Glutathionyl Hemoglobin Has Potential as a Biomarker of Iron Deficiency Anemia

Iron deficiency anemia (IDA) is a major health problem worldwide. Although a clinical diagnosis is relatively simple, specific laboratory markers of IDA are lacking especially in the setting of inflammation. Ferritin, the current standard to define IDA is an acute phase protein that is non-specifically elevated during inflammation. Serum transferrin receptor level measurements although available are not yet standardized as a clinical tool. Serum hepcidin is a recently developed novel marker that is currently neither available nor standardized sufficiently. Furthermore, such assays require instrumentation, technical sophistication, and are expensive.

We sought to identify novel markers of IDA using mass spectrometry based proteomics. Identifying such markers could yield targets that once validated could serve cost effective point of care assays to detect iron deficiency anemia. Since there is evidence for oxidative damage mediated by reactive oxygen species in IDA, as a first step, we characterized and quantified posttranslational oxidative modifications of hemoglobin and tested their utility as biomarkers.

We prospectively enrolled patients with IDA (defined as ferritin <12ng/ml in the presence of normal CRP and/or a bone marrow aspirate with “0” iron stores) and healthy controls (n = 23 and 15 respectively). Patients with diabetes, cardiovascular disease, renal disease, cerebrovascular disease and liver disease were excluded as these are conditions associated with preexisting oxidant stress. Erythrocytes from the blood of IDA patients and controls were isolated by centrifugation, washed in 0.9% saline, and lysed in distilled water to yield intracellular hemoglobin. Hemoglobin was then either studied further as an intact molecule or after digestion with trypsin. We used matrix assisted laser desorption ionization (MALDI - TOF) mass spectrometry to identify oxidative modifications of tryptic digested hemoglobin. We used electro spray ionization (ESI) mass spectrometry to identify and semiquantitate oxidative hemoglobin modifications by methods previously established and published by others and ourselves.

Using a combination of mass spectrometric methods, we identified 4 oxidative modifications of hemoglobin in patients with IDA and healthy controls (Table 1). Interestingly, a non enzymatic posttranslational modification of hemoglobin, glutathionyl hemoglobin, was found to be significantly increased in IDA patients compared with healthy controls (Glutathionyl hemoglobin % of beta chain; mean ± SD 0.169 ± 0.096 vs 0.077 ± 0.037; p = 0.001). Markers of oxidative stress (reduced RBC glutathione) were lower in IDA compared to healthy controls but the difference was not significant (mean ± SD 0.92 ± 0.53 vs 1.08 ± 0.52 mmol/L; p = 0.54). Glutathionyl hemoglobin levels correlated inversely with serum ferritin (Spearman rho -0.485; p < 0.05).

Using two distinct proteomic methods, we identified oxidative posttranslational modifications of hemoglobin in IDA and healthy controls. Glutathionyl hemoglobin, an established marker of oxidative stress was elevated in patients with IDA and correlated inversely with serum ferritin. Overall, these findings suggest that glutathionyl hemoglobin has potential as a biomarker of IDA.

No relevant conflicts of interest to declare.