RBC Physiology & Hemoglobin
How the oxygen curve shifts, and why Fe3+ and CO turn blood the wrong color
Erythropoiesis and the mature red cell
Erythropoietin (EPO). Peritubular interstitial cells of the renal cortex sense hypoxia through hypoxia-inducible factor (HIF-1α) and release EPO, which stimulates erythroid progenitors in the marrow to survive and mature. Low EPO explains the anemia of chronic kidney disease; hypoxia or an EPO-secreting tumor such as renal cell carcinoma causes secondary polycythemia.
Reticulocytes. Newly released RBCs retain residual ribosomal RNA, stain with new methylene blue, and appear slightly large and bluish on a routine smear. The normal count is about 0.5 to 2.5%. Because anemia inflates the percentage, it is corrected (retic % × patient Hct/45): a corrected count over about 2–3% means an appropriate marrow response (hemolysis or blood loss), and a low count means hypoproliferation.
The mature RBC. With no nucleus, mitochondria or other organelles, the RBC makes ATP only by anaerobic glycolysis (glucose enters by insulin-independent GLUT1) and uses the HMP (pentose phosphate) shunt for NADPH. It lives about 120 days; senescent cells are cleared by splenic macrophages (extravascular hemolysis), and heme is recycled to bilirubin.
Hemoglobin and the O2 dissociation curve
Structure. Hemoglobin is a tetramer of four globin chains, each holding a heme: protoporphyrin IX with ferrous iron (Fe2+). Adult HbA (α2β2) is about 97% of the total, HbA2 (α2δ2) 2–3%, and fetal HbF (α2γ2) dominates before birth but is under 1% in adults.
Fetal hemoglobin. γ Chains bind 2,3-BPG poorly, so HbF has HIGHER O2 affinity (left-shifted curve) and pulls O2 across the placenta from maternal HbA. The switch to HbA occurs by about 6 months of age, which is why β-chain diseases (sickle cell disease, β-thalassemia) present after infancy. Raising HbF with hydroxyurea dilutes HbS and lessens sickling.
Cooperative binding. The curve is sigmoidal because hemoglobin toggles between the taut T state (deoxy, low affinity) and the relaxed R state (oxy, high affinity), and each O2 bound eases binding of the next. Myoglobin, a monomer, has a hyperbolic curve. A right shift raises the P50, lowers affinity and unloads O2 to tissues; a left shift raises affinity and holds O2.
- Right shift ("CADET face Right"): ↑CO2, Acid (↓pH, the Bohr effect), ↑2,3-BPG (2,3-DPG), Exercise, ↑Temperature, plus high altitude.
- Left shift: the opposites (↓CO2, alkalosis, ↓2,3-BPG, hypothermia) plus HbF, carbon monoxide and methemoglobin.
2,3-BPG. Made in the Rapoport-Luebering side branch of glycolysis, 2,3-bisphosphoglycerate binds the central cavity of DEOXYhemoglobin (T state), lowering O2 affinity and promoting release. It rises with chronic hypoxia, high altitude and anemia to improve tissue delivery. Stored blood is low in 2,3-BPG, so transfused RBCs initially release O2 poorly.
Dyshemoglobins: methemoglobin and carbon monoxide
Methemoglobinemia. Heme iron oxidized to the ferric state (Fe3+) cannot bind O2, and it left-shifts the remaining heme, so delivery suffers twice. Oxidant triggers are dapsone, nitrites, benzocaine and other local anesthetics, and sulfonamides; the congenital form is cytochrome b5 reductase deficiency. Blood is chocolate-brown, cyanosis does not improve with O2, and there is a saturation gap: PaO2 is normal but measured SaO2 is low. Methylene blue, reduced by NADPH-dependent methemoglobin reductase, converts Fe3+ back to Fe2+.
Methylene blue is regenerated by NADPH, so it can fail (and cause hemolysis) in G6PD deficiency.
Carbon monoxide. CO binds hemoglobin with roughly 200–250 times the affinity of O2, forming carboxyhemoglobin (COHb) that cannot carry O2 and LEFT-shifts the curve, worsening delivery; CO also inhibits cytochrome c oxidase. Victims of fires, faulty heaters or exhaust have headache, nausea, confusion and cherry-red skin. PaO2 is normal, and standard pulse oximetry reads FALSELY normal because it cannot distinguish COHb from oxyhemoglobin; co-oximetry measures COHb directly. Treat with 100% O2, hyperbaric for severe cases.
| Feature | Methemoglobin | Carboxyhemoglobin |
|---|---|---|
| Heme iron | Oxidized to Fe3+ | Stays Fe2+, CO bound |
| Causes | Dapsone, nitrites, benzocaine, sulfonamides; cytochrome b5 reductase deficiency | Smoke, heaters, engine exhaust |
| Look | Chocolate-brown blood, cyanosis | Cherry-red skin |
| Saturation | Low measured SaO2, normal PaO2 (gap) | Pulse oximetry falsely normal; PaO2 normal |
| Curve | Left shift | Left shift |
| Treatment | Methylene blue | 100% or hyperbaric O2 |
Smoke inhalation can add cyanide, which also blocks cytochrome c oxidase; nitrites were classically used to induce methemoglobin that binds cyanide, and hydroxocobalamin binds it directly.
Red cell enzyme defects
G6PD deficiency. G6PD is the rate-limiting HMP-shunt enzyme, generating the NADPH that keeps glutathione reduced against oxidative stress. In X-linked G6PD deficiency, common in Mediterranean and African populations (it offers some protection against malaria), oxidants denature hemoglobin into Heinz bodies, which splenic macrophages pit out, leaving bite cells. Hemolysis is episodic and follows triggers: fava beans, infection, dapsone, primaquine, sulfonamides such as TMP-SMX, and nitrofurantoin.
Pyruvate kinase deficiency. Pyruvate kinase is the last glycolytic enzyme. Autosomal recessive deficiency starves the RBC of ATP, so cation pumps fail and rigid cells undergo chronic extravascular hemolysis (congenital nonspherocytic hemolytic anemia), often from birth. Upstream 2,3-BPG accumulates, right-shifting the curve and partly offsetting the anemia.
| Feature | G6PD deficiency | Pyruvate kinase deficiency |
|---|---|---|
| Missing product | NADPH (HMP shunt) | ATP (glycolysis) |
| Inheritance | X-linked | Autosomal recessive |
| Pattern | Episodic, oxidant-triggered | Chronic, from birth |
| Smear | Heinz bodies, bite cells | Rigid cells; no spherocytes |
| Hemolysis | Intravascular and extravascular | Extravascular; ↑2,3-BPG right-shifts the curve |
Heme synthesis and the porphyrias
Heme synthesis begins in mitochondria, moves to the cytosol, and returns to mitochondria for the final step.
- ALA synthase (mitochondria)Glycine + succinyl-CoA → ALA; rate-limiting, needs vitamin B6, inhibited by heme
- ALA dehydratase (cytosol)ALA → porphobilinogen (PBG); inhibited by lead
- PBG deaminase (cytosol)Deficient in acute intermittent porphyria
- Uroporphyrinogen decarboxylase (cytosol)Deficient in porphyria cutanea tarda
- Ferrochelatase (mitochondria)Inserts Fe2+ into protoporphyrin IX; inhibited by lead
Acute intermittent porphyria (AIP). PBG deaminase deficiency produces neurovisceral attacks, the 5 P's: Painful abdomen, Port-wine urine, Polyneuropathy, Psychiatric changes, and Precipitation by CYP450 inducers, alcohol or fasting. Urine ALA and PBG rise. Hemin and glucose treat attacks by suppressing ALA synthase. There is NO photosensitivity.
Porphyria cutanea tarda (PCT). Uroporphyrinogen decarboxylase deficiency is the most common porphyria, causing photosensitive blistering on sun-exposed skin such as the backs of the hands and tea-colored urine. It is linked to hepatitis C and alcohol. There are NO neurovisceral attacks.
Lead poisoning. By blocking ALA dehydratase and ferrochelatase, lead causes ALA and protoporphyrin to accumulate and produces a microcytic anemia with basophilic stippling. Vitamin B6 deficiency (for example from isoniazid) impairs ALA synthase and causes sideroblastic anemia.