Heme synthesis

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Heme Synthesis

Heme is an iron-containing porphyrin that serves as the prosthetic group for hemoglobin, myoglobin, cytochromes (including cytochrome P450), catalase, and peroxidases. Over 85% of all heme synthesis occurs in erythroid tissue (bone marrow); the liver accounts for most of the remainder.

Structure of Heme

Heme is protoporphyrin IX chelated with a ferrous iron (Fe²+) atom. Four pyrrole rings joined by methenyl bridges (=CH-) form the porphyrin macrocycle. Eight side chains substitute the ring - two per pyrrole - in the order M-V / V-M / V-P / P-M (methyl-vinyl/vinyl-methyl/vinyl-propionate/propionate-methyl), the signature of the type III porphyrin series.

Cellular Compartmentalization

The pathway shuttles between mitochondria and cytosol:

Location	Steps
Mitochondria	Steps 1 (ALA formation), 6-8 (coproporphyrinogen III → heme)
Cytosol	Steps 2-5 (PBG → coproporphyrinogen III)

The 8 Steps

Step 1 - ALA Formation (Mitochondria) - Rate-Limiting Step

Substrates: Succinyl-CoA + Glycine
Enzyme: δ-Aminolevulinic acid synthase (ALAS) - requires pyridoxal phosphate (PLP, vitamin B6) as cofactor
Product: δ-Aminolevulinic acid (ALA); releases CO₂ and CoA

Two isoforms exist:

ALAS1 - expressed in all tissues (especially liver); inhibited by heme (feedback)
ALAS2 - expressed only in erythroid cells; regulated by intracellular iron availability (stimulated by iron)

Step 1-2 pathway: Glycine + Succinyl-CoA → ALA → Porphobilinogen

Step 2 - Porphobilinogen (PBG) Formation (Cytosol)

Substrate: 2 × ALA
Enzyme: ALA dehydratase (PBG synthase) - a zinc-containing enzyme
Product: Porphobilinogen (PBG), a monopyrrole; releases 2 H₂O
Inhibited by: Lead (Pb²+) - replaces zinc, blocking this enzyme

Step 3 - Hydroxymethylbilane Formation (Cytosol)

Substrate: 4 × PBG
Enzyme: Hydroxymethylbilane synthase (also called porphobilinogen deaminase or uroporphyrinogen I synthase)
Product: Hydroxymethylbilane (a linear tetrapyrrole); releases 4 NH₃
Deficiency: Acute Intermittent Porphyria (AIP) - the most common acute porphyria

Step 4 - Uroporphyrinogen III Formation (Cytosol)

Substrate: Hydroxymethylbilane
Enzyme: Uroporphyrinogen III synthase
Reaction: Ring closure + isomerization (reverses the D ring, giving the asymmetric type III series)
Product: Uroporphyrinogen III
Deficiency: Congenital Erythropoietic Porphyria (CEP) - without isomerization, type I porphyrins accumulate

Steps 3-7: PBG → Uroporphyrinogen III → Coproporphyrinogen III → Protoporphyrinogen IX → Protoporphyrin IX

Step 5 - Coproporphyrinogen III Formation (Cytosol)

Substrate: Uroporphyrinogen III
Enzyme: Uroporphyrinogen III decarboxylase
Reaction: Decarboxylation of 4 acetate side chains → 4 methyl groups; releases 4 CO₂
Product: Coproporphyrinogen III
Deficiency: Porphyria Cutanea Tarda (PCT) - the most common porphyria overall

Step 6 - Protoporphyrinogen IX Formation (Mitochondria)

Substrate: Coproporphyrinogen III (re-enters mitochondria)
Enzyme: Coproporphyrinogen III oxidase
Reaction: Decarboxylation + oxidation of 2 propionate side chains → vinyl groups; releases 2 CO₂
Product: Protoporphyrinogen IX
Deficiency: Hereditary Coproporphyria (HCP)

Step 7 - Protoporphyrin IX Formation (Mitochondria)

Substrate: Protoporphyrinogen IX
Enzyme: Protoporphyrinogen oxidase
Reaction: Oxidation (removes 6 H); creates the fully conjugated, colored porphyrin ring
Product: Protoporphyrin IX
Deficiency: Variegate Porphyria (VP)

Step 8 - Heme Formation (Mitochondria)

Substrate: Protoporphyrin IX + Fe²+
Enzyme: Ferrochelatase (also called heme synthase)
Reaction: Chelation of Fe²+ into the porphyrin ring; releases 2 H+
Product: Heme (Fe²+-protoporphyrin IX)
Inhibited by: Lead (Pb²+) - inhibits ferrochelatase
Deficiency: Erythropoietic Protoporphyria (EPP)

Pathway Summary Table

Step	Location	Enzyme	Substrate → Product	Porphyria if Deficient
1	Mito	ALAS (PLP-dependent)	Succinyl-CoA + Gly → ALA	- (rate-limiting)
2	Cytosol	ALA dehydratase (Zn²+)	2 ALA → PBG	- (Pb toxicity target)
3	Cytosol	HMB synthase	4 PBG → HMB	AIP
4	Cytosol	Uroporphyrinogen III synthase	HMB → UPG III	CEP
5	Cytosol	Uroporphyrinogen III decarboxylase	UPG III → CPG III	PCT
6	Mito	Coproporphyrinogen III oxidase	CPG III → PPG IX	HCP
7	Mito	Protoporphyrinogen oxidase	PPG IX → PP IX	VP
8	Mito	Ferrochelatase	PP IX + Fe²+ → Heme	EPP

ALA = aminolevulinic acid; PBG = porphobilinogen; HMB = hydroxymethylbilane; UPG = uroporphyrinogen; CPG = coproporphyrinogen; PPG = protoporphyrinogen; PP = protoporphyrin; Mito = mitochondria

Regulation

ALAS1 (Hepatic)

Heme acts as a negative feedback regulator on ALAS1 via three mechanisms:

Represses transcription of the ALAS1 gene
Increases degradation of ALAS1 mRNA
Decreases mitochondrial import of the ALAS1 protein

When hepatic heme is consumed (e.g., by cytochrome P450 induction from drugs like barbiturates, phenytoin, rifampicin), free heme falls, ALAS1 is derepressed, and ALA production surges. This is the basis for drug-induced porphyria attacks.

ALAS2 (Erythroid)

Stimulated by iron availability - when intracellular iron is adequate, ALAS2 activity increases to match globin synthesis
Heme in erythroid cells also stimulates globin synthesis by maintaining the ribosomal initiation complex in an active state

Clinically Relevant Enzyme Inhibition by Lead

Lead (Pb²+) inhibits two enzymes:

ALA dehydratase (step 2) - accumulation of ALA
Ferrochelatase (step 8) - accumulation of protoporphyrin IX; zinc is substituted for iron → zinc protoporphyrin (ZPP) accumulates

Lab findings in lead poisoning: elevated urine ALA, elevated blood/urine ZPP, microcytic hypochromic anemia (resembles iron deficiency)

Porphyrias Overview

Porphyrias are inherited (usually autosomal dominant) or acquired defects in heme synthesis. They fall into two clinical categories:

Type	Deficient Enzyme	Features
Acute Intermittent Porphyria (AIP)	HMB synthase (step 3)	Neuropsychiatric attacks, abdominal pain, autonomic dysfunction; NO photosensitivity; urine turns red/port-wine on standing
Congenital Erythropoietic Porphyria (CEP)	UPG III synthase (step 4)	Severe photosensitivity, hemolytic anemia, skin fragility
Porphyria Cutanea Tarda (PCT)	UPG decarboxylase (step 5)	Most common porphyria; skin blistering/fragility in light-exposed areas; associated with alcohol, estrogens, hepatitis C
Hereditary Coproporphyria (HCP)	CPG III oxidase (step 6)	Acute attacks + photosensitivity
Variegate Porphyria (VP)	Protoporphyrinogen oxidase (step 7)	Acute attacks + skin lesions; dangerous with barbiturates
Erythropoietic Protoporphyria (EPP)	Ferrochelatase (step 8)	Photosensitivity; no acute attacks

Sources

Biochemistry, 8th ed. Lippincott Illustrated Reviews, Chapter 21 (Porphyrin Metabolism)
Basic Medical Biochemistry: A Clinical Approach, 6e, Chapter 42

Heme metabolism

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Heme Metabolism (Degradation)

Heme metabolism refers to the catabolism of heme - primarily from hemoglobin in senescent red blood cells (RBCs) - through a series of steps that produce bilirubin, which is then conjugated and excreted. Adults destroy roughly 200 billion RBCs per day, yielding ~250-350 mg of bilirubin daily.

Overview: Sources of Heme for Degradation

Source	Contribution
Senescent RBCs (120-day lifespan)	~75-80% of daily bilirubin
Ineffective erythropoiesis	~10-15%
Cytochromes, myoglobin, catalase	~5-10%

Mature RBCs are phagocytosed by macrophages of the mononuclear phagocyte system (MPS) - particularly in the liver (Kupffer cells), spleen, and bone marrow. Globin chains are hydrolyzed to amino acids; iron is recycled; the porphyrin ring of heme is degraded.

Step 1 - Heme → Biliverdin (Macrophages)

Enzyme: Heme oxygenase (microsomal; NADPH-dependent) Cofactors: O₂, NADPH Reaction: The α-methene bridge of the porphyrin ring is cleaved oxidatively

Heme-Fe³+ + 3 O₂ + 7 e⁻ → Biliverdin + CO + Fe²+ (or Fe³+)

Three products are released:

Biliverdin - a linear, green tetrapyrrole
Carbon monoxide (CO) - has signaling and anti-inflammatory roles; is why a bruise turns green-then-yellow
Fe²+ - returned to the iron pool (ferritin/transferrin)

Note: Heme oxygenase is substrate-inducible - heme itself induces its own oxygenase.

Step 1-2: Heme → Biliverdin → Bilirubin in macrophage

Step 2 - Biliverdin → Bilirubin (Macrophages)

Enzyme: Biliverdin reductase Cofactor: NADPH Reaction: Reduction of the central methylene bridge of biliverdin

Biliverdin + NADPH + H⁺ → Bilirubin + NADP⁺

Bilirubin is a yellow-orange pigment. It is the end product of heme catabolism in mammals. (Birds and amphibians excrete biliverdin directly without this step.)

Bilirubin as antioxidant: At low concentrations, bilirubin functions as an antioxidant - it is oxidized back to biliverdin, then regenerated by biliverdin reductase, forming a redox cycle that scavenges reactive oxygen species.

Step 3 - Transport to the Liver (Blood)

Bilirubin is poorly water-soluble (unconjugated/indirect bilirubin, UCB). It is transported in blood bound noncovalently to albumin:

Albumin has a high-affinity site (binds ~25 mg bilirubin/100 mL plasma) and a low-affinity site
This form is called unconjugated bilirubin (UCB) or indirect bilirubin
Certain drugs (salicylates, sulfonamides, some antibiotics) competitively displace bilirubin from albumin, risking CNS entry - especially dangerous in neonates (kernicterus)

Step 4 - Hepatic Uptake

At the sinusoidal surface of hepatocytes, bilirubin:

Dissociates from albumin
Enters via facilitated diffusion (saturable transport system)
Binds to intracellular proteins, especially ligandin (glutathione S-transferase) - this prevents back-diffusion into blood

Step 5 - Conjugation in the Liver (Hepatocytes)

Bilirubin UDP-glucuronosyltransferase in liver → Bilirubin diglucuronide → Bile

Enzyme: Bilirubin UDP-glucuronosyltransferase (bilirubin UGT) - located in the smooth ER Donor: UDP-glucuronic acid (×2) Reactions (sequential):

Bilirubin + UDP-glucuronate → Bilirubin monoglucuronide + UDP

Bilirubin monoglucuronide + UDP-glucuronate → Bilirubin diglucuronide + UDP

The product - bilirubin diglucuronide (conjugated bilirubin, CB / direct bilirubin) - is water-soluble. This is the predominant form secreted into bile. Note: In obstructive jaundice, the plasma form is predominantly monoglucuronide.

Induction: Phenobarbital and other drugs induce bilirubin UGT activity (clinically exploited in neonatal jaundice treatment).

Step 6 - Biliary Secretion

Transporter: MOAT (multispecific organic anion transporter), an ATP-binding cassette (ABC) transporter on bile canalicular membranes Direction: Active transport of CB into bile canaliculi - this is the rate-limiting step of hepatic bilirubin metabolism

Conjugated bilirubin passes from bile canaliculi → bile ducts → gallbladder → duodenum.

Step 7 - Intestinal Processing: Urobilinogen & Stercobilin

In the distal ileum and colon, gut bacteria:

Remove glucuronic acid (hydrolyze CB back to free bilirubin)
Reduce bilirubin → urobilinogen (colorless)
Oxidize most urobilinogen → stercobilin (brown) - gives feces their characteristic brown color

Fate of urobilinogen (enterohepatic circulation):

Full bilirubin metabolism overview - macrophage to liver to intestine to kidney

Fraction	Destination
~80-90%	Oxidized to stercobilin → excreted in feces
~10-20% reabsorbed	Enters portal blood → liver (enterohepatic urobilinogen cycle) → re-excreted in bile
Small remainder	Reaches kidney → oxidized to urobilin (yellow) → excreted in urine (gives urine its color)

Full Pathway at a Glance

Senescent RBC → Hemoglobin
                    ↓ (Macrophage/MPS)
              Heme (Fe²+-protoporphyrin IX)
                    ↓ Heme oxygenase (+O₂, NADPH)
                         → CO (gaseous signaling)
                         → Fe²+ (recycled)
              Biliverdin (green, linear tetrapyrrole)
                    ↓ Biliverdin reductase (+NADPH)
              Bilirubin (UCB) - yellow-orange
                    ↓ Bound to albumin → transported to LIVER
              Hepatocyte uptake → binds ligandin
                    ↓ Bilirubin UGT (+UDP-glucuronate ×2)
              Bilirubin diglucuronide (CB, "direct bilirubin")
                    ↓ Active secretion via MOAT → BILE
              Intestine: bacteria → Urobilinogen
                    ↓ (most)                ↓ (some reabsorbed)
              Stercobilin              Enterohepatic cycle / Kidney
              (feces, brown)           → Urobilin (urine, yellow)

Bilirubin Measurement: van den Bergh Reaction

Fraction	Also called	Behavior	Significance
Direct bilirubin	Conjugated bilirubin (CB)	Reacts rapidly in aqueous solution	Water-soluble; appears in urine when elevated
Indirect bilirubin	Unconjugated bilirubin (UCB)	Reacts only in methanol	Lipid-soluble; crosses BBB; bound to albumin
Total bilirubin	-	Reacts in methanol	CB + UCB

Normal total bilirubin: ≤1 mg/dL. Jaundice becomes visible at 2-3 mg/dL.

Jaundice - Types and Pathophysiology

1. Hemolytic (Pre-hepatic) Jaundice

Cause: Excessive RBC destruction (sickle cell, G6PD deficiency, hereditary spherocytosis, malaria)
Bilirubin: UCB (indirect) elevated; CB normal
Urine: Urobilinogen increased; no bilirubin in urine (UCB not water-soluble, cannot be filtered)
Stool: Dark (more stercobilin)
Key: Liver conjugates maximally but is overwhelmed

2. Hepatocellular (Hepatic) Jaundice

Cause: Hepatitis, cirrhosis, liver failure - impairs uptake, conjugation, and secretion
Bilirubin: BOTH UCB and CB elevated (mixed picture)
Urine: Dark urine (bilirubinuria from CB), elevated urobilinogen
Stool: Pale (reduced stercobilin)
Labs: ALT, AST elevated

3. Obstructive (Post-hepatic) Jaundice

Cause: Bile duct obstruction by stone, tumor (pancreatic/cholangiocarcinoma), stricture
Bilirubin: CB (direct) elevated - regurgitates into blood
Urine: Dark (bilirubinuria); urobilinogen absent (CB cannot reach intestine)
Stool: Pale/clay-colored (no stercobilin)
Key feature: Absent urinary urobilinogen distinguishes obstruction from hepatocellular disease

Summary Table

Feature	Hemolytic	Hepatocellular	Obstructive
Serum UCB	↑↑	↑	Normal
Serum CB	Normal/slight ↑	↑	↑↑
Urine bilirubin	Absent	Present	Present
Urine urobilinogen	Increased	Increased	Absent
Stool color	Dark	Pale	Pale/clay
ALT/AST	Normal	↑↑	Mildly ↑
ALP/GGT	Normal	Mildly ↑	↑↑

Hereditary Hyperbilirubinemias

Disorder	Defect	Bilirubin Type	Severity
Gilbert syndrome	Mild UGT reduction (~30%); often combined with reduced uptake	UCB ↑ (mild)	Benign; triggered by fasting/stress
Crigler-Najjar type I	Complete absence of bilirubin UGT	UCB ↑↑↑	Severe; fatal without liver transplant; phototherapy required continuously
Crigler-Najjar type II	Severely reduced UGT	UCB ↑↑	Moderate; responds to phenobarbital
Dubin-Johnson syndrome	Defective MOAT (MRP2) - cannot secrete CB into bile	CB ↑	Benign; black liver pigment
Rotor syndrome	Defective sinusoidal re-uptake of CB	CB ↑	Benign; no liver pigment

Neonatal Jaundice (Physiologic)

Affects ~60% of full-term and ~80% of preterm newborns in the first week of life
Cause: Bilirubin UGT activity is low at birth (reaches adult levels by ~4 weeks); high rate of fetal Hb turnover adds to the load
Risk: UCB > 20-25 mg/dL can exceed albumin binding capacity, cross the blood-brain barrier, and deposit in the basal ganglia causing kernicterus (toxic encephalopathy)
Treatment: Phototherapy with blue fluorescent light (wavelength ~460 nm) converts UCB to more polar, water-soluble photoisomers that can be excreted in bile without conjugation. Phenobarbital can also be used to induce UGT.

Sources

Biochemistry, 8th ed. Lippincott Illustrated Reviews, Chapter 21 (Porphyrin & Bile Pigment Metabolism)
Harper's Illustrated Biochemistry, 32nd ed., Chapter 31 (Catabolism of Heme)

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