14.1 Respiratory Biochemistry: Hemoglobin, Oxygen Binding, Hemoglobinopathies, Thalassemias, Methemoglobin, Buffers & Acid-Base Balance

Why this area matters

The TOS lists "chemistry of respiration, role of hemoglobin (oxygen-hemoglobin dissociation curve, hemoglobinopathies), acid-base balance/imbalance". Thalassemia and G6PD deficiency are common in Filipinos. Methemoglobinemia from dapsone or nitrites, and carbon monoxide from generators, are recurring emergencies.

Structure of hemoglobin and myoglobin

• Heme is a protoporphyrin IX ring with central ferrous iron (Fe2+). It is bound to the proximal histidine (F8) of each globin chain, and the distal histidine helps limit CO binding.
• Myoglobin has one chain and one heme. It has a hyperbolic dissociation curve and high affinity (P50 about 1 mmHg), which suits oxygen storage in muscle.
• Hemoglobin is a tetramer (adult HbA is alpha-2 beta-2). Its sigmoid curve suits loading oxygen in the lungs and unloading it in the tissues.
• Glycated hemoglobin (HbA1c) forms non-enzymatically in proportion to average glucose over the red-cell lifespan (about 3 months).
• Globin gene clusters:
  • alpha genes (two copies on each chromosome 16, four in total);
  • beta, delta and gamma genes on chromosome 11.
  • The switch from gamma to beta production occurs around birth, so beta-chain diseases appear after about 6 months.

The chemistry of respiration

• Oxygen: about 98% travels bound to hemoglobin. Each gram of Hb carries about 1.34 mL O2 when fully saturated.
• Carbon dioxide:
  • mostly as bicarbonate: red-cell carbonic anhydrase converts CO2 and H2O to H2CO3, which gives H+ and HCO3-, and HCO3- leaves the cell in exchange for Cl- (the chloride shift);
  • partly as carbamino-hemoglobin;
  • partly dissolved.
• Deoxyhemoglobin is a better buffer and binds more CO2 (the Haldane effect), while H+ and CO2 promote oxygen release (the Bohr effect).

Hemoglobin Molecular Biophysics and Pathologic Hemoglobinopathies

Hemoglobin is a globular heterotetramer composed of four globin subunits, each containing an iron-protoporphyrin IX (heme) moiety capable of binding one molecule of $O_2$. Normal adults express $96-98\%$ HbA ($\alpha_2\beta_2$), $1.5-3.5\%$ $\text{HbA}_2$ ($\alpha_2\delta_2$), and $<1\%$ HbF ($\alpha_2\gamma_2$).

Allosteric Cooperativity and the Bohr Effect

Hemoglobin exhibits a sigmoidal oxygen-dissociation curve reflecting positive cooperativity: oxygen binding to one heme subunit induces a quaternary conformational shift from the low-affinity T (Taut / Deoxygenated) state to the high-affinity R (Relaxed / Oxygenated) state, facilitating binding to remaining subunits.

• Rightward Shift of the $O_2$-Hb Curve (Increased $P_{50}$ / Facilitates Tissue Oxygen Unloading): Stabilized by factors favoring the T-state (Mnemonic: "CADET, face RIGHT"): Carbon dioxide ($PCO_2$), Acid (decreased pH / elevated protons, the Bohr Effect), 2,3-DPG (2,3-BPG), Exercise, and Temperature.
• Leftward Shift (Decreased $P_{50}$ / Tighter Oxygen Binding): Decreased $H^+$, decreased $PCO_2$, decreased 2,3-BPG, hypothermia, fetal hemoglobin (HbF), carboxyhemoglobin, and methemoglobin.
• Fetal Hemoglobin (HbF): $\gamma$-globin chains substitute neutral serine for positively charged histidine at position 143, eliminating positive charges in the central 2,3-BPG binding pocket. HbF binds 2,3-BPG poorly, resulting in higher affinity for $O_2$ than maternal HbA, facilitating transplacental oxygen transfer.

Hemoglobinopathies

• Sickle Cell Disease (HbS): Point mutation in the $\beta$-globin gene (codon 6: $\text{GAG} \to \text{GTG}$), replacing hydrophilic, negatively charged Glutamic Acid with hydrophobic, neutral Valine on the outer surface. In the deoxygenated (T) state, valine interacts with a hydrophobic receptor pocket on adjacent chains, polymerizing into rigid crystalline fibers that deform erythrocytes into sickled shapes, causing vaso-occlusive crises, autosplenectomy, and hemolysis.
• Hemoglobin C Disease (HbC): Point mutation at codon 6 of $\beta$-globin replacing Glutamic Acid with positively charged Lysine ($\text{GAG} \to \text{AAG}$; Mnemonic: "Lysine = C (Ly-C-ne)"). Produces mild hemolytic anemia and HbC crystals.
• Methemoglobinemia: Heme iron is oxidized from the normal ferrous ($Fe^{2+}$) state to the ferric ($Fe^{3+}$) state. Ferric iron cannot bind oxygen, and it allosterically shifts the remaining ferrous hemes to the high-affinity left state, preventing oxygen release to tissues.
  • Causes: Exposure to oxidizing drugs: nitrites/nitrates, sulfonamides, dapsone, local anesthetics (benzocaine, lidocaine). Congenital deficiency of NADH-cytochrome $b_5$ reductase.
  • Manifestations: Cyanosis unresponsive to supplemental $100\%$ oxygen; normal arterial $PO_2$ with severely reduced calculated oxygen saturation; pathognomonic "chocolate-brown" blood.
  • Treatment: Intravenous Methylene Blue, which is reduced by NADPH-dependent methemoglobin reductase to leucomethylene blue, which non-enzymatically reduces $Fe^{3+}$ back to $Fe^{2+}$. High-dose Ascorbic Acid (Vitamin C) acts as an adjunct.

Thalassemias: molecular basis

A microcytosis with normal or high RBC count and normal iron studies suggests thalassemia trait rather than iron deficiency. Iron does not help it and can harm.

Carbon monoxide and other abnormal hemoglobins

• Carboxyhemoglobin: CO binds about 200 to 250 times more avidly than oxygen and shifts the remaining curve left. Pulse oximetry falsely reads normal, so use CO-oximetry. Treat with 100% oxygen, which shortens the CO half-life from about 4 to 5 hours to about 1 hour; consider hyperbaric oxygen when severe.
• Methemoglobin:
  • Antidote: methylene blue needs NADPH (from G6PD) to work. In G6PD deficiency it is ineffective and can cause hemolysis, so use ascorbic acid or exchange transfusion instead.
  • Pulse oximetry tends to read about 85% regardless of the true value.
  • Common triggers in Philippine practice include dapsone (leprosy, dermatology), topical benzocaine, nitrites (well water, preserved meats) and aniline dyes.
• Sulfhemoglobin (from sulfur drugs) is irreversible.

Buffers and acid-base biochemistry

Buffers resist pH change best within about 1 unit of their pKa:

• Bicarbonate-carbonic acid (pKa 6.1). It is effective despite its low pKa because CO2 is regulated by the lungs and HCO3- by the kidneys, making it an "open" system.
• Hemoglobin and plasma proteins: histidine imidazole groups with pKa near 6 to 7. These are the main intracellular buffers.
• Phosphate (pKa about 6.8): important inside cells and in urine (titratable acid).
• Ammonia/ammonium: renal excretion of acid.

Henderson-Hasselbalch equation:

$$\text{pH} = 6.1 + \log\frac{[\text{HCO}_3^-]}{0.03 \times P_aCO_2}$$

With HCO3- of 24 mEq/L and PaCO2 of 40 mmHg, the ratio is 24/1.2 = 20, and log 20 is about 1.3. So pH = 6.1 + 1.3 = 7.4.

Biochemical causes of metabolic acidosis:

• High anion gap: acids are added.
  • Lactic acid: tissue hypoxia, shock, metformin, cyanide, mitochondrial disease.
  • Ketoacids: diabetic, starvation and alcoholic ketoacidosis.
  • Toxic alcohols: methanol forms formic acid; ethylene glycol forms glycolic and oxalic acid, with oxalate crystals.
  • Salicylates.
  • Uremia.
• Normal anion gap (hyperchloremic): bicarbonate is lost, as in diarrhea, renal tubular acidosis, and acetazolamide.

Other disturbances:

• Metabolic alkalosis: vomiting (loss of HCl), diuretics, mineralocorticoid excess.
• Respiratory acidosis: hypoventilation (opioids, COPD, neuromuscular weakness).
• Respiratory alkalosis: hyperventilation (anxiety, altitude, early salicylate toxicity, sepsis).

Compensation formulas are reviewed in renal physiology.