Haemoglobin Pigments

Introduction

• Degradation pigments of haemoglobin are coloured products formed during the normal breakdown of haemoglobin.

• Haemoglobin degradation occurs after the destruction of senescent red blood cells, which have an average life span of about 120 days.

• The process mainly takes place in the reticuloendothelial system (RES), especially in the spleen, liver, and bone marrow.

• During degradation, haemoglobin is first separated into globin and haem components.

• Globin part is broken down into amino acids, which are reutilized by the body.

• The haem portion undergoes a series of enzymatic reactions leading to the formation of bile pigments.

• The major degradation pigments formed are biliverdin and bilirubin.

• These pigments are responsible for the normal colour of bile, feces, and urine.

• Proper metabolism and excretion of these pigments are essential for normal physiology.

• Accumulation of degradation pigments, especially bilirubin, results in jaundice, indicating underlying pathological conditions.

Physiological Haemoglobin Pigments

Physiological or functional haemoglobin pigments are the normal forms of haemoglobin that actively participate in oxygen transport and delivery in the body. These pigments are formed reversibly under normal physiological conditions and maintain iron in the ferrous (Fe²⁺) state.

Oxyhaemoglobin (HbO₂)

1. Formation of Oxyhaemoglobin

• Occurs in the pulmonary capillaries where partial pressure of oxygen (pO₂) is high (~100 mmHg).

• Each haemoglobin molecule can bind four molecules of oxygen, one at each haem group.

• Oxygen binds to the ferrous (Fe²⁺) iron of haem without oxidation.

• Binding is reversible, allowing oxygen release in tissues.

Reaction:
Hb + 4O₂ ⇌ Hb(O₂)₄

2. Structural Basis of Oxygen Binding

• Haemoglobin is a tetrameric protein (α₂β₂).

• Each globin chain contains one haem group with Fe²⁺ at the center.

• Oxygen binds at the 6th coordination site of iron.

• Binding causes conformational change from:

  • T (tense) state → R (relaxed) state

• This structural change increases oxygen affinity of remaining haem groups (cooperative binding).

3. Oxygen Dissociation Curve (ODC)

• Oxyhaemoglobin shows a sigmoid-shaped oxygen dissociation curve.

• The sigmoidal shape is due to cooperative binding.

• Significance:

  • Upper flat portion: efficient oxygen loading in lungs.

  • Steep lower portion: efficient oxygen unloading in tissues.

4. Factors Affecting Oxyhaemoglobin Formation (Shift of ODC)

Right Shift (↓ affinity, ↑ oxygen release):

• Increased CO₂ (Bohr effect)

• Decreased pH

• Increased temperature

• Increased 2,3-BPG

• Exercise, anemia, high altitude

Left Shift (↑ affinity, ↓ oxygen release):

• Decreased CO₂

• Increased pH

• Decreased temperature

• Decreased 2,3-BPG

• Presence of fetal haemoglobin (HbF)

• Carbon monoxide (pathological left shift)

5. Colour and Optical Properties

• Oxyhaemoglobin is bright red in colour.

• Responsible for the colour of arterial blood.

• Absorbs light differently from deoxyhaemoglobin, which is used in:

  • Pulse oximetry

  • Spectrophotometric hemoglobin estimation

6. Physiological Functions

• Primary carrier of oxygen to tissues.

• Maintains adequate tissue oxygenation.

• Helps regulate blood pH indirectly via oxygen–carbon dioxide exchange.

• Essential for aerobic metabolism and ATP generation.

7. Clinical Significance

• Reduced formation leads to hypoxia.

• Normal oxygen saturation (SaO₂): 95–100%.

• Conditions affecting oxyhaemoglobin:

  • Lung diseases (COPD, pneumonia)

  • Anemia (normal SaO₂ but reduced oxygen content)

  • High altitude (reduced pO₂)

Reduced (Deoxy) Haemoglobin

1. Formation of Reduced Haemoglobin

• Formed in systemic capillaries where tissue pO₂ is low.

• Oxyhaemoglobin dissociates to release oxygen for cellular metabolism.

• Iron remains in the ferrous (Fe²⁺) state (no oxidation).

• Oxygen binding is reversible.

Reaction:
HbO₂ ⇌ Hb + O₂

2. Structural and Molecular Features

• Haemoglobin remains a tetrameric protein (α₂β₂).

• Loss of oxygen causes a shift from:

  • R (relaxed) state → T (tense) state

• T-state haemoglobin has:

  • Lower affinity for oxygen

  • Higher affinity for H⁺ and CO₂

• This facilitates oxygen unloading and CO₂ uptake.

3. Colour and Optical Properties

• Reduced haemoglobin has a dark bluish-red colour.

• Responsible for the darker colour of venous blood.

• The bluish appearance of skin and mucosa in high concentrations leads to cyanosis.

4. Role in Oxygen Dissociation Curve

• Predominant form on the steep portion of the oxygen dissociation curve.

• Small decreases in pO₂ cause large release of oxygen.

• Enhances efficient oxygen delivery during:

  • Exercise

  • Tissue hypoxia

  • Increased metabolic demand

5. Role in Carbon Dioxide Transport

Reduced haemoglobin facilitates CO₂ transport by two major mechanisms:

a) Haldane Effect

• Deoxygenated haemoglobin binds CO₂ more readily than oxyhaemoglobin.

• Promotes CO₂ uptake in tissues and release in lungs.

b) Buffering of Hydrogen Ions

• Reduced haemoglobin binds H⁺ ions formed during carbonic acid dissociation.

• Acts as an important blood buffer, helping maintain acid–base balance.

6. Clinical Significance

Cyanosis

• Occurs when reduced haemoglobin concentration exceeds 5 g/dL.

• Seen in:

  • Congestive heart failure

  • Chronic lung disease

  • Cyanotic congenital heart disease

• Absent in severe anemia despite hypoxia (low Hb).

Venous Blood Indicator

• High reduced haemoglobin indicates increased tissue oxygen extraction.

Abnormal / Pathological Hb Pigments

These pigments impair oxygen transport and are usually formed under pathological conditions.

Carboxyhaemoglobin

1. Formation of Carboxyhaemoglobin

• Formed by the binding of carbon monoxide (CO) to haemoglobin.

• CO binds to the ferrous (Fe²⁺) iron of haem.

• Affinity of CO for haemoglobin is 200–250 times greater than that of oxygen.

• Binding is reversible but extremely stable, displacing oxygen.

Reaction:
Hb + CO ⇌ COHb

2. Mechanism of Toxicity

Carboxyhaemoglobin causes hypoxia by two mechanisms:

a) Reduced Oxygen-Carrying Capacity

• CO occupies oxygen-binding sites on haemoglobin.

• Decreases the total amount of oxygen that can be transported.

b) Left Shift of Oxygen Dissociation Curve

• CO binding increases oxygen affinity of remaining haem groups.

• Impairs oxygen release to tissues.

• Results in functional anemia.

3. Structural and Molecular Effects

• Iron remains in ferrous (Fe²⁺) state.

• CO binds to the same site as oxygen but with much higher affinity.

• Stabilizes haemoglobin in the R (relaxed) state, preventing oxygen unloading.

4. Colour and Optical Properties

• COHb gives blood a bright cherry-red colour.

• This sign is rarely seen clinically and mostly described in textbooks.

• Pulse oximetry may show falsely normal oxygen saturation.

5. Sources of Carbon Monoxide Exposure

• Automobile exhaust

• Faulty gas heaters

• Fire smoke

• Coal or wood burning stoves

• Cigarette smoke (chronic low-level exposure)

6. Clinical Features of CO Poisoning

• Headache

• Dizziness

• Nausea and vomiting

• Confusion and altered consciousness

• Severe exposure: coma, arrhythmias, death

7. Diagnosis

• Measured as percentage of COHb in blood using co-oximetry.

• Normal:

  • Non-smokers: <1–2%

  • Smokers: up to 5–10%

• Symptoms correlate with COHb levels:

  • 10–20%: headache

  • 30–40%: severe headache, weakness

  • 50%: life-threatening

Methaemoglobin

1. Formation of Methaemoglobin

• Formed by oxidation of haem iron from Fe²⁺ to Fe³⁺.

• Normally, a small amount of MetHb (<1%) is continuously formed.

• It is rapidly reduced back to haemoglobin by methemoglobin reductase systems in RBCs.

Reaction:
Hb (Fe²⁺) → MetHb (Fe³⁺)

2. Enzymatic Reduction Systems

a) NADH–Cytochrome b₅ Reductase (Major Pathway)

• Converts MetHb back to functional haemoglobin.

• Accounts for ~95% of MetHb reduction.

b) NADPH–MetHb Reductase (Minor Pathway)

• Activated by methylene blue.

• Important in treatment of methemoglobinemia.

3. Causes of Increased Methaemoglobin

A. Congenital Causes

• Deficiency of NADH–MetHb reductase.

• Structural abnormalities of haemoglobin (Hb M).

B. Acquired Causes

• Drugs and chemicals:

  • Nitrates and nitrites

  • Aniline dyes

  • Sulfonamides

  • Local anesthetics (benzocaine, lidocaine)

• Contaminated well water (infants – “blue baby syndrome”).

4. Pathophysiology

Methaemoglobin causes hypoxia by:

1. Inability to bind oxygen (Fe³⁺ cannot carry O₂).

2. Left shift of oxygen dissociation curve of remaining normal Hb, reducing oxygen release.

This results in functional anemia despite normal haemoglobin concentration.

5. Colour and Optical Properties

• Blood appears chocolate brown.

• Cyanosis occurs at MetHb levels >10–15%.

• Cyanosis does not improve with oxygen therapy.

Sulphaemoglobin

1. Formation

• Formed when haemoglobin is exposed to sulphur-containing compounds.

• Sulphur is incorporated into the porphyrin ring of haem.

• Iron remains in ferrous (Fe²⁺) state, but haemoglobin becomes nonfunctional.

• Formation is irreversible.

2. Causes

• Chronic exposure to:

  • Sulfonamides

  • Phenacetin

  • Hydrogen sulphide

  • Certain laxatives and analgesics

• Often associated with drug-induced toxicity.

3. Pathophysiology

• Sulphaemoglobin cannot carry oxygen.

• Reduces effective oxygen-carrying capacity.

• Leads to chronic cyanosis.

4. Colour and Clinical Features

• Blood appears greenish-black or bluish-green.

• Cyanosis occurs even at low levels (≈0.5 g/dL).

• Cyanosis does not respond to oxygen therapy or methylene blue.

• Pigment persists until RBCs are destroyed (life span of RBC).

Cyanmethemoglobin

1. Formation

• Formed by reaction of haemoglobin with:

  • Potassium ferricyanide → converts Hb (Fe²⁺) to MetHb (Fe³⁺)

  • Potassium cyanide → combines with MetHb

• Results in formation of cyanmethemoglobin.

2. Importance in Laboratory Medicine

• Basis of Drabkin’s method for haemoglobin estimation.

• All forms of haemoglobin (except sulphaemoglobin) are converted to cyanmethemoglobin.

• The compound is stable and measured spectrophotometrically.

3. Iron State

• Iron is in the ferric (Fe³⁺) state.

4. Clinical Significance

• No physiological or pathological role in the body.

• Used only for quantitative estimation of haemoglobin.

Degradation Pigments of Haemoglobin

Degradation pigments of haemoglobin are formed during the physiological breakdown of haemoglobin after the destruction of senescent red blood cells (RBCs). These pigments are collectively known as bile pigments and are responsible for the normal colour of bile, feces, and urine. Their abnormal accumulation leads to jaundice.

Site of Haemoglobin Degradation

• Occurs mainly in the reticuloendothelial system (RES):
  • Spleen (major site)
  • Liver
  • Bone marrow
• RBC life span: ~120 days.

Steps in Haemoglobin Degradation

1. Breakdown of Haemoglobin

• Haemoglobin is split into:
  • Globin → amino acids (reused)
  • Haem → iron + porphyrin ring

2. Formation of Biliverdin

• Haem is converted to biliverdin by haem oxygenase.
• Requires:
  • Oxygen
  • NADPH
• Iron is released and reused or stored as ferritin.
• Biliverdin is a green pigment.

3. Formation of Bilirubin

• Biliverdin is reduced to bilirubin by biliverdin reductase.
• Bilirubin is yellow in colour.
• This bilirubin is:
  • Unconjugated
  • Lipid-soluble
  • Transported in plasma bound to albumin

Transport and Hepatic Handling of Bilirubin

1. Unconjugated (Indirect) Bilirubin

• Transported to liver bound to albumin.
• Cannot be excreted in urine.

2. Conjugated (Direct) Bilirubin

• In hepatocytes, bilirubin is conjugated with glucuronic acid by UDP-glucuronyl transferase.
• Becomes water-soluble.
• Excreted into bile.

Intestinal Metabolism of Bilirubin

• Conjugated bilirubin enters intestine via bile.
• Converted by intestinal bacteria into:
  • Urobilinogen

Fate of Urobilinogen

• Majority oxidized to stercobilin → gives brown colour to feces.
• Small amount reabsorbed:
  • Enterohepatic circulation
  • Excreted in urine as urobilin → yellow colour of urine.

Degradation Pigments 

1. Biliverdin

• Green pigment.
• First bile pigment formed from haem.
• Seen in resolving bruises.

2. Bilirubin

• Yellow pigment.
• Causes jaundice when accumulated.
• Exists as:
  • Unconjugated (indirect)
  • Conjugated (direct)

3. Urobilinogen

• Colourless.
• Formed in intestine.

4. Stercobilin

• Brown pigment of feces.

5. Urobilin

• Yellow pigment of urine.

Clinical Correlation – Jaundice