Non-transferrin bound iron (NTBI) is a form of plasma iron that appears when the iron-binding capacity of transferrin is increased. Although well characterized to be a toxic species of iron, NTBI is not routinely measured by major labs. The presence of NTBI is inferred, based upon the elevation of the Transferrin Saturation. What this threshold is, however, may be different from condition to condition. NTBI has been observed in several conditions, including: hemochromatosis, type 2 diabetes, cardiovascular disease, thalassemias, in patients receiving cancer chemotherapy, and in kidney dialysis. Additionally, NTBI may also form in people receiving both oral iron and intravenous iron infusions. In addition to NTBI’s potential for iron-mediated toxicity, it also may be involved in pathogenic microbial overgrowth, evidenced by the fact that several studies have investigated how microorganisms can readily obtain NTBI.

Iron: The Wrong Form, At the Wrong Time & In The Wrong Place Can Be Toxic

In the human body, iron exists primarily in 2 valence states: Fe2+, known as Ferrous iron, and Fe3+, known as Ferric iron. This difference of only 1 electron, significantly changes the form and function of iron. Fe3+ is the primary form that is bound to transferrin, and this is also the primary form that is bound to ferritin, which serves as the primary intracellular storage form of iron. Fe2+ on the other hand is the form of iron found within hemoglobin, and this valence state is necessary in order for hemoglobin to reversibly bind oxygen. Additionally, both the Fe2+ and Fe3+ forms are found within the mitochondrial cytochromes. Fe2+ is found in the ETC, complexes 1 and 2, and Fe3+ is located within ETC complex 3.

Any significant change in the gradient between Fe2+ and Fe3+ can lead to deleterious consequences. For example, in Methemoglobinemia, hemoglobin is bound to the Fe3+ form instead of Fe2+, and this prevents oxygen from being adequately transported by red blood cells, which leads to the accumulation of Methemoglobin.

The protein transferrin has the responsibility of serving as the primary plasma transport of Fe3+ iron. In this capacity, transferrin can transport newly absorbed Fe3+, derived from dietary iron throughout the body. Most importantly, transferrin also redistributes and recycles the iron that is derived from the continual degradation of hemoglobin and red blood cells in the spleen and liver. This recycled iron is actually the body’s primary way of obtaining iron on a daily basis, accounting for some 25-30mg of iron used.

One of the main problems with iron is that the Fe2+ form can readily react with hydrogen peroxide and generate hydroxyl radicals. Hydroxyl radicals (- OH) are highly reactive and toxic species that can cause extensive damage to biological systems, damaging virtually all types of macromolecules, such as carbohydrates, nucleic acids, lipids and proteins.

In order to maintain the proper gradient of iron between its two valence states, and to ensure that the wrong form is not used in the wrong place at the wrong time, the body uses Ferrooxidases (such as ceruloplasmin and hephaestin) to oxidize Fe2+ into the Fe3+, and Metalloreductases such as STEAP to reduce Fe3+ into Fe2+.
Under normal, healthy conditions, iron in the blood is either bound to transferrin, or it is found within the hemoglobin of circulating red blood cells. However, in certain pathological contexts and clinical situations, another species of iron can exist in the blood, and this is known as NTBI (non transferrin bound iron).

While iron is obviously essential for various physiological processes, its presence in unbound forms can lead to significant toxicity. As the name implies, non-transferrin bound iron (NTBI) is a form of iron that is not bound to transferrin. Instead, NTBI is loosely bound to organic molecules, such as citrate and acetate.

Unlike transferrin-bound iron, whose uptake is mediated via the interaction between TFR1 and TFR2 receptor on cells (transferrin-1 and 2 receptors), NTBI is labile iron that can exist in both valence states (Fe2+ and Fe3+), and can be readily taken up by cells via mechanisms independent of TFR1 or TFR2. An example of this is SLC39A14, a transporter of zinc, which has also been confirmed to import NTBI into hepatocytes (Liuzzi, et al; 2006). The ability for NTBI to bypass iron-sensing receptors, means that this form of iron can readily be taken up into cells, likely in an un-regulated manner. Moreover, it is likely to be the case that any Fe3+ fraction of NTBI must be reduced first to the more reactive Fe2+ upon cellular import.

NTBI In Iron Overload Disorders

The Transferrin Saturation is a percentage measurement of the total number of Transferrin sites bound by Iron. It is a key biomarker that provides clinicians with both qualitative and quantitative information. While there are no meaningful ways for quantifying tissue iron stores (outside of a biopsy), an elevation in the transferrin saturation is a significant indicator of iron overload. Most labs report the top of the transferrin saturation as 50%, or 55% in some instances.

While it has been generally accepted that NTBI is only found when the transferrin saturation is greater than 70%, there are conditions where NTBI can form even when transferrin is saturated under 50%.

Hereditary Hemochromatosis, Thalassemia & NTBI

Hereditary hemochromatosis and transfusion-dependent thalassemia are classic examples of iron overload conditions where NTBI plays a crucial role in the pathogenesis. In hereditary hemochromatosis, particularly among HFE C282Y homozygotes, NTBI becomes detectable at relatively low levels of transferrin saturation. A 2022 study found that NTBI was present in 124 out of 161 C282Y homozygotes where Transferrin Saturation only ≥ 50% (Ryan, et al; 2022). Importantly, all subjects with Transferrin Saturation ≥ 75% had detectable NTBI, with a median concentration of 2.21 µM11. Thalassemia patients, especially those receiving regular transfusions, are at high risk of NTBI-related complications. Piga et al. reported that all thalassemia patients with heart disease had transferrin saturation above 70% and detectable NTBI (Piga, et al; 2021).

Type 2 Diabetes & NTBI

A study by Lee et al. 2006 found that NTBI was commonly present in patients with type 2 diabetes, even without overt iron overload. This study evaluated NTBI among 48 well established diabetics, 49 newly diagnosed diabetics and 47 control subjects. The study found that NTBI was present in 92% of patients with longstanding diabetes and in 59% of newly diagnosed diabetics. Moreover, only 6 of 47 diabetics had a Transferrin Saturation >50%. This suggests that metabolic disturbances in diabetes affects iron homeostasis, leading to NTBI formation at much lower transferrin saturation levels than typically observed in primary iron overload disorders. This also implicates NTBI and iron toxicity as an important mechanism of ROS generation in diabetes.

Epilepsy, NTBI

Ounjaijean, et al, 2011 found that epileptic patients receiving valproic acid had elevated NTBI, despite having normal iron parameters: Ferritin, Serum Iron and Transferrin Saturation.

Cancer, NTBI & Chemotherapy

Chemotherapy is well established to increase NTBI, and it may be present in some patients when transferrin is not fully saturated. Bradley, et al; 1997 found that the elevation of NTBI in chemotherapy patients was inversely related to their fall in both reticulocytes and serum transferrin receptor levels. While most patients with NTBI had fully saturated Transferrin, four did not. In another study involving NTBI and cancer, Beloti, et al; 2015 concluded that NTBI emergence was likely due to “chemotherapy induced lysis of bone marrow cells and, partly, of hepatocytes after cytotoxic injury.”

NTBI & Chronic Kidney Disease (CKD)

Patients with CKD, particularly those on hemodialysis, are prone to developing NTBI. A study by Rostoker et al. showed that hemodialysis patients could develop iron overload and potentially NTBI even when following current anemia treatment guidelines.

NTBI & Neurodegenerative Diseases

An assortment of studies has described the possible presence of NTBI in both Parkinson’s and Alzheimer’s. Human studies however are lacking. Due to the known involvement of brain iron toxicity in both conditions, more research is warranted. A recent study found that oral iron chelation using the drug Deferiprone reduced iron accumulation in the basal ganglia, among patients with Parkinson’s (Grolez, et al; 2015). The patients with the greatest overall benefit, appeared to have lower ceruloplasmin levels at baseline.

NTBI & Cardiomyopathy

NTBI uptake by cardiomyocytes is a major contributor to cardiac iron loading and dysfunction, with NTBI entry exceeding transferrin-dependent pathways under conditions of iron overload. The resulting oxidative stress alters the pro-anti-oxidant balance, leading to increased free radical production and cellular damage, which are fundamental in the pathogenesis of iron-overload mediated heart disease.

NTBI Following Iron Administration

The appearance of NTBI following iron supplementation is a concern in both oral and intravenous (IV) administration.

Oral Iron Supplementation

Several studies have demonstrated NTBI elevation following oral iron supplementation:

• A study by Brittenham et al, 2014 found that oral ferrous sulfate (100 mg) led to significant NTBI levels in iron-adequate subjects, with peaks reaching 0.81 μmol/L at 4 hours post-dose when taken without food
• Research by Sharma et al, 2019 showed that oral ferrous salts caused NTBI values of 6-12 μM/L within the first eight hours after a 100 mg dose
• A study by Schumann, et al, 2012 reported that NTBI concentrations increased in a graded fashion in response to oral iron doses ranging from 15 to 240 mg in healthy men.

Intravenous Iron Administration

IV iron preparations pose a more significant risk of NTBI formation than oral. A study by Garbowski et al, 2021 found that different IV iron formulations led to varying levels of NTBI. The study reported:

• Iron sucrose rapidly increased NTBI
• Ferric carboxymaltose showed a delayed NTBI increase
• Iron isomaltoside-1000 resulted in lower NTBI levels

Blood Transfusion & NTBI

Blood transfusions are known to cause elevations of NTBI. In fact, it has been proposed that increased transfusion morbidity of both adults and babies may be due to high levels of NTBI. Collard, White, 2014 identified one source of NTBI: prolonged blood storage and subsequent heme oxidation.

Mechanisms of NTBI Toxicity

NTBI is particularly dangerous due to its high reactivity and ability to participate in redox reactions. It can lead to:

• Oxidative stress: NTBI catalyzes the formation of reactive oxygen species, including hydroxyl radicals, which can damage cellular components
• Mitochondrial Toxicity: Because NTBI is labile iron, its cellular uptake is considered un-regulated due to being imported independently of TFR1 and TFR2 receptors. NTBI is rapidly taken up by mitochondria, likely via mitoferrin. Mitochondrial iron overload can cause damage to mitochondrial DNA as well as the respiratory chain (Zhao, et al; 2024)
• Tissue iron loading: Unlike transferrin-bound iron, NTBI can be taken up by cells in an unregulated manner, leading to iron accumulation in organ parenchyma and tissues not adapted for iron storage.

NTBI & Microbial Growth

Some pathogens can utilize NTBI for growth, potentially increasing infection risk. For example, a study involving catheterized mice found that NTBI induced the formation of biofilm and increased the virulence of pseudomonas aeruginosa (La Carpia, et al; 2023). An ex vivo study analyzing blood of Hemodialysis patients receiving iron infusions who experienced elevations of NTBI found that staphylococcus aureus could grow with greater virulence (Pai, et al; 2006). An in vitro study confirmed that the fungal species aspergillus fumigatus can metabolize NTBI (Verena, et al; 2017)

Clinical Management of NTBI

Iron Chelators Known to Reduce NTBI

• Deferoxamine is an iron chelation drug that is known to reduce NTBI
• Deferiprone is an oral iron chelating drug, which initially raises NTBI, but consequently lowers it
• The polyphenol Curcumin, is an effective iron-chelating compound. A clinical trial in ß-Thalassemia found Curcumin was efficacious at significantly lowering NTBI & liver enzymes, in a randomized, double blinded placebo-controlled trial (Mohammadi, Tamaddoni, et al, 2018)
• Green tea derived EGCG prevents the formation of NTBI in vivo (Ounjaijean, et al; 2008), and has been trialed alongside other iron chelating compounds in thalassemia
• The beta amino acid, L-Carnosine has iron chelating properties, which have been confirmed in animal studies. To date, no studies have investigated its effects on NTBI, but it is a candidate due to its known ability to lower plasma iron among type 2 diabetic patients (Baye, Ukropec, et al; 2019)