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Metabolism of Iron and Laboratory Evaluation of Iron Deficiency

Overview

Iron is an essential micronutrient that plays crucial roles in oxygen transport, cellular respiration, and numerous enzymatic processes. The human body contains approximately 3-5 grams of total iron, with precise regulatory mechanisms maintaining this balance without a dedicated excretory pathway. Understanding iron metabolism is fundamental to diagnosing and managing iron deficiency, the most common nutritional disorder worldwide affecting over 2 billion people. The complex interplay between iron absorption, transport, storage, and utilization involves multiple proteins and regulatory molecules, with hepcidin serving as the master regulator. Laboratory evaluation of iron status requires comprehensive assessment using multiple parameters, as no single test perfectly reflects total body iron stores.

Iron Distribution and Functions

Body Iron Compartments

Functional Iron (70%): - Hemoglobin: 2,500 mg (65%) in red blood cells - Myoglobin: 400 mg (10%) in muscle tissue - Tissue enzymes: 150 mg (3%) - Cytochromes: Electron transport chain - Peroxidases: Antioxidant defense - Ribonucleotide reductase: DNA synthesis - Catalase: Hydrogen peroxide metabolism

Storage Iron (25%): - Ferritin: 700-900 mg in males, 200-300 mg in females - Hepatocytes: Primary storage site - Macrophages: Recycled iron storage - Bone marrow: Local storage for erythropoiesis - Hemosiderin: Degraded ferritin aggregates - Increases with iron overload - Visible on Prussian blue staining

Transport Iron (<1%): - Transferrin-bound iron: 3-4 mg - Non-transferrin bound iron (NTBI): Pathological - Labile plasma iron: Chelatable fraction

Iron Absorption

Intestinal Iron Uptake

Dietary Iron Forms: 1. Heme iron (10-15% of intake): - From meat, poultry, fish - 15-35% absorption efficiency - Heme carrier protein 1 (HCP1) mediated

  1. Non-heme iron (85-90% of intake):
  2. From plants, fortified foods
  3. 2-20% absorption efficiency
  4. Requires reduction to Fe²⁺

Molecular Mechanisms of Absorption

Apical Iron Uptake: 1. Duodenal cytochrome b (DcytB): Reduces Fe³⁺ to Fe²⁺ 2. Divalent metal transporter 1 (DMT1): - Transports Fe²⁺ into enterocyte - Co-transports H⁺ (pH dependent) - Also transports other divalent metals

Intracellular Processing: - Cytosolic iron: Bound to chaperone proteins - Storage option: Incorporated into ferritin - Export pathway: Via ferroportin

Basolateral Export: 1. Ferroportin (SLC40A1): Only mammalian iron exporter 2. Hephaestin/Ceruloplasmin: Oxidize Fe²⁺ to Fe³⁺ 3. Transferrin: Binds Fe³⁺ for transport

Regulation of Absorption

Factors Enhancing Absorption: - Vitamin C: Reduces Fe³⁺, chelates iron - Citric acid: Forms soluble complexes - Amino acids: Histidine, cysteine - Meat factors: Unidentified compounds - Low pH: Maintains iron solubility

Factors Inhibiting Absorption: - Phytates: In grains, legumes - Polyphenols: Tea, coffee tannins - Calcium: Competitive inhibition - Zinc: Shares DMT1 transporter - High pH: Promotes Fe³⁺ precipitation

Iron Transport

Transferrin System

Transferrin Characteristics: - Structure: 80 kDa glycoprotein, two binding sites - Synthesis: Primarily hepatic - Half-life: 8-10 days - Saturation: Normally 20-45% - Function: Safe iron transport, prevents free iron toxicity

Transferrin Receptor (TfR) Pathway: 1. TfR1 binding: High affinity for diferric transferrin 2. Receptor-mediated endocytosis 3. Endosomal acidification: pH 5.5 releases iron 4. STEAP3: Reduces Fe³⁺ to Fe²⁺ 5. DMT1: Exports iron to cytosol 6. Receptor recycling: Returns to surface

Tissue Distribution of TfR1: - Highest in erythroid precursors - Hepatocytes, placenta, rapidly dividing cells - Regulated by iron regulatory proteins

Non-Transferrin Bound Iron

NTBI in Pathological States: - Appears when transferrin saturation >45% - Causes oxidative damage via Fenton reaction - Preferentially taken up by 03 Spaces/Medical Hub/📝 Exam Prep/General Surgery SS Notes/SGE Notes INISS/HPB/Liver/Liver, heart, 04 Vault/Amboss Library/Basic sciences/By system/Endocrine system/Pancreas - Contributes to organ damage in iron overload

Iron Storage

Ferritin Biology

Structure and Function: - Apoferritin shell: 24 subunits (H and L chains) - H-chain: Ferroxidase activity for Fe²⁺ oxidation - L-chain: Iron nucleation and storage - Capacity: Up to 4,500 iron atoms per molecule

Serum Ferritin: - Origin: Primarily from macrophages - Glycosylation: Differs from tissue ferritin - Normal ranges: - Males: 30-300 ng/mL - Females: 15-200 ng/mL - Acute phase reactant: Increases with inflammation

Hemosiderin Formation

  • Composition: Partially degraded ferritin aggregates
  • Formation: In lysosomes under iron overload
  • Detection: Prussian blue stain positive
  • Clinical significance: Indicates tissue iron excess

Iron Utilization

Erythropoiesis

The major consumer of body iron:

Daily Requirements: - 20-25 mg for hemoglobin synthesis - 80% from macrophage iron recycling - 20% from transferrin-delivered iron

Erythroblast Iron Uptake: - High TfR1 expression - Iron responsive elements regulation - Coordinated with globin synthesis - Heme synthesis in mitochondria

Cellular Iron Utilization

Mitochondrial Iron: - Import: Via mitoferrin-1 and mitoferrin-2 - Iron-sulfur cluster synthesis: Essential for: - Electron transport chain complexes - Krebs cycle enzymes - DNA repair enzymes - Heme biosynthesis: 8-step pathway - Regulation: By iron regulatory proteins

Iron Recycling

Macrophage Iron Metabolism

Erythrophagocytosis Process: 1. Recognition of senescent RBCs 2. Phagocytosis by splenic macrophages 3. Heme oxygenase-1: Degrades heme 4. Release of Fe²⁺, CO, and biliverdin 5. Iron export via ferroportin or storage

Daily Recycling: - 20-25 mg iron from 200 billion RBCs - Efficient process (>95% recovery) - Critical for iron homeostasis

Regulation of Iron Metabolism

Hepcidin-Ferroportin Axis

Hepcidin Function: - 25-amino acid peptide hormone - Synthesis: Primarily hepatic - Action: Binds ferroportin → internalization → degradation - Effect: Decreases iron absorption and release

Regulation of Hepcidin: - Upregulated by: - High iron stores (via BMP-SMAD pathway) - Inflammation (IL-6 → JAK-STAT pathway) - Endoplasmic reticulum stress - Downregulated by: - Iron deficiency - hypoxia (via HIF) - Erythropoietic drive (erythroferrone) - Matriptase-2 activity

Iron Regulatory Proteins

IRP1 and IRP2: - RNA-binding proteins - Sense intracellular iron levels - Bind iron responsive elements (IREs) - Post-transcriptional regulation

Target Regulation: - 5' IRE (translation block when iron low): - Ferritin, ferroportin - ALAS2 (heme synthesis) - 3' IRE (mRNA stabilization when iron low): - Transferrin receptor 1 - DMT1

Laboratory Evaluation of Iron Status

Serum Iron Studies

Serum Iron: - Normal range: 50-170 μg/dL - Measurement: Colorimetric or atomic absorption - Variability: Diurnal (30-40%), dietary influence - Limitations: Poor indicator alone

Total Iron Binding Capacity (TIBC): - Normal range: 250-450 μg/dL - Represents: Total transferrin binding sites - Calculation: TIBC ≈ Transferrin (mg/dL) × 1.25 - Increases: Iron deficiency - Decreases: Inflammation, malnutrition

Transferrin Saturation: - Calculation: (Serum iron/TIBC) × 100 - Normal range: 20-45% - <16%: Suggests iron deficiency - >45%: Suggests iron overload - Most useful: For monitoring iron status

Ferritin Assessment

Clinical Utility: - Best single test for iron stores - 1 ng/mL ≈ 8-10 mg storage iron - <15 ng/mL: Diagnostic of iron deficiency - Caveats: Acute phase reactant

Interpretation Challenges: - Inflammation: Falsely elevates ferritin - C-reactive protein: Check concurrently - Adjusted thresholds: - With inflammation: <70-100 ng/mL suggests deficiency - Chronic disease: Higher cutoffs needed

Advanced Iron Parameters

Soluble Transferrin Receptor (sTfR): - Normal: 3-7 mg/L - Increases: Iron deficiency, increased erythropoiesis - Advantage: Not affected by inflammation - sTfR/log ferritin ratio: Improved diagnostic accuracy

Reticulocyte Hemoglobin Content (CHr): - Normal: >29 pg - Reflects: Recent iron availability (3-4 days) - <29 pg: Functional iron deficiency - Useful: Monitoring iron therapy response

Zinc Protoporphyrin (ZPP): - Increases: When iron unavailable for heme synthesis - Normal: <40 μmol/mol heme - Advantages: Stable, reflects chronic status - Limitations: Also elevated in lead poisoning, inflammation

Specialized Testing

Bone Marrow Iron Staining: - Gold standard historically - Prussian blue stain - Assessment: - Macrophage iron (0-6 scale) - Sideroblast percentage (normal 30-50%) - Rarely needed with modern assays

Hepcidin Assays: - Methods: ELISA, mass spectrometry - Clinical use: Limited currently - Research applications: Iron disorder classification - Future potential: Personalized therapy

Staging of Iron Deficiency

Three Stages of Development

Stage 1: Iron Depletion: - Ferritin: <20-30 ng/mL - Serum iron: Normal - TIBC: Normal - Transferrin saturation: Normal - Hemoglobin: Normal - No clinical symptoms

Stage 2: Iron-Deficient Erythropoiesis: - Ferritin: <15 ng/mL - Serum iron: Decreased - TIBC: Increased - Transferrin saturation: <16% - Hemoglobin: Normal or slightly decreased - CHr: <29 pg - ZPP: Elevated

Stage 3: Iron Deficiency Anemia: - All above abnormalities plus: - Hemoglobin: Below normal range - MCV: <80 fL (eventually) - MCH: Decreased - RDW: Increased - Blood smear: Hypochromic, microcytic RBCs

Differential Diagnosis of Abnormal Iron Studies

Pattern Recognition

Iron Deficiency Pattern: - ↓ Iron, ↓ Ferritin, ↑ TIBC, ↓ Saturation - ↑ sTfR, ↓ CHr - Microcytic, hypochromic anemia (late)

Anemia of Chronic Disease: - ↓ Iron, Normal/↑ Ferritin, ↓ TIBC, ↓ Saturation - Normal sTfR (usually) - Normocytic or mildly microcytic

Combined Iron Deficiency and ACD: - ↓ Iron, Variable ferritin, ↓ TIBC - ↑ sTfR, ↑ sTfR/log ferritin ratio - Most challenging diagnosis

Thalassemia Trait: - Normal/↑ Iron, Normal/↑ Ferritin - Normal TIBC and saturation - Very low MCV with mild anemia - Mentzer index <13

Special Considerations

Population-Specific Evaluation

Pregnancy: - Hemodilution: Affects all parameters - Increased requirements: 1000 mg total - Modified thresholds: Ferritin <30 ng/mL - CDC recommendations: Routine screening

Elderly: - Occult GI bleeding: Common cause - Chronic disease: Confounds diagnosis - Multiple medications: May affect absorption - Comprehensive evaluation: Essential

Athletes: - Sports anemia: Dilutional - Foot-strike hemolysis - GI bleeding: From exercise - Higher requirements: Increased turnover

Challenges in Diagnosis

Inflammation Effects: - Ferritin elevation - Decreased serum iron - Reduced TIBC - Consider multiple parameters

Functional Iron Deficiency: - Adequate stores but poor utilization - Common in CKD with ESA therapy - Normal ferritin, low saturation - CHr most useful marker

Iron Studies in Clinical Practice

Initial Evaluation Algorithm

  1. Screen high-risk populations:
  2. Menstruating women
  3. Children, adolescents
  4. Frequent blood donors

  5. Vegetarians/vegans

  6. Basic panel:

  7. CBC with indices
  8. Ferritin

  9. Consider CRP if inflammation suspected

  10. Extended testing if unclear:

  11. Add TIBC, calculate saturation
  12. Consider sTfR
  13. CHr if available

Monitoring Iron Therapy

Oral Iron Response: - Reticulocytosis: 3-7 days - Hemoglobin rise: 2 g/dL in 3 weeks - CHr increase: Within 1 week - Ferritin normalization: 3-6 months

IV Iron Monitoring: - Wait 2-4 weeks: Before rechecking ferritin - Transferrin saturation: May transiently exceed 100% - Hemoglobin response: Similar timeline - Safety parameters: Phosphate (with certain formulations)

Future Directions

Emerging Biomarkers

Erythroferrone: - Links erythropoiesis to iron regulation - Suppresses hepcidin - Potential diagnostic marker

Non-coding RNAs: - MicroRNAs regulating iron genes - Circulating biomarkers - Personalized medicine applications

Advanced Diagnostics

Mass Spectrometry: - Hepcidin quantification - Multiple analyte panels - Improved standardization

Genetic Testing: - TMPRSS6 mutations - Iron-refractory iron deficiency anemia - Personalized supplementation

Clinical Pearls

References