Mass Spectrometry–Based Hepcidin Measurements in Serum and Urine: Analytical Aspects and Clinical Implications

Abstract

Background: Discovery of the central role of hepcidin in body iron regulation has shed new light on the pathophysiology of iron disorders. Information is lacking on newer analytical approaches to measure hepcidin in serum and urine. Recent reports on the measurement of urine and serum hepcidin by surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) necessitate analytical and clinical evaluation of MS-based methodologies. Methods: We used SELDI-TOF MS, immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, we investigated analytical reproducibility and biological and preanalytical variation for both serum and urine on Normal Phase 20 and Immobilized Metal Affinity Capture 30 ProteinChip arrays. We obtained samples from healthy controls and patients with documented iron-deficiency anemia, inflammation-induced anemia, thalassemia major, and hereditary hemochromatosis. Results: Proteomic techniques showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. The interarray CV was 15% to 27%, and interspot CV was 11% to 13%. Preliminary studies showed that hepcidin-25 differentiated disorders of iron metabolism. Urine hepcidin is more affected by multiple freeze-thaw

Serum hepcidin level and erythropoietic activity after hematopoietic stem cell transplantation

“…Recently, several investigators have indicated the existence of an erythropoiesis-associated humoral factor that negatively regulates hepcidin synthesis, mainly based on animal experiments and in vitro experiments using sera of β-thalassemia patients. [9][10][11][12][13][14] These patients usually show severe microcytic anemia, increased iron stores and ineffective erythropoiesis with relatively low hepcidin production, and a clear inverse correlation was observed between the urinary hepcidin level and levels of both erythropoietin and sTfR. 20 Kemna et al demonstrated that patient sera containing high sTfR levels could suppress hepcidin mRNA expression in human hepatoma cells.…”

“…In addition to these signals, the existence of erythropoiesis-associated regulatory factors of hepcidin has been hypothesized. [9][10][11][12] Consistent with this hypothesis, hepcidin expression increased when hematopoiesis in mice was blocked by irradiation or chemotherapeutic agents. However, this increase was not suppressed by erythropoietin administration or anemia caused by phenylhydrazine or phlebotomy, indicating that neither erythropoietin nor anemia per se was the hepcidin regulator.…”

Peering into the future: Hepcidin testing

“…There are numerous variations and modifications of MS (e.g., HPLC-MS/MS, MALDI-TOF MS, and SELDI-TOF) that have been employed by different laboratories to measure hepcidin levels in biological samples, however, the details of these modifications are beyond the scope of this paper [8][9][10][11]. One of the major benefits of measuring hepcidin by MS methodologies is the ability to distinguish and quantitate the bioactive hepcidin-25 [11].…”

Immunoproteomics: From biomarker discovery to diagnostic applications

“…A general advantage of MS-based approaches is that it allows the visualisation of different isoforms of the same protein, which are otherwise difficult to distinguish by ELISA-based methods [53,54]. A limitation of antigen profiling is that MS-based assays provide qualitative data, whereas quantification still needs much improvement.…”