Hemoglobin (Hb) Derivatives

Hemoglobin (Hb) is the chief protein of the red blood cells. A single red blood cell contains about 280 million molecules of hemoglobin (Hb). Hb can give rise to many derivatives which are discussed below.

Hemoglobin exists in multiple structural and functional forms, and its derivatives are broadly classified into:

1. Functional derivatives: HbO₂, HbCO₂, HHb are oxygenated, carbamino, and deoxygenated forms of Hb involved in normal physiology.
2. Dysfunctional derivatives: COHb, MetHb, SulfHb, HbNO, and HbA1c are abnormal forms that impair oxygen transport or alter Hb function.

1. Action of Acids

Acids split Hb into globin and heme (containing ferrous iron, Fe²⁺, therefore also called ferroheme). In the presence of O₂, the ferrous iron may be oxidized to ferric iron (Fe³⁺), forming ferriheme, which can bind negatively charged acid anions.

Reaction (Sahli’s method principle):

• Hb + 2HCl → Globin + Hematin (acid hematin)
• (Fe²⁺ → Fe³⁺ oxidation may occur depending on conditions)

Key points:

1. Acid hematin is a brown-colored compound that is responsible for the color change seen in acid hemoglobin estimation.
2. This reaction forms the basis of Sahli’s method for estimation of hemoglobin, which is a simple colorimetric method used in basic laboratories.
3. Acid hematin formation is an irreversible denaturation process under strong acidic conditions in which the protein structure is destroyed.
4. Hematin formed is also referred to as the acid hematin chloride complex in older literature because it represents the Hb + acid combination product.

2. Action of Alkalies

Alkalies also split Hb into globin and ferroheme. The heme iron is oxidized to ferric form (Fe³⁺), which combines with hydroxyl groups to form alkaline hematin (hematin hydroxide complex).
Reaction:

• Hb + NaOH → Globin + Alkaline hematin (hematin-OH complex)

Key points:

1. This compound is brownish in color and produces a pigment similar to acid hematin.
2. Alkaline hematin formation represents irreversible denaturation of hemoglobin under alkaline conditions, leading to loss of oxygen-binding structure.
3. Alkaline hematin is also referred to as hematin hydroxide complex or alkaline hematin base in different literature sources.

3. Reaction with CO₂

This reaction leads to the formation of carbamino compounds.

Reaction:

Hb-NH₂ + CO₂ ⇌ Hb-NH-COO⁻ + H⁺
(formation of carbaminohemoglobin, HbCO₂)

Key points:

1. CO₂ binds to the terminal amino groups of globin rather than to heme, meaning that the globin chains participate in binding.
2. This is a reversible reaction that is important for CO₂ transport, and about 10–20% of CO₂ is carried in this form while the rest is mainly transported as bicarbonate.
3. This reaction contributes to the Haldane effect, in which deoxygenated hemoglobin binds more CO₂, an important mechanism in tissue gas exchange.
4. It also contributes indirectly to acid–base balance through the formation of H⁺ ions, thereby helping to buffer blood pH.
5. The majority of CO₂ transport occurs as bicarbonate (HCO₃⁻) in plasma through the action of carbonic anhydrase in RBCs.

4. Reaction with CO

This reaction results in the formation of carboxyhemoglobin (HbCO or CO-Hb).

Reaction:

Hb(Fe²⁺) + CO ⇌ HbCO

Key points:

1. Endogenous CO is produced during heme degradation by the enzyme heme oxygenase:
   • Heme + O₂ + NADPH → Biliverdin + Fe²⁺ + CO
   Biliverdin is then converted to bilirubin by biliverdin reductase, and this pathway operates continuously during normal RBC turnover.
2. In heavy smokers, up to 5–10% COHb may be present, whereas in non-smokers about 1–2% COHb is normally detectable because baseline exposure varies with the environment.
3. The affinity of Hb for CO is about 200–250 times higher than its affinity for O₂, which explains the strong toxicity of CO.
4. COHb is highly stable and dissociates slowly, leading to prolonged hypoxia.

Toxic effects of COHb:

1. COHb cannot carry oxygen, resulting in decreased O₂-carrying capacity of blood and producing a functional anemia state.
2. COHb causes a left shift of the O₂-Hb dissociation curve, thereby impairing oxygen release to tissues so that tissues receive less oxygen despite normal oxygen levels.
3. COHb allosterically stabilizes the R-state of hemoglobin, which further reduces O₂ unloading and prevents adequate oxygen release in tissues.
4. CO inhibits cytochrome c oxidase (Complex IV), causing histotoxic hypoxia by blocking cellular respiration at the mitochondrial level.
5. CO exposure can also cause delayed neurological injury because oxidative stress and lipid peroxidation may occur after reperfusion during recovery from severe poisoning.

Clinical features:

• Cherry-red coloration of blood is a classic finding, although it is not always clinically visible and is more evident post-mortem than in living patients.
• Headache, dizziness, confusion, and loss of consciousness may occur, with symptoms progressing according to the level of exposure.

Treatment:

Treatment consists of 100% oxygen or hyperbaric oxygen therapy to rapidly displace CO from Hb.
Reaction:

• HbCO + O₂ → HbO₂ + CO

Hyperbaric oxygen increases dissolved plasma oxygen independent of Hb, thereby supporting tissue oxygenation even when Hb is saturated with CO.

5. Reaction with Oxidizing Reagents

Oxidizing agents such as ferricyanide [Fe(CN)₆]³⁻, nitrites (NO₂⁻), chlorates, and peroxides convert hemoglobin to methemoglobin (MetHb).

Reaction:

• Hb(Fe²⁺) → MetHb (Fe³⁺)

Key points:

1. Ferric iron (Fe³⁺) cannot bind oxygen, leading to loss of oxygen-carrying ability.
2. Methemoglobin is dark brown in color, producing the characteristic chocolate-brown appearance.
3. MetHb is physiologically present in small amounts and is normally kept low by enzymatic reduction systems.

Reduction back to Hb:

• MetHb + NADH + H⁺ → Hb + NAD⁺
  

This reaction occurs via the cytochrome b5 reductase system, which is the major protective pathway in RBCs.

Additional pathways:

1. The glutathione-dependent reduction system acts as a secondary antioxidant defense mechanism.
2. The NADPH-dependent methemoglobin reductase system is enhanced by methylene blue and serves as a drug-assisted pathway.

Normal physiology

1. About 1–2% of Hb is continuously oxidized to MetHb, but this oxidation is normally controlled.
2. The major reducing system is the NADH-dependent cytochrome b5 reductase pathway, which is the most important enzyme system in RBCs.
3. The minor reducing system is the NADPH-dependent pathway, which becomes especially important during therapy.
4. The normal MetHb level is less than 1–2% (approximately 1%), and it is maintained at this stable level under healthy conditions.

(a) Methemoglobinemia

Causes:

1. Congenital deficiency of cytochrome b5 reductase is an inherited enzymatic defect that predisposes to methemoglobinemia.
2. Hemoglobin M variants (HbM disease) contain abnormal globin chains that stabilize the Fe³⁺ state.
3. Oxidizing agents such as nitrites, nitrates in well water, sulfonamides, aniline dyes, dapsone, and benzocaine are common drug-induced causes.

Effects:

1. Methemoglobinemia reduces oxygen delivery and produces a functional anemia because Hb is present but non-functional.
2. It causes a left shift of the O₂-Hb dissociation curve, thereby impairing oxygen release to tissues.
3. Patients may have normal PaO₂ despite tissue hypoxia, which is an important diagnostic clue.

Clinical features:

• Cyanosis unresponsive to oxygen is a classic sign of methemoglobinemia.
• Chocolate-brown blood is a characteristic diagnostic appearance.
• Dyspnea, headache, and dizziness occur because of tissue hypoxia.

Treatment

1. Methylene blue is given in a dose of 1–2 mg/kg IV.
2. It acts through the NADPH-dependent reduction system and accelerates the conversion of MetHb back to Hb.

Reaction:

• Methylene blue (oxidized) → Leucomethylene blue (reduced) → reduces MetHb to Hb

Ascorbic acid acts as an adjunct antioxidant and slowly supports the reduction of MetHb.

Important caution:

In G6PD deficiency, methylene blue may be ineffective or may even cause hemolysis because reduced NADPH availability increases the risk of oxidative RBC damage.

(b) Cyanide binding

MetHb binds cyanide to form cyanomethemoglobin:

• MetHb + CN⁻ → CyanometHb

Nitrite therapy basis:

1. Nitrites induce the formation of MetHb as a therapeutic mechanism.
2. MetHb then binds cyanide and detoxifies it, thereby protecting the cytochrome oxidase system.

Final detoxification:

• CN⁻ + S₂O₃²⁻ → SCN⁻
  

This reaction occurs through the action of the mitochondrial enzyme rhodanese, and the thiocyanate formed is less toxic and excreted in urine.

Key mechanism:

• Cyanide inhibits cytochrome c oxidase (Complex IV), causing cellular hypoxia despite normal oxygen content in blood because cellular respiration fails.

6. Formation of Sulfhemoglobin

Sulfhemoglobin can be formed by incorporation of sulfur into Hb in the presence of H₂S or sulfur-containing drugs.

Reaction:

Hb + sulfur compounds (H₂S / drugs) → Sulfhemoglobin (SulfHb), which is an irreversible modified Hb pigment.