Ferrozine: Laboratory Reagent for Iron Measurement
Ferrozine is a chromophoric chelating agent used in colorimetric assays to quantify ferrous iron (Fe²⁺) concentrations in biological samples, forming a colored complex that absorbs light at 562 nm and enables spectrophotometric measurement of iron status. 1
Chemical Properties and Mechanism
Ferrozine functions as a high-affinity chelator that binds specifically to ferrous iron (Fe²⁺), creating a Fe(ferrozine)₂³⁺ complex with maximum absorbance at 562 nm. 1 This colorimetric reaction forms the basis for quantitative iron determination in various biological and clinical specimens.
Key Characteristics:
- Specificity: Detects ferrous iron with minimal interference from other divalent metal cations 2
- Sensitivity: Capable of measuring iron concentrations ranging from 0.2 to 30 nmol 2
- Accuracy: Demonstrates 99.3-101.4% accuracy with precision of 0.73% within-run and 1.1% day-to-day variability 3
Clinical Applications in Iron Status Assessment
While ferrozine itself is not a routine clinical test ordered for patients, it serves as the analytical reagent in laboratory assays that measure serum iron concentrations. However, clinicians should recognize that serum iron alone is unreliable for assessing iron status due to substantial diurnal variation and increases after meals. 4
Recommended Clinical Approach:
For diagnosing iron deficiency, prioritize serum ferritin and transferrin saturation (TSAT) rather than serum iron measurements alone. 5, 6, 4
Primary Diagnostic Tests:
- Serum ferritin <15 μg/L: 99% specificity for iron deficiency in absence of inflammation 4
- Serum ferritin <30 μg/L: Generally indicates low body iron stores 5, 4
- TSAT <16-20%: Confirms iron deficiency when combined with low ferritin 5, 4
In Inflammatory Conditions:
- Use ferritin <100 μg/L as diagnostic threshold when inflammation, chronic kidney disease, heart failure, or inflammatory bowel disease is present, as ferritin acts as an acute-phase reactant and may be falsely elevated 5, 6, 4
- TSAT remains more reliable than ferritin in inflammatory states as it is less affected by acute-phase reactions 5, 6
Laboratory Methodology Considerations
Sample Preparation:
Iron must be liberated from protein complexes before ferrozine can bind it. This is typically achieved through:
- Proteolytic disruption using pepsin in hydrochloric acid medium 3
- Acidic KMnO₄ treatment to make protein-bound iron accessible to ferrozine 2
- Reductive agents (ascorbate, cysteine, glutathione) to convert ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) for detection 1
Critical Pitfalls in Ferrozine Assays:
Ferric ions (Fe³⁺) can interfere with ferrous iron measurements when assays are performed in the dark, causing linearly increasing absorbance with incubation time. 7 This represents a previously unrecognized source of error in iron quantification.
Highly crystalline iron minerals (magnetite, goethite) require dissolution in 6M HCl at 60°C for accurate total iron determination, but this heating renders subsequent ferrous iron measurement impossible due to rapid abiotic oxidation. 8
Clinical Monitoring After Iron Supplementation
Do not check iron parameters within 4 weeks of intravenous iron administration, as circulating iron interferes with assays and produces falsely elevated results. 6, 4
Recheck iron parameters 4-8 weeks after initiating supplementation to assess therapeutic response, expecting hemoglobin to increase by 1-2 g/dL if treatment is effective. 6, 4
Practical Clinical Recommendations
For routine clinical practice, order:
- Complete blood count (CBC) with hemoglobin, hematocrit, and MCV 6, 4
- Serum ferritin as the primary marker of iron stores 5, 6, 4
- Transferrin saturation (TSAT) to assess immediately available iron for hemoglobin synthesis 5, 6, 4
Do not rely on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), or serum iron alone for diagnosing iron deficiency, as these are late indicators or have significant variability. 5, 4