Serum Iron, Ferritin and Transferrin

-Gel-O-Fury- V. Saavedra, RMT

MS Medical TechnologyUST Graduate School

Importance of Iron

1. Serve as both electron donor and acceptor in the Electron Transport Chain.

2. Needed by peroxidase enzymes as catalysts to convert harmful peroxides into water.

3. Needed in oxidative phosphorylation (oxidation of nutrients into ATP) by iron-sulfur proteins.

4. Main Importance: Oxygen Transport (incorporated in heme in hemoglobin)

Distribution of Iron3-5 g of iron is in the body (total)

2-2.5 g of which is in the hemoglobin.

Some (about 130 mg) are in myoglobin (oxygen carrier in tissues).

Little (about 8 mg) is bound to enzymes like peroxidases, cytochromes and other enzymes involved in the Krebs Cycle.

Some are stored in ferritin and hemosiderin.

Little (3-5 mg) is in plasma in transferrin.

Storage Iron and Transferrin

2 forms of storage iron:

Ferritin – water-soluble complex of ferric salt (Fe+3) and the protein apoferritin. Present in blood.

- a positive acute phase reactant/protein – increases in inflammation.

Hemosiderin – water-insoluble. Present in tissues.

Storage Iron and Transferrin

Transferrin – main protein for iron transport.

- a negative acute phase reactant/protein – decreases in inflammation.

Transferrin + iron = serum iron

Iron Metabolism

1.Iron (Ferric) from foods is ingested.2.To be absorbed by the intestinal cells, ferric

ions (Fe+3) must be reduced to ferrous ions (Fe+2) by agents like Vitamin C (ascorbate).

3. Ferrous ions (Fe+2) are bound to apoferritin then oxidized to ferric ions (Fe+3) by ceruloplasmin to become bound as ferritin.

4. Ferritin is carried into blood (plasma) and releases its ferric ions (Fe+3). 2 ferric ions (Fe+3) are then absorbed by the protein apotransferrin to become transferrin.

5. Ferric (Fe+3) ions are then incorporated into the bone marrow for hemoglobin production.

* methemoglobin reductase – reduces (Fe+3) to (Fe+2)

6. RBC’s are degraded by the spleen, liver and macrophages. Iron leftovers from RBC’s are carried by transferrin and recycled.

However, some iron is lost and excreted in the feces or urine. Women lose 20-40 mg of iron due to menstruation...

Implication

Diagnosis of conditions/diseases- sort out the diseases- most common: iron

deficiency anemia

Assess nutritional status of the patient.

Specimen and Patient Preparation

Hemolyzed specimens must be rejected.

Specimen must be collected as serum: - Oxalate, citrate and EDTA as anticoagulant is unacceptable – chelators that can bind to iron.

Early morning samples are preferred – diurnal variation in iron concentration. 25% lower in the evening.

Fasting specimen is required – diet may contain iron.

Patient must not be in iron medication.

Serum Iron Measurement

Spectrophotometry - very sensitive test

- Iron is first removed from transferrin by acidification (HCl).

- Ferric (Fe+3) ions are reduced to ferrous (Fe+2) ion with a reducing agent (ascorbate)

- Ferrous (Fe+2) ion is complexed with a color reagent (ferrozine, ferene or bathophenanthroline). Measure spectrophotometrically.

Serum Iron MeasurementAtomic Absorption Spectroscopy – used to measure concentration by detecting absorption of electromagnetic radiation by atoms.

- specimens are burned to break the bonds and liberate free unexcited atoms.

- light from the hollow-cathode lamp passes through the atoms to shift into excited state.

- the excited atoms then release light and shifts to ground state again – measured by light detectors.

Ferritin MeasurementRadioimmunoassay (RIA) - highly sensitive test

- uses radioisotopes as labels to detect the presence of antigen (ferritin) or antibody in a sample.

- Unlabeled ("cold") antigens compete for the antibodies. Radioactive antigens are displaced from the antibodies.

- The antibody-bound antigen is separated from the free antigen in the supernatant fluid.

- The radioactivity of each bound antigen is measured.

Ferritin Measurement

Enzyme-linked immunosorbent assay (ELISA)

- Patient’s serum containing the antigen (ferritin) is added in Microwell strips (which contains the antibody)

- 2nd antibody coupled to an enzyme is then added (forming a “sandwich”)

- substrate (chromogen) solution is added and will be cleaved by the enzyme to form a colored product.

- measured quantitatively by a spectrophotometer.

Transferrin MeasurementTotal Iron Binding Capacity (TIBC) – ability of transferrin to bind iron in a saturated state.

- Indirect measurement of transferrin.

- Ferric (Fe+3) ions are bombarded to serum.

- All excess iron is removed (by the precipitant MgCO3) and the serum is analyzed for iron using serum iron methods.

- Iron measured here reflects the total ability of the transferrin to bind iron (TIBC).

TIBC (μg/dL) = transferrin (mg/dL) x 1.25

Transferrin MeasurementPercent/Transferrin Saturation – ratio of the serum iron to the TIBC.

% Saturation = Total Iron (μg/dL) x 100%

TIBC (μg/dL)

Nephelometry – directly measures transferrin.

- a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than absorbing it.

- The amount of light scatter is determined by collecting the light at an angle (usually about 70-75°) by detectors.

FEP MeasurementFree Erythrocyte Protoporphyrin (FEP) – protoporphyrin IX in which ferrous (Fe+2) iron is added to form heme.

FEP MeasurementZinc protoporphyrin (ZPP) - compound found in RBCs when heme production is inhibited or if iron is deficient.

- Instead of incorporating a ferrous ion, to form heme, protoporphyrin IX, incorporates a zinc ion, forming ZPP.

- normally present in trace amounts.

Measured by:

- High Performance Liquid Chromatography (HPLC) - FEP is extracted from the heme and measured.

- Hematofluorescence – ZPP fluorescence is measured using fluorescent spectrophotometer (Protoporphyrin + Iron will not fluoresce).

Reference ValuesPatient

Population

Serum Iron

(μg/dL)

Transferrin

(mg/dL)

Ferritin(μg/L)

% Saturatio

n

TIBC(μg/dL)

Adult Male 50-160 200-380 20-250 20-55 250-425

Adult Female 45-150 200-380 10-120 15-50 250-425

Newborn 100-250 130-275 25-200 12-50 100-400

Infant 40-100 200-360 200-600 12-50 100-400

Child 50-120 200-360 7-140 12-50 100-400

Condition Ferritin%

Saturation

Transferrin

TIBC Serum IronFEP/ZPP

Iron Deficiency Anemia

Thalassemia Major N / N N N N N

Anemia of Chronic Disease N N / N /

Sideroblastic Anemia N / N / Variabl

e

Lead Poisoning N N / N N N /

Hemochromatosis / Iron Overload N N N

Malnutrition Variable N

Malignancy N

Iron Deficiency Anemia

- An impaired production disease.

- Exists when there’s an increased need for iron or when excessive blood loss has reduced the body's iron reserves.

- Insufficient iron is available for normal hemoglobin production.

- Most common cause of anemia on the planet, affecting at least 1/3 of the world's population

Iron Deficiency AnemiaThe sequence of events in developing iron deficiency anemia:

Stage 1: Iron Depletion – when blood loss exceeds absorption, iron is mobilized from stores, ferritin decreases, iron absorption increases, and plasma iron-binding capacity (transferrin) increases.

Stage 2: Iron-Deficient Erythropoiesis – after iron stores are depleted, the plasma iron concentration falls, percent saturation falls and the percentage of sideroblasts decreases in the marrow. As a result of lack of iron for heme synthesis, protoporphyrin also increases.

Stage 3: Iron Deficiency Anemia – in addition to the above abnormalities, microcytic, hypochromic anemia is present.

ConditionIron

Replete (normal)

Stage 1 (Iron

Depletion)

Stage 2 (Iron-

Deficient Erythropoiesi

s)

Stage 3 (IDA)

Iron Overloa

d

Ferritin > 12 < 12 < 12 < 12 > 300

TIBC300-360

360 390 410 < 300

Serum Iron 65-165 115 < 60 < 40 > 175

% Saturation 20-50 30 < 15 < 10 > 60

FEP(NV = 17-77 μg/dL)

< 50 < 50 100 200 < 50

Iron Deficiency AnemiaThe mechanisms of Iron Deficiency include:

Increased physiologic demand:Rapid growth of infants and children.Pregnancy, lactation.

Inadequate intake:Iron-deficient diet Inadequate absorption (achlorhydria, decreased

absorptive surface)

Blood loss:MenstruationGastrointestinal bleedingHemorrhoidsRegular blood donationHemolysis

Iron Deficiency AnemiaClinical Presentation:

Fatigue, breathlessness and dizziness – due to reduced oxygen delivery.

Pica – persistent compulsive desire of eating substances like ice, clay, plaster, dirt and even insects.

Disturbances in the gastrointestinal system – swallowing difficulties (due to webbing of esophageal tissue), stomatitis (cracks in the mouth corners), tongue abnormalities (soreness and papillary atrophy) and gastritis (may progress to gastric atrophy which results in achlorhydria).

Koilonychia – spooning of nails

Plummer-Vinson SyndromePlummer-Vinson syndrome (US) or Paterson-Brown Kelly

syndrome (UK) is a rare disease defined by severe, long-term iron deficiency anemia, which causes swallowing difficulty (dysphagia) due to web-like membranes of tissue growing in the throat (esophageal webs).

Currently, the cause and the epidemiology of this syndrome are unknown, however, genetic factors and nutritional deficiencies were pointed out to play a role.

Clinical findings include almost all those mentioned in iron deficiency anemia. This may progress to squamous cell carcinoma of the oral cavity, esophagus and hypopharynx.

The 1st step in the management of the disease is to clarify the cause of iron deficiency in order to exclude active hemorrhage, malignancy or celiac disease.

Iron Deficiency in Children With Attention-Deficit/Hyperactivity Disorder by Eric Konofal, MD, et al. (From Archives of Pediatrics and Adolescent Medicine, December 17, 2004)

Background: Iron deficiency causes abnormal dopaminergic neurotransmission and may contribute to the physiopathology of attention-deficit/hyperactivity disorder (ADHD).

Objective: To evaluate iron deficiency in children with ADHD VS iron deficiency in an age and sex-matched control group.

Design: Controlled group comparison study.

Setting: Child and Adolescent Psychopathology Department in European Pediatric Hospital, Paris, France.

Patients (Subjects): 53 children with ADHD aged 4 to 14 years (mean ± SD, 9.2 ± 2.2 years) and 27 controls (mean ± SD, 9.5 ± 2.8 years).

Journal

Main Outcome Measures: Serum ferritin levels evaluating iron stores and Conners’ Parent Rating Scale scores measuring severity of ADHD symptoms have been obtained.

Results: The mean serum ferritin levels were lower in the children with ADHD (mean ± SD, 23 ± 13 ng/mL) than in the controls (mean ± SD, 44 ± 22 ng/mL; P < 0.001).

Serum ferritin levels were abnormal (<30 ng/mL) in 84% of children with ADHD and 18% of controls (P < 0.001).

In addition, low serum ferritin levels were correlated with more severe general ADHD symptoms measured with Conners’ Parent Rating Scale (Pearson correlation coefficient, r = -0.34; P < 0.02) and greater cognitive deficits (r = -0.38; P < 0.01).

Conclusion: The results suggest that low iron stores contribute to ADHD and that ADHD children may benefit from iron supplementation.

Journal

Double Burden of Iron Deficiency in Infancy and Low Socioeconomic Status: A Longitudinal Analysis of Cognitive Test Scores to Age 19 Years by Betsy Lozoff, MD, et al. (From Archives of Pediatrics and Adolescent Medicine, July 8, 2006)

Objective: To assess change in cognitive functioning after iron deficiency in infancy, depending on socioeconomic status (SES; middle VS low).

Design: Longitudinal study.

Setting: Urban community in Costa Rica (infancy phase [July 26, 1983, through February 28, 1985] through 19-year follow-up [March 19, 2000, through November 4, 2002]).

Participants: A total of 185 individuals enrolled at 12 to 23 months of age (no pre-term or low-birth-weight infants or infants with acute or chronic health problems).

Journal

The participants were assessed by various cognitive assessment score tests in infancy and at 5, 11 to 14, 15 to 18, and 19 years of age. A total of 97% were evaluated at 5 or 11 to 14 years and 78% at 15 to 18 or 19 years.

Iron status in infancy was determined by venous concentrations of hemoglobin, % saturation, FEP, and serum ferritin. Individuals who had chronic iron deficiency in infancy (iron deficiency with hemoglobin concentrations ≤10.0 g/dL or, with higher hemoglobin concentrations, not fully corrected within 3 months of iron therapy) were compared with those who had good iron status as infants (hemoglobin concentrations ≥12.0 g/dL and normal iron measures before and/or after therapy).

Main Outcome Measures: Cognitive change over time (composite of standardized scores at each age).

Journal

Results:

For middle-SES participants, scores averaged 101.2 in the group with chronic iron deficiency VS 109.3 in the group with good iron status in infancy and remained 8 to 9 points lower through 19 years (95% confidence interval [CI], -10.1 to -6.2).

For low-SES participants, the gap widened from 10 points (93.1 VS 102.8; 95% CI for difference, -12.8 to -6.6) to 25 points (70.4 VS 95.3; 95% CI for difference, 20.6 to 29.4).

Conclusions: The group with chronic iron deficiency in infancy did not catch up to the group with good iron status in cognitive scores over time. There was a widening gap for those in low-SES families. The results suggest the value of preventing iron deficiency in infancy.

Journal

Bishop, M. L., et al. (2006) Clinical Chemistry: Principles, Procedures, Correlation (2nd Edition). USA: Lippincott Williams and Wilkins.

Hillman, R. S., et al. (2005) Hematology in Clinical Practice (4th Edition). USA: McGraw-Hill

Lehman, C. A. (1998) Saunders Manual of Clinical Laboratory Science. USA: W.B. Saunders

McPherson, R. A., Pincus, M. R. (2006) Henry’s Clinical Diagnosis and Management by Laboratory Methods (21st Edition). USA: Elsevier

Stiene-Martin, E. A., et al. (1997) Clinical Hematology: Principles, Procedures, Correlation (2nd Edition). USA: Lippincott.

References

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