Serum Iron, Ferritin and Transferrin

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

MS Medical Technology UST 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 Iron
3-5 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.

tissues.

Hemosiderin water-insoluble. Present in

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. RBCs are degraded by the spleen, liver and macrophages. Iron leftovers from RBCs 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 Measurement

Atomic Absorption Spectroscopy used to measure
concentration by detecting electromagnetic radiation by atoms. absorption of

- 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 Measurement
Radioimmunoassay (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)
- Patients 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 Measurement
Total 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 (g/dL) = transferrin (mg/dL) x 1.25

Transferrin Measurement
Percent/Transferrin Saturation ratio of the serum iron to
the TIBC.

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

TIBC (g/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 Measurement
Free
protoporphyrin IX in which ferrous (Fe+2) iron is added to form heme.

Erythrocyte

Protoporphyrin

(FEP)

FEP Measurement
Zinc 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.
measured using fluorescent spectrophotometer (Protoporphyrin + Iron will not fluoresce).

- Hematofluorescence ZPP fluorescence is

Reference Values
Patient Population Adult Male Serum Iron Transferrin (g/dL) (mg/dL) Ferritin (g/L) % Saturation TIBC (g/dL)

50-160 45-150 100-250

200-380 200-380 130-275

20-250 10-120 25-200

20-55 15-50 12-50

250-425 250-425 100-400

Adult Female Newborn

Infant

40-100

200-360

200-600

12-50

100-400

Child

50-120

200-360

7-140

12-50

100-400

Condition Iron Deficiency Anemia Thalassemia Major Anemia of Chronic Disease Sideroblastic Anemia Lead Poisoning Hemochromatosis / Iron Overload Malnutrition

Ferritin

% Transferrin Saturation

TIBC

Serum Iron

FEP/ ZPP

N/ N

N N N/
Variable

N N/ N/ N N

N N/ N/ N N

N N/

N
Variable

N N

Malignancy

Iron Deficiency Anemia

- An impaired production disease. - Exists when theres an increased need for iron or when excessive blood loss has reduced the body's iron reserves. - Insufficient iron is hemoglobin production. available for normal

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

Iron Deficiency Anemia

The 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.

Condition

Iron Stage 1 Stage 2 (IronReplete (Iron Deficient (normal) Depletion) Erythropoiesis)

Stage 3 (IDA)

Iron Overload

Ferritin

> 12

< 12

< 12

< 12

> 300

TIBC

300-360

360

390

410

< 300

Serum Iron

65-165

115

< 60

< 40

> 175

% Saturation FEP

20-50

30

< 15

< 10

> 60

(NV = 17-77 g/dL)

< 50

< 50

100

200

< 50

Iron Deficiency Anemia

The mechanisms of Iron Deficiency include:

Increased physiologic demand:

Rapid growth of infants and children. Pregnancy, lactation. Iron-deficient diet Inadequate absorption (achlorhydria, decreased absorptive surface)
Menstruation Gastrointestinal bleeding Hemorrhoids Regular blood donation Hemolysis

Inadequate intake:

Blood loss:

Iron Deficiency Anemia

Clinical Presentation: reduced oxygen delivery.

Fatigue, breathlessness and dizziness due to 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 Syndrome
Plummer-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.

Journal
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 attentiondeficit/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.

References
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) Henrys 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.

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