Erythrocyte morphology and hemoglobin

Erythrocytes, or red blood cells (RBCs), are the most common blood cells. Normal RBCs have a biconcave shape and contain hemoglobin but no nucleus or organelles. Dysmorphic RBCs (e.g., sickle cells, target cells) have an altered form and are often a sign of an underlying condition. Hemoglobin (Hb) is composed of heme and globin subunits and is responsible for transporting oxygen and carbon dioxide throughout the body. Hb can undergo conformational changes (e.g, depending on its oxygenated state), which influence how it binds and releases O₂ and CO₂. Deficient Hb (anemia), genetic Hb variants (e.g., HbS, HbC), and certain substances (carbon monoxide, nitrates that form methemoglobin, cyanide) affect the affinity for and ability to transport O₂, resulting in decreased tissue oxygenation.

Normal morphology

• No nucleus, cell organelles, or granules
• Biconcave shape
  • Maintained by spectrin, a cytoskeletal protein
  • Leads to a large surface area to volume ratio, enabling rapid gas exchange
  • Provides great flexibility, allowing for easy movement through narrow vessels and capillaries
• Contain hemoglobin, which leads to acidophilic cytoplasm

Dysmorphic RBCs

• Anisocytosis: the presence of RBCs of varying sizes
• Poikilocytosis: the presence of RBCs of abnormal shapes

HALT when you see a Target! Associated etiologies of target cells are: H – Hemoglobinopathies (HbC/HbS), A – Asplenia, L – Liver disease, T – Thalassemia

Dacryocytes (teardrop cells) are formed via extramedullary erythropoiesis: “Don't cry, we can play outside to-marrow.”

RBCs with inclusion bodies

The lifespan of RBCs is about 120 days. After this time, macrophages in the reticuloendothelial system of the bone and the spleen phagocytose RBCs. They are then broken down and their parts recycled.

Globin metabolism

Globin chains are released and converted into amino acids.

Hemoglobin metabolism

Process: heme (red) → biliverdin (green pigment) → bilirubin (yellow pigment)

1. Heme is converted to biliverdin by heme oxygenase.
   • Requires NADPH + H⁺
   • Carbon monoxide (CO) is released.
   • Iron is released, oxidized, and taken up by transferrin to be recycled.
2. Biliverdin is converted to bilirubin by biliverdin reductase (requires NADPH + H⁺)

Heme breakdown is responsible for the color changes in hematomas.

Bilirubin metabolism

1. Unconjugated bilirubin (insoluble in water) is released into the blood by macrophages → binds to albumin and reaches the liver
2. Unconjugated bilirubin is converted into bilirubin via the enzyme UDP-glucuronosyltransferase in the liver.
   • Bilirubin is conjugated with glucuronic acid → bilirubin diglucuronide = conjugated bilirubin (water soluble)
   • Most conjugated bilirubin is excreted into the GI tract via bile, while some is released into the blood.
3. Conjugated bilirubin excreted in bile is broken down by GI bacteria into urobilinogen
   • Most urobilinogen is converted to stercobilin → excreted in feces (brown color).
   • Part of urobilinogen oxidates to urobilin → excreted in the urine (yellow color).

Indirect (unconjugated) bilirubin is insoluble in water.

2,3-bisphosphoglycerate shunt

• 2,3-bisphosphoglycerate mutase is vital for the formation of 1,3-bisphosphoglycerate (intermediate in glycolysis) → 2,3-BPG
  • 2,3-BPG mutase is unique to erythrocytes and placental cells.
• 2,3-BPG binds to hemoglobin → conformational change → release of oxygen into tissue
• 2,3-BPG binds with greater affinity to deoxygenated hemoglobin than oxygenated hemoglobin.

Energy production

• Mature RBCs are unable to generate energy (ATP) via the Krebs cycle because they lack mitochondria.
• Glucose is the main source of energy for RBCs.
• Biochemical energy-producing pathways available in RBCs
  • Glycolysis (catabolizes 90% of the available glucose) ^[1]
  • Pentose-phosphate pathway
  • Glutathione reduction (a reaction that converts oxidized glutathione to its reduced form)

Overview

Hb is a circulating globular protein composed of a heme moiety with a central iron ion and four subunits of globin.

• The main function of Hb is to take up O₂ from the lungs and deliver it to tissues.
• It can undergo conformational changes (e.g., depending on its state of oxygenation), which influence how it binds and releases O₂ and CO₂.
• Deficient or defective Hb can ultimately affect the transport of O₂ (see “Hemoglobin variants” below for details).
• For more information about disorders of Hb, see “Anemia.”

Heme synthesis

• Heme is synthesized from protoporphyrin, a porphyrin.
• Porphyrins: a group of intermediates in the heme synthesis pathway that is composed of four subunit rings
  • Includes porphobilinogen and uroporphyrinogen
  • Binding of a central ferrous iron (Fe²⁺) forms a porphyrin complex (i.e., heme).
• The steps of heme synthesis occur both in the cytoplasm and the mitochondria (the first and final steps occur in mitochondria).

Sideroblastic anemia is due to ineffective heme synthesis, which may be congenital (X-linked defect in the δ-ALA synthase gene) or acquired (e.g., vitamin B₆ deficiency, or lead poisoning leading to sequential inhibition of δ-ALA dehydratase and ferrochelatase).

Globin

• Globin is an integral part of the Hb molecule.
• Tetramer consisting of four individual polypeptide subunits that bind heme; composed of amino acids that fold to form 8 alpha helices
• There are 6 types of globin chains.
  • β, δ, γ, ε are encoded on chromosome 11.
  • α and ζ are encoded on chromosome 16.
• The combination of subunits in the Hb molecule determines the type of Hb (e.g., embryonic, fetal, newborn, or adult Hb); each subunit is able to bind one O₂ molecule
• Mutations in genes encoding globin results in Hb variants.

Hemoglobin patterns

For genetic variants of hemoglobin patterns, see “Hemoglobin variants” below.

Sequence of switching from fetal to adult hemoglobin (γ-globin in HbF is replaced by β-globin in HbA, and α-globin is always present in both HbA and HbF): Gamma goes, Beta becomes, Alpha always.

Overview

• O₂, CO₂, and protons all bind Hb and influence one another's affinity to Hb, which is important for gas exchange.
  • Hb releases CO₂ more easily with increasing pO₂ (Haldane effect).
  • Hb binds CO₂ more easily if the pO₂ is low.
  • Hb releases O₂ easier with increasing H⁺(Bohr effect).
• CO₂ is mainly carried in three forms in the body:
  • Plasma bicarbonate, HCO₃^- (70%)
  • Bound to Hb (carbamino-Hb, HbCO₂) at the N-terminus of globin (does not compete with O₂ at heme)
  • Dissolved in blood (5%)

Oxygenation and deoxygenation of Hb

• Oxyhemoglobin
  • Hemoglobin with oxygen bound to its heme component (oxygenated) → bright red blood
  • Oxygenated hemoglobin has approximately 300 times higher affinity for oxygen compared to deoxygenated hemoglobin
  • Exhibits positive cooperativity and positive allostery
• Deoxyhemoglobin
  • Hemoglobin with no oxygen bound to its heme component (deoxygenated) → dark red blood
  • Blue to purple appearance of tissue during hypoxia → cyanosis
  • Deoxygenated hemoglobin has a low affinity for oxygen → release of oxygen is promoted

Bicarbonate buffer system

• RBCs carry carbonic anhydrase, which converts HCO₃^- and H⁺ to H₂O and CO₂ in the following steps: HCO₃⁻ + H⁺ ⇄ H₂CO₃ ⇄ H₂O + CO₂
• Ultimately, excess H⁺ during acidic states is eliminated through conversion to CO₂, which can be exhaled.
• During basic states, the bicarbonate buffer system can reverse so that CO₂ is converted to HCO₃⁻ + H⁺.
  • Chloride shift: Excess intracellular HCO₃⁻ produced this way is released into the plasma in exchange for Cl^-.
• This phenomenon makes HCO₃^- the most important buffer in the body.
• For more details on the buffering mechanisms of the body, see “Compensation.”

Bohr effect

• The O₂ affinity of Hb is inversely proportional to the CO₂ content and H⁺ concentration of blood.
• High CO₂ and H⁺ concentrations (from tissue metabolism) cause decreased affinity for O₂ → O₂ that is bound to Hb is released to tissue (the O₂-Hb dissociation curve is shifted to the right).
  • HbO₂ + H⁺ ⇄ H⁺Hb + O₂
  • HbO₂ + CO₂ ⇄ Hb-COO^- + H⁺ + O₂

Haldane effect

• The CO₂ affinity of Hb is inversely proportional to the oxygenation of Hb.
• When Hb is deoxygenated (typically in peripheral tissue), uptake of CO₂ is facilitated.
• When Hb is oxygenated (in high pO₂, for example, in the lungs):
  • Oxygenated Hb has a decreased affinity for CO₂ → CO₂ that is bound to Hb is released in the pulmonary arteries to diffuse into the alveoli (the O2-Hb dissociation curve is shifted to the left).
  • Hb releases bound H⁺; → ↑ H⁺ shifts equilibrium to CO₂ production (see equation above) → CO₂ is exhaled in lungs

Overview

• The O₂-Hb dissociation curve shows the arterial partial pressure of O₂ (PaO₂) in relation to the percentage saturation of Hb, i.e., the binding affinity of Hb for O₂.
• Relationship between saturation and partial pressure of O₂ is not linear
• Reflected in the sigmoidal shape (S-shape) of the curve, which begins flat, then rises steeply before plateauing.
• Due to positive cooperativity (the phenomenon by which, e.g., binding of one O₂ molecule to a single heme increases the O₂ affinity of hemoglobin, facilitating the binding of subsequent O₂ molecules).
• Myoglobin is composed of a single polypeptide chain that is associated with a monomeric heme molecule.
• Therefore, it cannot show positive cooperativity and its curve is not sigmoidal.
• The binding affinity of Hb is influenced by external factors that may lead to a left or right shift of the O₂ dissociation curve (ODC).

Shift to the right of the oxygen dissociation curve

• ↓ Hb affinity for O₂ → ↑ O₂ dissociation from Hb → ↑ tissue oxygenation → ↑ P₅₀ (the partial pressure of oxygen needed to maintain 50% of Hb saturated)
• Causes of shift to the right include:
  • ↑ Partial pressure of carbon dioxide (↑ PCO₂)
  • ↑ Body temperature (e.g., fever)
  • ↑ H⁺ (↓ pH)
  • ↑ 2,3-BPG (generated by 2,3-BPG mutase during erythrocyte glycolysis)
  • ↑ Exercise
  • ↑ Altitude

Shift to the left of the oxygen dissociation curve

• ↑ Hb affinity for O₂ → ↓ O₂ dissociation from Hb → ↓ tissue oxygenation → ↑ erythropoietin synthesis (due to renal hypoxia) → compensatory erythrocytosis
• Causes of shift to the left include:
  • ↓ Partial pressure of carbon dioxide (PCO₂)
  • ↓ Body temperature
  • ↓ H⁺ (↑ pH)
  • ↓ 2,3-BPG (e.g., due to mutations in the BPGM gene on chromosome 7) ^[4]
  • ↑ CO
  • ↑ Methemoglobin
  • ↑ Fetal hemoglobin (HbF)

Overview

Arterial oxygen content (CaO₂)

• Definition: the sum of oxygen bound to hemoglobin and dissolved in plasma within arterial blood
• Formula: arterial oxygen content (CaO₂, mL of oxygen per 100 mL of blood) = (1.34 x Hb x SaO₂) + (0.003 x PaO₂)
  • Hb (g/dL) = hemoglobin concentration
  • SaO₂ (%) = arterial oxygen saturation in hemoglobin
  • PaO₂ (mm Hg) = arterial oxygen saturation in plasma (partial pressure of oxygen)
  • 1.34 (mL) = maximum oxygen binding capacity of 1 g of hemoglobin. Normal blood Hb concentration is approximately 15 g/dL. The oxygen-binding capacity of blood is, therefore: 15 g/dL x 1.34 mL ≈ 20 mL of oxygen per dL of blood.
  • 0.003 = solubility coefficient of oxygen in plasma
• Arterial oxygen content is directly proportional to the Hb, SaO₂, and PaO₂.

Oxygen delivery (DO₂)

• Definition: the rate at which oxygen is transferred from the lungs to the peripheral circulation
• Formula: oxygen delivery (DO₂, mL O₂/min) = cardiac output (CO) x arterial oxygen content (CaO₂)
  • Oxygen delivery to an organ = organ blood flow rate x arterial oxygen content (CaO₂). For example, renal oxygen delivery (renal DO₂) = renal blood flow rate (RBF) x arterial oxygen content (CaO₂).
  • In an adult with a cardiac output of 5 L/min, the oxygen delivery is ∼ 1000 mL/min.
• Oxygen delivery is directly proportional to cardiac output and arterial oxygen concentration.

Carbon monoxide poisoning

• Formation of carboxyhemoglobin: hemoglobin that is removed from oxygen transportation because its oxygen binding site is occupied by carbon monoxide
• See “Carbon monoxide intoxication.”

Methemoglobinemia ^[5][6]

• Methemoglobin
  • A form of hemoglobin that contains iron in its oxidized (ferric = Fe³⁺) rather than its reduced state (ferrous = Fe²⁺) and therefore cannot bind oxygen.
  • Ferric iron binds less readily to oxygen compared to ferrous iron, leading to a decrease in blood oxygen saturation and total oxygen content (can lead to tissue hypoxia).
  • Ferric iron has a high affinity for cyanide (therefore, in cyanide poisoning, amyl nitrite is given → Fe²⁺ turns into Fe³⁺→ formation of methemoglobin → binding of free cyanide in the blood to methemoglobin → less cyanide binding to cytochrome c oxidase)
• Methemoglobin levels
  • 0–3% of total hemoglobin: physiological
  • > 3%: visible cyanosis (skin and membranes are brownish-blue or gray)
  • > 20%: clinical symptoms of oxygen deprivation; brown blood ("chocolate-colored blood")
  • > 70%: fatal
• Causes of methemoglobinemia
  • Exposure to substances that increase methemoglobin levels (most common cause)
    • Nitrate/nitrite-containing compounds (e.g., drinking water, meat and cheese preservatives)
      • These compounds are partially converted to nitrite in the gastrointestinal tract → nitrite oxidizes iron from its reduced state to its oxidized state, catalyzing the conversion of hemoglobin to methemoglobin.
    • Drugs: nitroglycerin, sulfonamides, dapsone, inhaled nitric oxide, topical anesthetics such as lidocaine or benzocaine, and aniline derivatives
    • Nitro and amino compounds of benzene
  • In newborns and infants, the enzyme activity of methemoglobin reductase (NADH-cytochrome b5 reductase) is low → ↑ risk of methemoglobinemia
  • Congenital (hereditary) methemoglobinemia: autosomal recessive deficiency of methemoglobin reductase
  • Glucose-6-phosphate dehydrogenase deficiency
• Diagnosis
  • Dark brown (“chocolate”) discoloration of the blood
  • Reduced oxygen saturation and total oxygen content with normal PaO₂ on arterial blood gas analysis despite clinical signs of cyanosis
  • Confirm with CO-oximetry.
• Treatment
  • Methylene blue
    • At lower concentrations, it is highly effective at reducing methemoglobin to hemoglobin.
    • Contraindications: G6PD deficiency , concomitant use of serotoninergic drugs
  • Reducing agents such as ascorbic acid (vitamin C) and riboflavin may sometimes help to reduce methemoglobin to hemoglobin.
  • In acquired methemoglobinemia: Assess for pharmaceutical triggers and cease therapy with any agents thought to be the cause.
  • Last resort for patients who do not respond to methylene blue: exchange transfusion

Because MetHb artificially increases pulse oximeter readings, oxygen concentration measured via pulse oximetry remains high (> 80%) even if methemoglobin levels are very high!

To remember that ferrous iron (Fe2+) is the reduced form of iron, think: “Joe reduced 2 ferocious animals from his iron cage.

• Anemia
• Iron deficiency anemia
• Hemolytic anemia
• Vitamin B₁₂ deficiency
• Folate deficiency
• Thalassemia
• Sickle cell anemia
• Hemolytic anemia
• Hereditary spherocytosis
• Glucose-6-phosphate dehydrogenase deficiency