Unit 6: Hemoglobin Derivatives, Heme and Globin Defects

Hemoglobin Derivatives
Oxyhemoglobin
o Oxygenated form of Hemoglobin
o Scarlet Red color
o Hemoglobin before the release of oxygen
o Oxygen can freely bind and diffuse
Deoxyhemoglobin
o AKA: Reduced hemoglobin
o Hemoglobin after the release of oxygen
o Dark Red Color
o Binds Carbon Dioxide
Carbaminohemoglobin
o CO2 bound to free amino acid groups of Hemoglobin

Dyshemoglobins
o Dysfunctional hemoglobins are hemoglobin forms that are unable to transport oxygen
o Most of dyshemoglobins are Acquired
o These hemoglobins are different from Abnormal Hemoglobins (Inherited globin defects)
o Dyshemoglobins are: Sulfhemoglobin, Methemoglobin, Carboxyhemoglobin

Carboxyhemoglobin
o Problem: Carbon monoxide + Hemoglobin (COHb)
o Carboxyhemoglobinemia is always Acquired
o Affinity of CO is 210x to 240x higher than that of Oxygen
o COHb causes a Shift to the Left (Increased Affinity)
o Carbon Monoxide has been called “Silent Killer” because it is odorless and colorless causing victims to quickly
become hypoxic
o CO poisoning is the most common type of accidental poisoning in US
o Sources of Carbon Monoxide:
 Car Exhaust, Tobacco Smoke, Coal and Charcoal Burning, Smoking
o Levels of Carboxyhemoglobin

% of Total Effects
Hemoglobin
10% Shortness of breath on exertion
20 % to 30% Headache, Dizzines, Disorientation, Muscle
Weakness
40% Coma, Seizure, Lung Edema, Death
50% to 70% Asphyxiation and Death

o COHb can be detected using spectral absorption at 540 nm

o COHb blood is Cherry Red color
o Reference Value:
 Endogenous Carboxyhemoglobin = 1% - 3% (Turgeon)
 0.5% Non-Smoker (Steininger)
 5% Smoker (Steininger)
o Diagnosis needed when:
 Non-smokers = >3%
 Smokers = >10%
o Treatment :
 100% Oxygen/Hyperbaric O2
 Start to administer if COHb exceeds 25%

Methemoglobin
o Fe3+ Hemoglobin (instead of Fe2+)
o AKA: Hemiglobin (Hi)
o Intermediary in the Cyanmethemoglobin Method for Quantitation of Hemoglobin
o REVIEW:
 Ferric = Fe 3+
 Ferrous = Fe 2+
o Problem: Reversible oxidation of Heme Iron
o Causes a Shift to the Left (Increased Affinity)
o Methemoglobin cannot carry oxygen because of oxidized ferric iron

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 o Methemoglobin reduction system in the body:
 NADH-Methemoglobin Reductase Pathway
 Main Enzyme = NADH-Cytochrome b5 Reductase 3
 Limits Methemoglobin to only 1% of total Hemoglobin
 Reduced Triphosphopyridine nucleotide – Glucose-6-phosphate Dehydrogenase Pathway
 Glutathione and its related enzymes
o Types:
1. Acquired Methemoglobinemia / Toxic Methemoglobinemia
 Occurs in Nitrite poisoning
 Aniline Dye Poisoning (Shoe Dye)
 Primaquine (Anti-Malarial Drugs)
 Dapsone , Trimethoprim, Sulfonamide (Antibiotics)
 Benzocaine (Local Anesthetics)
 Treatment: Methylene Blue
2. Hereditary Methemoglobinemia
 Rare
 Results of Mutations either of the TWO genes:
 NADH-Cytochrome B5 Reductase 3 gene (CYB5R3 gene)
o Autosomal Recessive
o AKA: Diaphorase Deficiency
o First described in Europe (Inbreeding)
o Methemoglobin = >50% of Total Hb
o Treatment :Methylene Blue
 Alpha, Beta or Gamma Globin Gene
o Autosomal Dominant
o AKA: Hemoglobin M disease
o Produces M Hemoglobin or Hb M
o Hb M is 30 to 50% of Total Hb
o No Treatment possible nor is it necessary
o Spectral Absorption = 630 nm
o High Hemiglobin/Methemoglobin = Chocolate Brown Color (cannot revert to regular color)
o Cyanosis is a sign of high Hemiglobin in the blood
o Levels of Hemiglobin/Methemoglobin
% of Total Effect % of Total Hemoglobin Effect
Hemoglobin
Rhodak Turgeon and Steininger
Less than 25% Asymptomatic >10% Cyanosis starts
>30% Cyanosis, Hypoxia >35% Shortness of
breath,Tachycardia
>50% Coma and Death >60% to 70% Hypoxia and Death
o Reference value:
 1% - 2%
o Treatment :
 Oral or IV Methylene Blue (1% solution) OR Ascorbic Acid
 Methylene Blue = Methylthioninium chloride
 Methylene Blue is more effective
 Methylene Blue +NADPH –---( NADPH Methemoglobin Reductase)---Leukomethylene Blue
 Turns Urine to Blue
o Side notes;
 Methemoglobinemia mostly affects infants of 6 months of age (“Blue babies”)
 Infants are more susceptible to Methemoglobin production because
 Hemoglobin F is easily converted to Methemoglobin
 Infants are deficient in required reducing enzymes
 “Blue Fugates”
 Family in Kentucky with both parents having Recessive Methemoglobinemia gene
 “Blue Men of Lurgan”
 “Familial Idiopathic Methemoglobinemia”

Sulfhemoglobin
o Problem: Irreverible oxidation of Hemoglobin by oxidizing drugs
o Causes:
 In vivo
 Drugs: Sulfonamides, Phenacetin, Nitrites, Phenylhydrazine
 Clostridium welchii (Bacteremia)
 In vitro
 Hydrogen Sulfide + Hb
o Forms a greenish pigment
o Spectral Absorption = 630nm
o Cannot be measured in Cyanmethemoglobin Method for Total Hemoglobin
o It is ultimately precipitated as Heinz Bodies
o Sulfhemoglobin + Carbon Monoxide = Carboxysulfhemoglobin
o Rarely exceeds >20%
o Cyanosis is the only significant effect of Sulfhemoglobin
o Reference Value:
 1% to 10 %
o Treatment
 Removal of offending agent

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 Abnormal Heme Synthesis
Porphyrias:
o Disorders of heme synthesis
o Derived from the greek word “Porphyra” = Purple
o Purple-red pigment in the urine (“Wine-red Urine)
o Porphyrins are excreted (urine and feces) in Porphyrias and Lead Poisoning
o Accumulation of Porphyrinogen in the
 Erythropoietic Porphyrias
 Accumulation in Bone Marrow
 Enzyme Deficiency occurs in the RBC
 Hepatic Porphyrias
 Accumulation in Liver
 Enzyme Deficiency occurs in the Liver
o Result = Decreased Hb production
o Types:
 Primary Porphyrias
 Inherited disorders
 Hepatic Porphyria
 Secondary Porphyria
 Acquired disorders (Drugs/Chemicals)
 Hepatic Porphyria ,rarely, Erythropoietic Porphyria
 Erythropoietic Porphyria
 Lead Posioning
o Formation of Ringed Sideroblasts
 (+) Perl’s Prussian Blue
 Bright Red under Fluorescent Microscope
o Lead interferes with ALA-dehydrase and Ferrochelatase
o Basophilic Stippling in PBS
o S/s = Asymptomatic or with skin lesions or neurologic disturbances
 Skin lesions
 Photocutaneous lesions upon exposure to sunlight
 Caused by increased porphyrin levels
 Porphyrins strongly absorb Soret Band Wavelengths (400-430nm)
o Laboratory Findings
o Hallmark : Increase in Free erythrocyte Protoporphyrin (FEP)

Porphyria Enzyme Defect Inheritance Clinical Presentation Laboratory Findings

Hepatic Porphyria
Acute Intermittent PBG deaminase Autosomal Dominant (+) Neurologic Darkened urine upon
Porphyria Disturbances stainding
>Exacerbated by
Steroid hormones
Porphyria Cutanea Uroporphyrinogen Autosomal Dominant (+) Photocutaneous Most common
Tarda Decarboxylase lesions Porphyria in US
>Urine +Acid =Pink
Urine
Hereditary Protoporphyrinogen Autosomal Dominant (+) Photocutaneous Red Urine
Coproporphyria Oxidase lesions
Variegate Protoporphyrinogen Autosomal Dominant (+) Photocutaneous Red urine
Porphyria/South Oxidase lesions
American Porphyria
Hereditary PBG Porphobilinogen Autosomal Dominant (+) Neurologic Rarest Porphyria
Synthase Deficiency (PBG) Synthase/ ALA Distrubances (Henry’s)
/ALA Dehydratase dehydratase > must be
Deficiency differentiated with
/Doss Porphyria Tyrosinemia and
/Plumboporphyria Heavy Metal
Intoxication
Erythropoietic Porphyria
Congenital Uroporphyrinogen III Autosomal Recessive (+) Photocutaneous > Rarest
Erythropoietic Cosynthase lesions Porphyria(Steininger)
Porphyria (CEP) > Teeth Fluoresce > Hemolytic Anemia
/ Gunther’s Disease under UV > Polychromasia
light(“Erythrodontia”) > Increased
> Patients rarely Coproporphyrin (Red
survive past middle Urine)
age
Erythrocytic Ferrochelatase Autosomal Dominant (+) Photocutaneous > Second Most
Protoporphyria (EPP) lesions common Porphyria
> Mild Hepatobiliary > Increased FEP
Disease
X-linked EPP Increased ALA- X-Linked Dominant (+) Photocutaneous > High FEP and Zinc
synthase 2 activity lesions PP
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 Abnormal Globin Synthesis
Structural Globin Defects
o Hemoglobinopathies
o Mainly due to amino acid substitutions and deletions
o Defects can be: Substitutions, Deletions, Elongations
o Variants can be produced by defects in
 Alpha Globin Chain
 Hemoglobin G-Philadelphia
 Beta Globin Chain
 Hemoglobin S
 Hemoglobin C
 Hemoglobin D
 Hemoglobin E
 Hemoglobin O-Arab
 Multiple Amino Acid Substitutions
 Hemoglobin C-Harlem
 Hemoglobin S-Antilles and Hemoglobin S-Oman
 Deletions of Amino Acid
 Hemoglobin Gun Hill
 Elongation of Polypeptide Chain
 Hemoglobin Constant Spring
 Other Variants
 Hemoglobin I
 Hemoglobin Barts
 Hemoglobin H
Basic Terminologies
o Hemoglobin Variant
 Abnormal Hemoglobin produced by a globin chain defect
 Example: Hemoglobin S
o Hemoglobinopathy
 Condition diagnosed when the presence of a variant is confirmed in the blood by laboratory test
 Sickle Cell Disease, Sickle Cell Trait
o Qualitative Globin Chain Abnormality
 Abnormality in the Amino Acid sequence of the globin chain, not the amount of globin produced
o Structural Globin Chain Abnormality
 Abnormality in the Globin Chain composition that produce hemoglobin variants

Beta Chain Substitutions

Hemoglobin S
o Sickle Hemoglobin
o Substitution of Glutamic Acid in the 6th position of Beta Chain with Valine [B6 glu val]
o Effects of Oxygenation on Sickle Cell
 Oxygenated = Soluble
 Non-Oxygenated = Non-soluble, forms crystals or tactoid inclusion bodies (allows addition of valine)
o First Most Common Hemoglobin Variant (in US)
o Highest frequency of Sickle Cell Gene is found in
 Sub Saharan Africa > Arab-India >America >Eurasia >SEA
o Sickle Cell Disease is mostly found in individuals with African Descent
o Sickle cells leads to hemolysis and culling
o “Gardos Effect” = Sickle cells contain high calcium levels, which stimulate potassium and water efflux and
exaggerate cell dehydration caused by Gardos channel
o Hemoglobin S gene mutation provides resistance, not immunity, to P.falciparum infections
 P.falciparum dies if the RBC sickles
o Forms:
 Homozygous :
 Sickle Cell Disease/Anemia
 Hb SS
 Most Severe Hemoglobinopathy
 Heterozygous:
 Sickle Cell Trait
 Hb AS
o Cells that can be confused with Sickle cells
 Ovalocytes
 Schistocytes
 Burr Cells
o Copolymerization of Hb S (in decreasing order) (increase in “crystal” formation)
 Hb C > Hb D > Hb O-Arab > Hb A > Hb J > Hb F (blocks polymerization of HbF)
o Sickle Cell Crises (6 Crises)
1. Hand-Foot Syndrome/ Sickle Cell Dactylitis
 Bilateral painful swelling of hands and feet as a result of sickling and capillary stasis

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 Splenic Complications (3 Crises)

2. Splenic Sequestration Crises

 Sudden pooling of blood and rapid enlargement of spleen resulting to hypovolemic shock
 Often happens after infections
3. Functional Asplenia/Infectious Crises
 Inadequate antibody response with inability of RES to clear bacteremia due to splenic blockade
 Primary cause of death in sickle cell anemia
 Salmonella spp and Streptococcus pneumoniae infections are usually prevalent in children with
sickle cell anemia
 Staphylococcus aureus and Haemophilus influenzae are also reported
4. Autosplenectomy
 Progressive fibrosis and contraction of the spleen

5. Vaso-occlusive crises/ Painful crises

 Debilitating episodes of abdominal and bone or joint pain accompanied by fever due to plugging of
small vessels by sickle
 Hallmark of Sickle Cell Disease
6. Aplastic Crises
 Fever and infection
 Temporary failure of RBC production
 Usually results from infections of Parvovirus B19, Folic deficiency

o Organs affected in Sickle Cell Anemia

 Liver (Hepatomegaly)
 Spleen (Splenomegaly)
 Extramedullary Erythropoiesis is seen in severe cases
o Sickle Cell types:
 Reversible Sickle Cells
 Changes shape with regards to oxygen tension
 Irreversible Sickle Cells
 Does not change shape
 Stays elongated (“Banana shaped”) throughout the RBC’s life
 Causes vasoocclusion
o Diagnosis of Sickle Cell Disease
 Two Step Process
1. Demonstration of Insolubility of Deoxygenated Hemoglobin S
2. Hemoglobin Electrophoresis, HPLC or Capillary Electrophoresis
o Treatment
 Keep patient warm
 Prevent excessive sweating (Dehydration can also cause sickling)
 Prevent Hypoxic conditions
 Prevent acidic pH of blood

Hemoglobin C
o Crystal Hemoglobin
o Substitution of Glutamic acid at 6th position to Lysine [B6 glulys]
o 2nd most common Hemoglobin Variant
o Commonly found in people of African descent
o Heterozygous (Hb AC) or Homozygous (Hb CC)
o Hb CC tends to crystallize upon Dehydration
 Hence C=Crystal
 Older RBCs are the usual victims
o Hb CC crystals are
 “Hexagonal or Rod-shaped intracellular crystals, some, usually with both ends are blunt”
o Hb SC crystals are
 “ Finger-like Projections” or “Washington Monument crystals” usually seen with “Pocketbook cells”
 Most common heterozygous syndrome of structural defects in hemoglobin molecule
 Patients are prone to S.pneumoniae infections

Hemoglobin D
o Heterozygous (Hb AD) or Homozygous (Hb DD)
o May be found in combination with Hb S (Hb SD)
 Hb S/D-Los Angeles
 Hb S /O Arab
o Rare
o Variants:
 Glycine substituted at 121 th position for Glutamic Acid [B121 glyglu]
 Hb D-Los Angeles / Hb D-Punjab
 Hb D-Los Angeles
o Most common Hb D variant in US
 Hb D-Punjab
o Found in Pakistan and NW India

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Hemoglobin E
o Lysine is substituted at 26th position for glutamic acid [B26 lysglu]
o Most common Hemoglobin variant in the WORLD
o 3rd most common hemoglobin variant in the US
o Causes Microcytic anemia
o Found primarily in people of Southeast Asian descent
o High prevalence in Thailand
o Malaria prevalent area
o Hb EE slows malarial multiplication

Hemoglobin O-Arab
o Substitution of glutamic acid at 121th position to lysine [B121 glulys]
o Rare
o Can combine with Hb S (HbOS)

Alpha Chain Substitutions

Hemoglobin G-Philadelphia
o Only alpha globin chain variant of significance in US
o {A68 asnlys) *asparagine to lysine
o Homozygous HbG-Philadelphia is incompatible with life

Multiple Amino Acid Substitutions

Hemoglobin C-Harlem
o AKA: Hb C-Georgetown
o Two AA substitutions
 [B6 glu val]
 [B73 aspasn] *Aspartic Acid Asparagine

Hemoglobin S-Antilles and Hemoglobin S-Oman

o Hb S-Antilles
 [A23 isoleucinevaline] + [B6 glutamic acid valine]
o Hb S-Oman
 [B6 glutamic acid  valine] + {B121 lysineglutamic acid]

Deletions of Amino Acid

Hemoglobin Gun Hill
o Deletions of 5 amino acids from 91st position to 95th position
o No heme can be bound to Beta chains of this hemoglobin
o One of the unstable hemoglobins

Elongation of Polypeptide Chain

Hemoglobin Constant Spring (Hb CS)
o Addition of 31 amino acids at the end of the alpha chain
o Often inherited with Hb H (Hb H Constant Spring)
o Prevalent in Chinese and Greeks

Other Variants
Hemoglobin I
o Rare
o Affects either Alpha or Beta chain
o Lys  glutamic acid
Hemoglobin Barts and Hemoglobin H
o Variants found in alpha Thalassemia
o Hemoglobin Barts
o occurs in Newborns
o Deletion of all alpha genes
o Comprises of four gamma chains
o Hemoglobin H
o Occurs in older children
o Consists of 4 normal beta chains (instead of 2 alpha and 2 beta chains)

Afternote:
o The most clinically significant hemoglobinopathies are Hb SS, Hb SC, and Hb S–b-thalassemia

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 Hemoglobin M and Pseudocyanosis
o Pseudocyanosis = no enzyme abnormality in the red cell nor a presence of toxic drugs nor cyanotic heart
disease
o 9 Variants:
o Alpha Chain substitutions
 Hb M Iwate
 Hb M Boston
 Hb Auckland
o Beta Chain substitutions
 Hb Chile
 Hb M Saskatoon
 Hb M Milwaukee-1
 Hb M Milwaukee-2 (AKA: Hb M Hyde Park)
o Gamma chain variants (eventually disappears as Hb A transitions in)
 Hb FM Osaka
 Hb FM Fort Ripley

Unstable Hemoglobins
o AKA: Congenital Heinz Body Hemolytic Anemia, Congenital non-spherocytic hemolytic anemia
o Very rare disease
o Autosomal dominant
o Homozygous fetuses do not survive
o Most commonly reported unstable hemoglobin = Hb Koln

Variant Increased Decrease Methemoglobinemia Degree of Hemolytic

Oxygen Oxygen Disease
Affinity Affinity
Casper/Southhampton  Severe
Genova  Mod. Severe
Gun Hill  Mild
Koln  Mild
M-Saskatoon   Mild
Bristol  Severe
Torino  Mod. Severe
Seattle  Mild
Constant Spring Mild
Tacoma No Clinical
Abnormalities

Hemoglobins with High Oxygen Affinity

o Familial erythrocytosis
o Over 90 different variants
Hemoglobins with Low Oxygen Affinity
o Familial Cyanosis
o Variants:
o Hb Kansas
o Hb Beth Israel
o Hb Yoshizuka
o Hb Agenogi
o Hb Titusville
o Hb Providence
Thalassemias

o Abnormally low production rate of globins due to gene deletions

o Different from hemoglobinopathy (abnormal globin structure due to abnormal genes)
o First reported Thalassemia is in Cypress (1944)

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