For clinical purposes, total iron binding capacity (TIBC) is considered as a measure of transferrin (Tf) concentration in serum or plasma (albeit indirect). Normally, about 1/3 of transferrin (33%) has iron bound to it. There are more direct methods of measuring transferrin, i.e. immunologic-based techniques, but these are not available in veterinary medicine. Studies in humans have shown that TIBC correlates well to immunologic measurement of transferrin (correlation coefficient of 0.70) (Tietz, Textbook of Clinical Chemistry).
Apotransferrin is a β-1 globulin that is produced in the liver. When iron (ferric form or Fe3+) binds to apotransferrin, it is called transferrin. Transferrin (Tf) is the iron transport protein in plasma. It is responsible for shuttling iron between sites of absorption, storage and utilization for the biosynthesis of iron-containing macromolecules. Donation of iron by Tf requires the interaction with specific receptors on the recipient cells. Such transferrin receptors have been identified on the surface of reticulocytes, hepatocytes, lymphocytes, fibroblasts and rapidly proliferating cells (that require iron for metabolism).
Transferrin concentrations are influenced by the following:
- Production: the following factors affect hepatic production of apotransferrin.
- Hepatocyte iron content: Increased iron reduces apotransferrin synthesis while decreased iron stimulates transferrin synthesis. Therefore higher transferrin levels are expected in animals with iron deficiency.
- Inflammatory cytokines: Transferrin is a negative acute phase reactant protein and will be downregulated by the hepatocyte in favor of acute phase reactant proteins after cytokine stimulation.
- Hepatic insufficiency or portosystemic shunts. A decrease in TIBC may occur (this is not a consistent feature in dogs with shunts).
- Protein intake: Protein synthesis depends on adequate intake of amino acids.
- Loss: Transferrin is a small protein and can be lost with albumin in protein-losing or exudative disorders.
- Catabolism: Transferrin is a protein and can be catabolized for energy in states of negative energy balance.
The measurement of TIBC actually requires assessment of the amount of transferrin in plasma or serum that has not bound iron, i.e. the unbound iron binding capacity (UIBC). TIBC is then calculated from the sum of UIBC + iron. It is expressed as μg/dL of iron binding capacity.
In the procedure, a known amount of iron is added in excess to the sample. This saturates transferrin with iron (transferrin is normally composed of 33% iron). This is accomplished at an alkaline pH (because an acidic pH would liberate the iron of transferrin) and ensures that the excess iron binds to the transferrin that lacks iron (unbound), i.e. assesses the iron binding capacity of transferrin. The iron remaining in the sample from that added is then measured using the standard dye-binding assay for iron measurement. This gives a value called the UIBC. The reactions are given below:
Serum + excess iron (added iron binds to parts of transferrin that have not already bound iron) in an alkaline pH
Measure UIBC or remaining (unbound iron) in presence of vitamin with Ferrozine®, i.e. UIBC = iron added – iron remaining
TIBC = UIBC + iron (already measured using an acidic pH to release the iron that is prebound to transferrin)
Units of measurement
The concentration of TIBC is measured in μg/dL (conventional units) and μmol/L (SI units). The conversion formula is shown below:
μg/dL x 0.179 = μmol/L
Reference intervals for TIBC concentration in domestic species have been established by the Clinical Pathology Laboratory in the Animal Health Diagnostic Center at Cornell University.
Lithium heparin is the preferred anticoagulant use in plasma samples for TIBC measurement. Anticoagulants with chelating agents such as EDTA, oxalate, and fluoride should not be used (they will chelate the excess iron resulting in very high TIBC).
TIBC is quite stable, like iron.
- Lipemia: Severe lipemia (>200 lipemia index) may increase TIBC.
- Hemolysis: Can affect both iron and UIBC, therefore variable unpredictable effect. Internal studies show that TIBC may be falsely increased with markedly hemolyzed samples in dogs (>400 hemolysis index).
- Icterus: No known effect.
This is relatively uncommon. Frequently, high TIBC results are not explainable.
- Artifact: High serum iron (e.g. iron overload), possibly severe lipemia.
- Iron deficiency: From testing in our laboratory, we have found that TIBC is high in woodchucks with iron deficiency. Presumably, woodchucks behave like humans in which iron deficiency stimulates apotransferrin synthesis, causing an increased TIBC. We have not seen any consistent increase in TIBC in dogs, cats or camelids with iron deficiency, but a high TIBC may occur in pigs, horses and cattle with iron deficiency (Smith J, personal communication).
- Necrotizing hepatitis in dogs: (Stockham and Scott, Fundamentals of Veterinary Clinical Pathology). The mechanism unclear – this be an artifact from release of high concentrations of storage iron from hepatocytes.
The most common causes of a low TIBC are decreased production secondary to inflammation and loss.
- Decreased production by the liver:
- Inflammatory cytokines: Transferrin is a negative acute phase reactant protein and will be downregulated by the hepatocyte in favor of acute phase reactant proteins (ceruloplasmin, serum amyloid A, haptoglobulin, α1-antitrypsin, α2-macroglobulin) after cytokine stimulation (IL-1, IL-6, TNFα). Transferrin has a longer half-life (7 days in man) than iron and may decrease slightly slower than iron in acute inflammation. Thus, with acute inflammation, we expect to see low iron and % saturation but a normal TIBC. With longer inflammation (>24-48 hours), both iron and TIBC may be low, resulting in a normal % saturation.
- Hepatic insufficiency or portosystemic shunts: This can decrease TIBC values, especially if there is hypoproteinemia.
- Protein intake: Malnutrition in ruminants can also result in decreased transferrin. In humans, low transferrin is a more sensitive marker of malnutrition than albumin.
- Loss of transferrin: Transferrin is a small protein and can be lost with albumin in protein-losing disorders, e.g. protein-losing nephropathy, protein-losing enteropathy, burns. Low albumin and TIBC would be expected under these conditions.
- Catabolism of transferrin: Transferrin is a protein and can be catabolized for energy in states of negative energy balance.
- Clinical Pathology Laboratory in the Animal Health Diagnostic Laboratory at Cornell University website: Information on testing for iron panels (iron, TIBC, % saturation). These are included on our small and large animal chemistry panels.