduction of heme precursors in bone marrow or liver

Diagnosis is made by combination of history & physical & laboratory findings.

Types associated with neuropsychiatric symptoms (these 3 conditions are associated with excess of early precursors (ALA, phorphobilinogen or both)

ALA dehydratase (ALAD)

Deficiency porphyria (ADP)

Acute intermittent porphyria (AIP)

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Porphyrins

Clinical Significance and Disease Correlation

Types associated with cutaneous symptoms (photosensitivity, blisters, excess facial hair, and hyperpigmentation). The following are due to excess later intermediates:

Porphyria cutanea tarda (PCT)

**increased fragility to light-exposed skin

Hepatoerythropoietic porphyria (HEP)

Erythropoietic porphyria (EP)

Congenital erythropoietic porphyria (CEP)

Types associated with neurocutaneous symptoms – due to excess in both early and later intermediates.

Hereditary coproporphyria (HCP)

Variegate porphyria (VP)

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Summary of

Porphyrias

Porphyria

Enzyme Deficiency

Symptoms

Plumboporphyria

(PP)

ALA

dehydratase

Neuropsychiatric

Acute intermittent

porphyria

(AIP)

Porphobilinogen

deaminase

Neuropsychiatric

Porphyria

cutanea

tarda

(PCT)

Uroporphyrinogen

decarboxylase

Cutaneous

Hepatoerythropoietic

porphyria

(HEP)

Uroporphyrinogen

decarboxylase

Cutaneous

Erythropoietic

porphyria

(EP)

Ferrochelatase

Cutaneous

Congenital

erythropoietic

porphyria

(CEP)

Uroprophyrinogen

III

cosynthase

Cutaneous

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Porphyrins

Methods of Analyzing Porphyrins

Tests for urinary PBG (porphobilinogen) & ALA

Watson-Schwartz

Hoesch

Hoesch sometimes used to confirm results of Watson-Schwarts due to Hoesch not having interference with uronbilinogen

Bo

th of above use

Ehrlich’s reagent

. When PBGs mixed with Ehrlich’s – form a red-orange color.

Tests for porphyrins

Enhanced fluorescence of compounds in acidic solution

Chromatic separation & quantitation with spectrophotometry or fluorometry

Molecular diagnostic techniques

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Hemoglobin

Role in Body: transports oxygen to tissue & CO

2

to lungs

Structure

A large, spherical, complex protein molecule (molecular weight is 64,000)

Comprises heme (3%) & globin proteins (97%)

Contains 4 heme groups attached to 4 globin chains

Each globin chain consists of 141 or more amino acids & has 4-fold structure.

Majority of hemoglobin in normal adults is hemoglobin A

(or A

1

).

Hemoglobinopathies:

diseases related to defects in hemoglobin structure

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Hemoglobin (cont’d)

Hemoglobin A: structure of the hemoglobin molecule

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Hemoglobin

Synthesis and Degradation of Hemoglobin

Synthesis occurs in immature RBCs in bone marrow:

65% in nucleated cells

35% in reticulocytes

Normal synthesis depends on adequate iron supply & heme & protein synthesis to form globin portion.

Two pathways degrade hemoglobin

:

Extravascular

(80–90%): occurs outside circulatory system within

phagocytic cells of spleen

, liver, & bone marrow

Intravascular

(10–20%): released directly into bloodstream &

dissociated into

 &  dimers

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Hemoglobin

Clinical Significance and Disease Correlation

Hemoglobin Qualitative Defects: The Hemoglobinopathies

Hemoglobin S

Amino acid defect is at 6th position on

-chain – glutamic acid substituted by valine.

Most common hemoglobinopathy in U.S

.

HbAS (heterozygous = trait) and HbSS (homozygous = disease)

Heterozygous state offers protection from parasites (plasmodium falciparum)

Hemoglobin C

Glutamic acid in 6th position of

-chain is replaced by lysine, resulting in net positive charge

.

Found in West Africa (North Ghana) in 17–28% of population & in 2–3% of African Americans

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Hemoglobin

Clinical Significance and Disease Correlation

Hemoglobin Qualitative Defects: The Hemoglobinopathies

Hemoglobin SC

Most common mixed hemoglobinopathy

-genes code for -S chains & -C chains, leaving no normal  chains to produce hemoglobin A.

Less severe than HbSS disease but still has similar clinical symptoms to HbSS disease

.

Hemoglobin E

An amino acid substitution of

lysine for glutamic acid in 26th position of

 chain

, resulting in net positive charge

Found in Asia in about 20 million people, 80% of whom live in Southeast Asia

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Hemoglobin

Clinical Significance and Disease Correlation

Hemoglobin Qualitative Defects: The Hemoglobinopathies

Hemoglobin D

Variant with electrophoretic mobility on cellulose acetate, like that of hemoglobin S but with negative solubility test

Hemoglobin Quantitative Defects: The Thalassemias

Thalassemias:

group of diseases in which defect causes reduced synthesis of 1 or more of hemoglobin chains

-Thalassemias – defective production of alpha chains

-Thalassemias – defective production of beta chains

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Hemoglobin

-

Thalassemias

2 alpha genes located on each chromosome 16, yielding a total of 4

 genes.

Hydrops

fetalis

– (4 gene deletion) total absence of alpha chain synthesis

Make hemoglobin

Barts

(four gamma chains)

Die before birth or upon birth

Hemoglobin H disease (3 gene deletion)

Hgb

H = beta chain tetramers (due to excess beta chains and lack of alpha chains).

-Thalassemia minor (2 gene deletion)

Mild, microcytic, hypochromic anemia.

Silent carrier (1 gene deletion) – sufficient alpha chains for normal amounts of hemoglobin

Often discovered by expression of 1 to 2%

Hgb

Barts

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Hemoglobin

-

Thalassemias

(point mutations vs. gene deletions)

Many possible expressions of this condition/disease

-Thalassemia Major – aka Cooley’s anemia (severe anemia present)

In -Thalassemia, there is only one gene inherited from each parent – however – there can be a gene expression – which correlates to varying degree of severity



0

= no beta chain production



+

= decreased beta chain production

 = normal beta chain production

Clinical findings of Beta Thalassemia

Increased

Hb

F (2 alpha/2 gamma)

Varied increases

in HbA

2

(2 alpha/2 delta)

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Hemoglobin

Methodology

Solubility test (screening test for sickling hemoglobins)

Based on principle that sickling hemoglobin, in deoxygenated state, is relatively insoluble & forms precipitate in solution

If sickling hemoglobin present – can’t see through.

If No sickling hemoglobin present – see through

.

Cellulose acetate (alkaline) hemoglobin electrophoresis

Fresh hemolysate made from packed RBC sample is applied to a cellulose acetate plate using a buffer of alkaline pH (8.4–8.6); electrophoresis is performed.

Citrate agar (acidic) electrophoresis

Performed at acid pH (6.0–6.2) after abnormal hemoglobin is detected on cellulose-acetate electrophoresis

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Hemoglobin

Methodology

Hemoglobin A

2

quantitation

(2 alpha / 2 delta)

Best accomplished by microcolumn chromatography or high-performance liquid chromatography

(HPLC)

Acid elution stain for hemoglobin F

(2 alpha / 2 gamma)

Distinguishes erythrocytes containing increased amount of hemoglobin F from normal adult cells

Hemoglobin F quantitation

Based on principle that fetal hemoglobin is resistant to alkali denaturation in 1.25 mol/L NaOH for 2 minutes

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Hemoglobin (cont’d)

DNA Technology

Definitive diagnosis of some hemoglobinopathies & thalassemias that involve combinations of genetic defects may require DNA analysis.

DNA sequence of interest may be easily analyzed from whole blood or spots of dried blood on filter paper.

Advantages:

provides definitive info on genotype of individuals tested & sometimes direct detection of molecular lesions

Disadvantages:

higher cost & lack of availability in many labs

Especially useful in prenatal diagnosis of thalassemia major & sickle cell anemia

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Myoglobin

Structure and Role in the Body

A simple heme protein found in skeletal & cardiac muscle

Can reversibly bind oxygen, similar to hemoglobin molecule

Unable to release oxygen, except under low oxygen tension

Acts as oxygen carrier in cytoplasm of muscle cell

Serves as an extra reserve of oxygen to sustain activity in exercising muscle

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Myoglobin

Clinical Significance

Elevated levels in serum & urine often

indicate muscle damage.

Combination of high serum myoglobin & low clearance rate indicates high risk for acute renal failure.

Primary use of serum myoglobin is investigation of chest pain to rule out acute myocardial infarction

.

Also has been investigated to aid in diagnosis & differentiation of types of hereditary progressive muscular dystrophy

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Myoglobin

Methodology

Several immunoassay methods are used to measure & identify myoglobin (fluorescence, chemiluminescence, immunochromic).

Procedures incorporate binding of specific antibodies to myoglobin.

Resulting chemical or physical change can be measured & correlated to myoglobin concentration.

Methods have been adapted to point-of-care devices for rapid assessment of chest pain.