Red Blood Cell

Structure, Physiology,
Metabolism and
Destruction

Ma. Christy V. Gonzales, RMT, MPH

School of Medical Technology
Emilio Aguinaldo College
MCC4 31 (Hematology 1) Lecture
The life cycle of a red blood cell
a. Kidneys respond to a lower than normal
oxygen concentration in the blood by
releasing the hormone
erythropoietin.
b. Erythropoietin travels to the red bone
marrow and stimulates an increase in
the production of red blood cells
(RBCs).
c. The red bone marrow manufactures RBCs
from stem cells that live inside the
marrow.
d. RBCs squeeze through blood vessel
membranes to enter the circulation.
e. The heart and lungs work to supply
continuous movement and
oxygenation of RBCs.
f. Damaged or old RBCs are destroyed
primarily by the spleen.
Reticulocyte

Membrane Composition and Characteristics:

• in erythroblastic island surrounding a central macrophage
• BM and PC
• tubulin and actin
• important during terminal erythroid differentiation in terms of cell
division and cell motility
• Changes:
• Increase in shear resistance
• Loss of surface area due to loss of membrane lipid
• Acquisition of a biconcave shape
Mature Red Blood Cells

• no nucleus or cytoplasmic organelles

• limited in metabolic activity
• metabolism of FA and AA
• oxidative metabolism
• Erythrocyte glycolysis
• source of energy through breakdown of glucose
• major pathway: ____________
• other supplementary pathways: ______________
• Hemoglobin
• Main cell component
• Membrane
• survival for 120 days in circulation
Mature Red Blood Cells
I. Shape
• Biconcave
• facilitates O2-CO2 transport function
• maximize the ratio of the surface area to volume
• allows cell flexibility (RBC deformability)
• allows cell to adjust to small vessels and still maintain cell viability
• Alteration in ratio
• less deformable à __________________________
• spheroid shape
• due to:
• membrane loss due to fragmentation à
__________________________
• increased uptake of cations and H2O à
__________________________
Mature Red Blood Cells
II. Membrane Composition and Structure
• composition:
• proteins: _____
• lipids: _____
• carbohydrates: _____
• allows to function:
• separate IC fluid env. of cytoplasm from the EC fluid env. of the plasma
• allow nutrient and ion passage selectively into and out of the cell
• allow the cell to deform when required
• Lipids and Proteins
• asymmetric arrangement from cytoplasmic side to membrane interior
or from plasma side
• allows selective passage of mol. into and out of the cell
Mature Red Blood Cells

II. Membrane Composition and Structure

A. Lipids
• equal proportions of phospholipids & unsterified cholesterol
1. Phospholipids
• arranged in bilayer lipid
• phospholipid polar groups allows membrane to
act as liquid sealer
• nonpolar fatty acids
• fluid because FA are free to move laterally w/in the membrane,
allowing lipids to interact with other lipids and membrane proteins
• maintains extreme differences in in osmotic pressure, cation
concentrations, and gas concentrations between plasma and
cytoplasm
Mature Red Blood Cells

II. Membrane Composition and Structure

A. Lipids
1. Phospholipids
• asymmetrically distributed
• outer: phosphatidylcholine and sphingomyelin
• inner: phosphatidylserine (PS) and phosphatidylethanolamine
• distribution is energy dependent
• flippase, floppase, scramblase (membrane-associated enzymes)
• disrupted distribution
• conditions (thalassemia or sickle cell anemia) PS flips to
outer layer
• aging RBCs
Mature Red Blood Cells

II. Membrane Composition and Structure

A. Lipids
2. Cholesterol
• regulates membrane fluidity
• membrane permeability to electrolytes and non-electrolytes
• confers tensile strength to the lipid bilayer
• 98% of membrane: unesterified
• 70% of plasma: esterified
Mature Red Blood Cells

II. Membrane Composition and Structure

B. Proteins
• Bound to lipids throughout the membrane
• acts as receptor for various molecules & skeletal structure
1. Peripheral protein
• contain 2 skeletal proteins
• spectrin • underlie the lipid bilayer on
• actin cytoplasmic side
• regulate the membrane shape and
deformability
• provide viscoelastic properties
Hematology

• stud
Mature Red
Blood Cells
II. Membrane
Composition and
Structure
B. Proteins
1. Peripheral protein
Mature Red Blood Cells

II. Membrane Composition and Structure

B. Proteins
2. Integral protein
• band 3
• functions:
• transport sites
• adhesions sites
• contain sialic acid
• zeta potential
• negativity between cells
• causes cells to repel one another
• decreased zeta potential: _______
• signaling receptors
Mature Red
Blood Cells
II. Membrane
Composition and
Structure
B. Proteins
2. Integral protein
Mature Red Blood Cells

II. Membrane Composition and Structure

B. Proteins
3. Membrane enzyme
• Na+, K+ - ATPase
• Controls active transport of Na and K
• Na+ w/o loss of K + : ____________ __________
• K + : _____________ ______________
• Ca+2, Mg+2 – ATPase
• Moves calcium out of the cell to the plasma against high
concentration gradient
• Calcium
• Involve in regulating and stabilizing membrane
phospholipid structure
Mature Red Blood Cells

II. Membrane Composition and Structure

C. Carbohydrates
1. Glycolipids
• sugar bearing lipids
• associate in clumps or rafts
• support carbohydrate side chains that extend into the aqueous plasma
to anchor the glycocalyx
• layer of carbohydrates
• net (-) charge prevents microbial attack & mechanical damage
caused by adhesion to neighboring RBCs or to the endothelium
• bears antigens of the ABH and the Lewis blood group systems
Mature Red Blood Cells

III. Hemoglobin Viscosity

• Nomal RBC Hgb concentration has low viscosity and is fluid
• Causes of deformable RBC’s:
• Loss of water
• Precipitated Hgb
• Heinz bodies
• Polymerized Hgb
• Hgb S
• Crystallized Hgb
• Hgb C
Mature Red Blood Cells

IV. Energy Metabolism

• Energy is required to:
• maintain components of RBC
• preserving membrane shape
• perform enzymatic reactions
• movement of Ca2+, Na+, K+
• reduce oxidized proteins
• hgb must be maintained in reduced state
Mature Red Blood Cells

IV. Energy Metabolism

• Hgb site prone to oxidation:
• Iron atom in the heme ring
• Methemoglobin
• formed from oxidation of normal Fe2+ state to Fe3+ state
• normally occurs daily
• hereditary unstable hgb
• methemoglobin reductase deficiency
• Exposure to oxidant drugs
• Sulfhydryl (-SH) groups on the globin chains
• Heinz bodies
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Glucose
• principal source of energy
• quickly pass through membrane without expense of energy
• Galactose, fructose and mannose
• Pentose and disaccharides lactose, sucrose and maltose
• Not metabolized and cannot pass through the membrane
• Glycogen
Mature Red
Blood Cells
IV. Energy Metabolism
Sources of Energy aldolase
Pathways for energy
metabolism
PGK
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Embden-Meyerhof Pathway
• metabolize 90-95% of glucose used by the cell (anaerobic)
• glucose catabolized to lactate
• same as other EMP except for ____________________
• O2 tension, 2,3-DPG binds to deoxyhemoglobin form
• causes Hb to resist deoxygenation à ____________________
• ATP and nicotinamide adenine dinucleotide (NAD+ & NADH)
• important intermediates in EMP
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Embden-Meyerhof Pathway
• 2,3-DPG
• generated by Rapoport-Luebering shuttle
• bypass pathway in the EMP
• 1,3-DGP can be catabolized to 3-PG
• or directly by path w/c high energy ATP is generated
• Hypoxia
• shifts catabolism through RLS
• ATP levels: cause direct metabolism of 1,3-DGP
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Embden-Meyerhof Pathway
• ATP
• maintains membrane’s shape
• provides energy for transport of cations
• assist in modulating the amount of 2,3-DPG
• 2 mol of ATP per mol of glucose during the first 3 steps
• 4 mol of ATP generated
• Net yield ATP= ________
Mature Red Blood Cells
IV. Energy Metabolism
Sources of Energy
• Pathways for energy metabolism
Embden-Meyerhof Pathway
• NAD+ (oxidized form) & NADH (reduced form)
• Involved in 2 steps:
1. NAD+ as coenzyme with glyceraldehyde-3-phosphate
dehydrogenase to form 1,3-DPG
2. NADH as coenzyme in with LDH to reduce pyruvate to lactate
• NADH
• coenzyme w/ methemoglobin reductase
• reduce methemoglobin to Hb
• Pyruvate
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Embden-Meyerhof Pathway
• Rate of glycolysis
• pH dependent
• phosphofructokinase & hexokinase: pH above 7.0
• during hypoxia due to _____________________
• Glycolysis affected by ATP
• ATP: glycolysis
• ATP: glycolysis
• Pyruvate kinase deficiency
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Hexose Monophosphate Shunt needed to reduce glutathione
• metabolize 5-10% of glucose used by the cell (aerobic)
• provide reducing potential for the cell
• by generating reduced NADPH
• G6P is catabolized (G6PD) to 6-PG instead of passing EMP
• activity: reduced glutathione (reduction of NADP+ to yield NAPH)
• defective: decreased reduced glutathione to neutralize oxidants
Mature Red Blood Cells

IV. Energy Metabolism

Sources of Energy
• Pathways for energy metabolism
Hexose Monophosphate Shunt
• Reduced glutathione (GSH)
• principal reducing agent n the cell
• reduced oxidized sulfhydryl groups in hgb and other proteins
• yields reduced sulfhydryl groups and oxidized glutathione (GSSG)

• Glutathione reductase & NADPH

• reduce GSSG to GSH
Mature Red Blood Cells

Sources of Energy
• Pathways for energy metabolism
Hexose Monophosphate Shunt
• R-5-P Ribose-5-phosphate
• generated by HMP
• used by nucleated cells during nucleic acid metabolism
• recycled back to EMP
• glyceraldehyde-3-phosphate & fructose-6-phosphate
• Common reactants in both pathways
Mature Red Blood Cells

Sources of Energy
• Pathways for energy metabolism
Methemoglobin Reductase Pathway
• prevent oxidation of the heme iron
• by reducing peroxide
Mature Red Blood Cells

Erythrokinetics
• Erythropoiesis
• Release from BM to PC
• Destruction and death
• Requirements for balanced erythron model:
• Normally functioning BM
• Normal EPO level
• Adequate nutrients
• Anemia
• Erythrocytosis
 Erythropoiesis
BONE
KIDNEY LIVER MARROW

Store Iron, Site of RBC’s

EPO
formation
Store protein,
Vit. B12 & Folic

Synthesize
globin

Produce
eryhtropoietin
(10%)
Mature Red Blood Cells

Erythrocyte Destruction
• Aging RBC
• Loss of sialic acid and lipids Liver
• Decreased ATP levels - Greater blood flow
• Increased calcium levels - More active role in the removal of
• RES severely damaged cells
• Intravascular and extravascular hemolysis
• Phagocytic cells (histiocyes, monocytes & macrophages)

Spleen
- Principal sites of RBC phagocytosis by tissue macrophages
after damage by normal aging
Mature Red Blood Cells

Erythrocyte Destruction
• Infants: 35-50 days
• Fetus: 60-70 days
• As RBC ages:
• membrane becomes less flexible
• concentration of cellular Hb increases
• enzyme activity (glycolysis) diminishes
Mature Red Blood Cells

EPO Production and Regulation

Production sites
• Kidney
• Liver
Regulation
• Hb O2 saturation
• 2,3-DPG levels
• pO2 of the plasma
• Hb concentration
• Erythrocyte mass
• Basal metabolic rate
Mature Red Blood Cells

EPO Production and Regulation

Regulation
• Prostaglandins
• Helps regulate EPO
• Enhance effect of EPO in CFU-E
• Estrogen
• Inhibit EPO production
References:
• Lotspeich-Steininger e.t al; Clinical hematology
principles, procedures, correlations, Lippincott
Company, 1992
• Turgeon, Clinical Hematology: Theory and Procedures
5ht ed., Lippincott Williams & Wilkins, 2012
• Keohane et. al, Rodak’s Hematology: Clincal Principles
6th ed., Elsevier, 2020
• DOH Dept Circ 2017-0173 Schedule of EQAS Application
 HEMOGLOBIN AND
HEMOGLOBINOMETRY
Hematology 1 Lecture
Hemoglobin
• Comprises 95% of the cytoplasmic
content of RBCs
• Concentration of Hemoglobin within
RBCs: ~34g/dL
• Molecular Weight: ~64,000 Daltons
• Main function: Transport Oxygen from
the lungs to tissues and transport CO2
from the tissues to the lungs
• Acid-base balance
Hemoglobin
• Red pigment
• Conjugated protein and a
Chromoprotein
Hemoglobin Structure
• Structure was described using X-
Ray Crystallography
• Heme Structure
• Protoporphyrin IX: Ring of Carbon,
Hydrogen, and Nitrogen atoms with
a central atom of divalent Fe2+
• Fe2+ reversibly combines with one O2
molecule
• Fe2+ Irons  Fe3+ they no longer can
bind O2.
• Position: In pockets of the
polypeptide chain
Hemoglobin Structure
• Globin Structure
• Four Globin chains in each
Hemoglobin molecule with 2
identical pairs of unlike Polypeptide
chains, 141 to 146 amino acids each
• Each Globin chain is divided into
eight helices separated by 7 non-
helical segments (Helices A to H)
Hemoglobin Molecule
• Structure of Hemoglobin
• Primary structure
• Amino Acid sequence of the Polypeptide
chains
• Secondary structure
• Chain arrangements in helices and non-
helices
• Tertiary structure
• Arrangement of the helices into a pretzel-
like configuration
• Quaternary structure (Tetramer)
• Describes the complete Hemoglobin,
spherical
Hemoglobin Molecule
• HEME IS A METALOPORPHYRINE
(Cyclic Tetrapyrrole)
• Structure of Iron-Protoporphyrin
IX
• Porphyrin Nucleus
• 4 Pyrrole Rings (Tetrapyrrole)
• Bridges: Methine (CH)
• Side chains (8)
• 4: Methyl (CH3)
• 2: Vinyl (CH=CH2)
• 2: Propionic Acid (CH3CH2COOH)
Hemoglobin Molecule
• Complete Hemoglobin
• Four Heme groups attached to four polypeptide chains, which contains 4 Iron
atoms, and can carry up to four molecules of Oxygen
• Hemoglobin A: Predominant adult Hemoglobin
• Composed of 2 α-globin chains and two β-globin chains
• Bonds between the α1-β2 and α2-β1 important to stabilize the quaternary structure in the
Oxygenated and deoxygenated form
• HbA1c: Glycated Hemoglobin A
• Glycation: Posttranslational modification formed by the nonenzymatic binding of various sugars to
globin chain amino groups over the lifespan of the RBC
• HbA1C: Glucose attaches to the N-terminal Valine of the β chain
• Normal value: 4-6% (Mean blood glucose level over the preceding 2-3 months)
Hemoglobin Biosynthesis
• Heme Biosynthesis
• Heme group: Protoporphyrin IX + Fe2+
• Steps in Heme biosynthesis:
1. Mitochondria: Glycine + Succinyl CoA (ALA
Synthase)  ALA
2. Cytoplasm: ALA dehydratase (Porphobilinogen
synthase) convert ALA to PBG
3. Cytoplasm: PBG  Hydroxymethylbilane 
Uroporphyrinogen III  Coproporphyrinogen III
4. Mitochondria: Coproporphyrinogen III 
Protoporphyrinogen IX  Protoporphyrin IX
5. Mitochondria: Protoporphyrin IX (Ferrochelatase
+ Iron)  Heme
Hemoglobin Biosynthesis
• Heme Biosynthesis
• Transferrin: Protein that carries Iron in Fe3+
to developing Erythroid cells
• Binds to transferrin receptors on erythroid
precursor cell membranes, then brought into
the cell in an endosome
• Acidification of endosome  Release of Iron
• Iron transported into the Mitochondria 
Reduced to Fe2+  United with Protoporphyrin
IX  Heme
Hemoglobin Biosynthesis
• Globin Synthesis
• Globin: Protein (Start 3rd week of
Gestation)
• Six structural genes code for six globin
chains
• α ζ globin genes: Ch 16p
• β γ δ ε genes: Ch 11p
• Production of globin genes:
Pronormoblast up to Polychromatic
Erythrocytes
• Production of α and β chains are in equal
amounts
Hemoglobin Biosynthesis
• Hemoglobin Assembly
• Affinity of globin chains
• α chain: positive charge, highest affinity
for β chains (negative charge)
• Affinity: γ, followed by δ
Hemoglobin Biosynthesis
• Hemoglobin Assembly
• Concentration of Hemoglobin
• HbA: Major Hemoglobin present from 6
months of age through adulthood
• HbA2: 3.5% of total Hgb in adults
• Low concentration due to mutation in the
δ globin chain
• HbF: 1-2% total Hgb in adults
Regulation of Hemoglobin Production
• Heme Regulation
• Limiting step: Negative Feedback
Mechanism by the production of Heme
• Heme inhibits:
• ALA synthase (PRIMARY)
• ALA dehydrase
• PBG deaminase
• Ferrochelatase
• ↑ demand for Heme = ↑ synthesis of ALA
synthase
Regulation of Hemoglobin Production
• Heme Regulation
• Lead Ions (Lead Poisoning)
• Inhibition of Porphobilinogen
synthase
• Inhibition of Ferrochelatase
Regulation of Hemoglobin Production
• Globin Regulation
• Critical to regulate:
• ↑ Globin chains, Protoporphyrin IX, Iron  Accumulation and Cell Damage  ↓ lifespan
• Main control: Transcription (complex interaction of DNA sequences and soluble
transcription factors that bind to DNA or to one another to promote or suppress
transcription)
Regulation of Hemoglobin Production
• Globin Regulation
• Requirements to initiate globin gene transcription:
1. The promoter DNA sequences should be immediately before the 5’ end or the beginning of
the gene
2. Presence of Krüppel-like Factor 1 (KLF1)
3. Presence of other transcription factors (GATA1, Ikaros, TAL1, p45-NF-32, LDB1)
4. Locus Control Region - Enhancer region of DNase 1 hypersensitive nucleic acid sequences
(20 kilobases upstream before the 5’ start site of the gene)
• Krüppel-like Factor 1 (KLF1)
• Regulation of switching from γ chain to β chain (γ- β switching)
• Regulation of expression of repressors of γ-globin gene transcription (BCL11A and MYB)
Regulation of Hemoglobin Production
• Globin Regulation
• Regulated during translation when mRNA coding for Globin chains associates
with ribosomes to produce Globin
• Heme: Important regulator of globin mRNA translation
• High heme: Promote activation of a translation initiation factor and inactivating its repressor
• Low heme: Repressor accumulates and inactivates initiation factor  Blocked translation of globin
mRNA
Systemic Regulation of Erythropoiesis
• Hypoxia: Decreased oxygen transport in tissues due to insufficient Hgb
quantity or defective transport system
• Detected by the peritubular cells of the Kidney  Increased production of EPO
• Reference Values
• Men : 13.5-18.0 g/dL (135-180 g/L)
• Women : 12.0-15.0 g/dL (120-150 g/L)
• Newborns : 16.5-21.5 g/dL (165-215 g/L)
• Variations in values
• Age Group
• High Altitudes
Hemoglobin Function
• Oxygen Transport
• Primary function of Hemoglobin
(High Oxygen affinity)
• ~1.34 mL of Oxygen is bound by
each gram of Hemoglobin
• Affinity of Hemoglobin for Oxygen =
Partial Pressure of Oxygen (Amount
of Oxygen needed to saturate 50%
of Hgb or P50 value)
• Relationship described by the Oxygen
Dissociation Curve of Hemoglobin
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Proportion of Hemoglobin in its
saturated form on the vertical axis
against the prevailing Oxygen tension
on the Horizontal axis
• Relates between:
• % Oxygen carrying capacity of
Hemoglobin
• Partial Pressure of Oxygen
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Description:
• Represents the relationship between
O2 concentration and the percentage
saturation of Hgb.
• Sigmoidal curve: Low Hgb affinity for
O2 at low O2 tension and high affinity
for O2 at high O2 tension
• Plateau portion: range that exists at
the pulmonary capillaries
• Steep portion: range that exists at the
systemic capillaries
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Horizontal Axis: pO2 (Amount of
Oxygen available)
• Normal 27 mmHg pO2 = 50% O2
saturation
• <27 mmHg: Shift to the Left
• >27 mmHg: Shift to the Right
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Vertical Axis: sO2 (Amount of
Hemoglobin saturated with Oxygen)
• Arterial Oxygen saturation
• Reference Interval: 96-100%
• Shift to the left: Higher %
Oxygen saturation and a higher
affinity for Oxygen
• Shift to the right: Lower Oxygen
affinity
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Changes in the pH of the blood
• Bohr Effect: Shift in the curve because of
pH change (or H+ concentration change)
• Lower pH: Shift to the right, reduced
Oxygen affinity, Hemoglobin more
readily releases Oxygen
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Curve starts when:
• pH 7.4
• 37˚C
• pCO2 at 40 mmHg
• Changes from these values are
called “shifts”
• Affinity: Oxygen’s attraction to
hemoglobin binding sites. Changes
with pH variations, Temperature,
CO2, and 2,3-DPG concentration
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Shift to the Left (B)
• Increased affinity for Oxygen and
release less to the tissues
• Acute Alkalosis
• Decreased pCO2
• Decreased temperature
• Low 2,3-DPG levels
• Carboxy-, Methemoglobins,
other abnormal Hemoglobins
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Shift to the Right (C)
• Decreased affinity for Oxygen
• Acute Acidosis
• Increased pCO2
• Increased temperature
• High levels of 2,3-DPG
• Abnormal Hemoglobin
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Factors affecting the curve
• Hydrogen Ion concentration
• Changes in concentration
• Bohr Effect: Describes
hemoglobin's lower affinity for
oxygen secondary to increases in
the partial pressure of carbon
dioxide and/or decreased blood
pH
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Factors affecting the curve
• Hydrogen Ion concentration
• Haldane Effect: Oxygenation of
blood in the lungs displaces
carbon dioxide from hemoglobin,
increasing the removal of carbon
dioxide.
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Factors affecting the curve
• Carbon Dioxide
• Affects the curve in 2 ways:
• Influences Intracellular pH
• CO2 accumulation causes
Carbamino compounds to be
generated
• Low Carbamino: Left
• High Carbamino: Right
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Factors affecting the curve
• 2,3-DPG
• Shifts:
• High Levels: Right
(Hypoxemia, Chronic Lung
Disease, Anemia, CHF)
• Low Levels: Left (Septic
Shock, Hypophosphatemia)
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of Hemoglobin
• Factors affecting the curve
• 2,3-DPG
• Tense or T Confirmation
• Deoxygenated state
• Stabilized by 2,3-DPG between the β
Globin chains
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of Hemoglobin
• Factors affecting the curve
• 2,3-DPG
• Relaxed or R State
• Oxygenated state
• What happens:
• Hydrophobic interaction at the
contact point
• Release of 2,3-DPG
• 15-degree rotation
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Factors affecting the curve
• Temperature
• Hyperthermia: Rightward
• Hypothermia: Leftward
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Effects of Methemoglobinemia and
Carboxyhemoglobinemia
• Methemoglobin – Hgb containing Fe3+
• Carboxyhemoglobin – CO in the blood
and attaches to Hemoglobin
• Fe3+ does not effectively bind to Oxygen
 Shift to the Left
• CO2  Hypoxemia  Normal pO2, shift
to the left
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of
Hemoglobin
• Myoglobin
• Oxygen-binding Heme protein in
tissues
• Has greater affinity to O2 than Hgb
• Curve indicates it released O2 at very
low partial pressures
• Seen in plasma during: Myocardial
Infarction, Trauma, Severe Muscle
Injury (Rhabdomyolysis)
Hemoglobin Function
• Oxygen Transport
• Oxygen Dissociation Curve of Hemoglobin
• Fetal Hemoglobin (HbF)
• Has a P50 of 19-21 mmHg (Left)
• Advantages:
• Provide more effective Oxygen
withdrawal from the maternal circulation
• Disadvantages:
• Low Fetal Arterial Oxygen pressure
• ↑ Affinity to O2 due to ↓ ability to bind
2,3-DPG
• RBC ct, Hgb, Hct, higher in Newborn than in
adults
 Different analytes
Hemoglobin Function in play:
CO2, H2O, H+,
H2CO3, Cl-
• Carbon Dioxide Transport
Hemoglobin Function
• Nitric Oxide Transport
• Nitric Oxide: Secreted by Vascular
Endothelial cells
• Relaxes vascular wall smooth muscle
• Vasodilation
• Hemoglobin: Preserve and transports NO
to hypoxic microvascular areas to
stimulate vasodilation and increase blood
flow (Hypoxic vasodilation).
Dyshemoglobins
• Dysfunctional Hemoglobins that are unable to transport Oxygen
• Methemoglobin
• Sulfhemoglobin
• Carboxyhemoglobin
• May accumulate to toxic levels
• Modifies Hemoglobin structure  Prevent O2 binding
Dyshemoglobins
• Methemoglobin
• Fe2+  Fe3+
• ↑ Methemoglobin, ↓ Decreased delivery of O2
• Accumulation limited to 1% only by
NADH-Methemoglobin reductase
pathway (NADH-Cytochrome b5
reductase)
• <25% Methemoglobin: Asymptomatic
• >30% Methemoglobin: Cyanosis, Hypoxia
• >50% Methemoglobin: Comatose, Death
Dyshemoglobins
• Methemoglobin
• Methemoglobinemia
• Acquired: Toxic Methemoglobinemia
• Exposure to Exogenous oxidants (Nitrites,
Primaquines, Dapsone, Benzocaine)
• Treatment:
• <25%: Withdrawal of the offending oxidant
• >30%: Intravenous Methylene Blue (Fe2+ 
Fe3+ by NADPH-Methemoglobin reductase
and NADPH produced by G-6-PD)
Dyshemoglobins
• Methemoglobin
• Methemoglobinemia
• Hereditary causes: Rare
1. NADH-cytochrome b5 reductase 3 gene
mutations (CYB5R3)  Diminished capacity to
reduce Methemoglobin
• Autosomal recessive
2. M Hemoglobin (HbM) Structurally abnormal
chain that favors Fe3+ than Fe2+
• Autosomal dominant pattern (30-50%
Methemoglobin)
• No effective treatment
Dyshemoglobins
• Methemoglobin
• Methemoglobinemia
• Testing
• CO-Oximeter (Spectrophotometer)
• Absorption peak at 630 nm
• Blood takes Chocolate Brown color and
does not revert back to Normal red color
after O2 exposure.
• Hemoglobin Electrophoresis
• HPLC
• DNA Mutation Testing: HbM variants
• Enzyme Assays and DNA mutation testing:
Cytochrome b5 reductase 3 deficiency
Dyshemoglobins
• Sulfhemoglobin
• Irreversible Oxidation of Hemoglobin by drugs
(Sulfanilamides, Phenacetin, Nitrites,
Phenylhydrazine) or exposure to sulfur chemicals
• Cannot be converted to HbA  Persists for the life of the
cell
• Normal concentration: <0.37 g/L
• Addition of sulfur to the pyrrole ring of Heme
(Greenish pigment)
• Ineffective for Oxygen transport  Cyanosis
• ↑ Sulfhemoglobin, ↓ Decreased delivery of O2
Dyshemoglobins
• Sulfhemoglobin
• Treatment: Prevention by avoidance of the offending
agent
• Detection: CO-Oximeter
• SulfHb concentration must be > 5 g/L to be clinically
detectable
• Similar peak with MethHb (630 nm)
• Differentiate with Methemoglobin by:
• Checking spectral curve
• Sulfhemoglobin: does not shift when Cyanide is added
• Methemoglobin: shifts when Cyanide is added
Dyshemoglobin
• Carboxyhemoglobin (COHb)
• CO + Heme  COHb
• Affinity of CO is 240x to Hemoglobin than
O2
• Once associated  Left shift
• “Silent Killer” – Odorless, Colorless
(Hypoxia)
• Produced endogenously (<2% of total Hgb)
• Exogenous CO: Exhaust of cars, tobacco
smoking, coal gas, charcoal burning
• Tobacco smokers: Up to 15% COHb
• Higher Hct, Polycythemia (compensation)
Dyshemoglobin
• Carboxyhemoglobin (COHb)
• CO exposure: Coincidental, Accidental,
Intentional
• Symptoms:
• 20-30% COHb: Headache, Dizziness,
Disorientation
• >40% COHb: Coma, Seizure, Hypotension,
Cardiac Arrhythmias, Pulmonary Edema,
Death
Dyshemoglobin
• Carboxyhemoglobin (COHb)
• Testing
• Spectral Absorption of 540 nm
• Blood: Cherry Red Color, may be imparted to
victim’s skin
• Diagnosis for CO poisoning if:
• >3% COHb if nonsmoker
• >10% COHb if smoker
• Treatment:
• Remove CO source and administer 100% O2
• Prevent neurologic and cognitive impairment after
acute CO poisoning if COHb is >25%
Hemoglobin quantification and
identification
• Methods
• High Performance Liquid
Chromatography
• Electrophoresis
• Used for:
• Fractionation
• Presumptive identification
• Quantification
Hemoglobin Determination
• Cyanmethemoglobin Method
• Reference method for Hemoglobin assay
• 𝐻𝑏 𝐹𝑒 + 𝐾 𝐹𝑒(𝐶𝑁) → 𝑀𝑒𝑡ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 + 𝐹𝑒 𝐾𝐶𝑁 → 𝐶𝑦𝑎𝑛𝑚𝑒𝑡ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛
• Absorbance: 540 nm (Directly proportional to Hemoglobin concentration)
• Sulfhemoglobin: Not converted to Cyanmethemologin (Not measured)
• Carboxyhemoglobin, Methemoglobin: Can be converted
Hemoglobin Determination
• Cyanmethemoglobin Method
• Sources of Errors:
• Light sensitivity
• Store in brown bottle or dark place
• Cell constituents
• High WBC count >20 x 109/L
• High Platelet count >700 x 109/L
• Turbidity and a Falsely high result
• Lipemia
• Turbidity and Falsely high results
• Correction: Add 0.01 mL of patient’s plasma to 5 mL
of Cyanmethemoglobin reagent as the reagent blank
Hemoglobin Determination
• Cyanmethemoglobin Method
• Sources of Errors:
• Hemoglobin S, Hemoglobin C
• Resistant to Hemolysis (Turbidity)
• Correction: 1:2 dilution sample:dH2O and multiply results from the standard curve by 2
• Abnormal Globulins
• Seen in: Plasma Cell Myeloma, Waldenström’s Macroglobulinemia
• May precipitate in the above conditions
• Correction: Add 0.1 g Potassium Carbonate to the Cyanmethemoglobin reagent
Hemoglobin Determination
• Cyanmethemoglobin Method
• Testing comments:
• Carboxyhemoglobin: 1 hour conversion to
Cyanmethemoglobin
• Can cause erroneous results in smokers’
specimens
• Handheld systems for Hgb measurement
• HemoCue: Lesser interference from turbidity
• Hemoglobin  Azidemethemoglobin
• Read photometrically at 570 and 880 nm
• Sodium Lauryl Sulfate addition
• Hemoglobin  SLS-Hemoglobin
• Does not generate toxic wastes
• AVOXimeter 1000E: Measure total Hgb
spectrophotometrically
THANK YOU!!!