Heterogeneous expressions of hepcidin isoforms in hepatoma‐derived cells detected using simultaneous LC‐MS/MS

Hosoki, Takaaki; Ikuta, Katsuya; Shimonaka, Yasushi; Sasaki, Yoshiyuki; Yasuno, Hideyuki; Sato, Kazuya; Ohtake, Takaaki; Sasaki, Katsunori; Torimoto, Yoshihiro; Saito, Keiji; Kohgo, Yutaka

doi:10.1002/prca.200900112

Cited by 12 publications

(8 citation statements)

References 25 publications

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“…To overcome this intrinsic problem for high cysteine containing peptides, some researchers utilize the immonium ion for SRM transitions with high cleavage energy, which results in reduced sensitivity [27]. In this study, we found that the N-terminal residue of hepcidin, namely, H 2 N-Asp-Thr-His-Phe-Pro-Ile, which is free of cysteines, demonstrated a linear dissociation efficiency by collision-induced dissociation and SRM transitions.…”

Section: Resultsmentioning

confidence: 74%

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method

Hwang¹,

Lee²,

Park³

et al. 2011

Clinica Chimica Acta

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BACKGROUND The measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. METHODS In order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8 h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease. RESULTS The linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r2 = 0.9512 ± 0.0202 (n=5) for urine and r2 = 0.9709 ± 0.0291 (n=5) for serum [r2= mean ± SD]. Compared to baseline, the levels of urinary hepcidin between 2-4 h and 4-8 h of both women and men showed significant increases with p < 0.05 and p < 0.001, respectively. The levels of serum hepcidin between 4 h and 8 h in both women and men showed significant increases, compared with baseline values, with both p < 0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points. CONCLUSIONS We have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men.

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Section: Resultsmentioning

confidence: 74%

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method

Hwang¹,

Lee²,

Park³

et al. 2011

Clinica Chimica Acta

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“…This LC-tandem MS method was useful because it is able to measure all three hepcidin isoforms in serum simultaneously [22]. We also investigated how the three hepcidin isoforms are processed, as well as the role hepatocytes may play in hepcidin processing.…”

Section: Introductionmentioning

confidence: 99%

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS)

et al. 2015

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Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

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“…25 While physiological roles of the other two isoforms (hepcidin-20 and -22) have been detected in human urine and hepatomaderived cells, they are poorly understood. 25,26 As hepcidin mRNA expression in peripheral leukocytes was found to be associated with neither serum ferritin nor iron in the present study, and its regulatory mechanism was different from that in hepatocytes, it is reasonable to speculate that hepcidin expressed in peripheral leukocytes in response to inflammation may exert immune modulating functions beyond iron metabolism. Further studies testing the protein levels of hepcidin via validated methodology, such as mass spectrometric assay, 26 are warranted to verify whether the hepcidin proteins expressed in peripheral immune cells are the same as those from hepatoma-derived cells.…”

mentioning

confidence: 59%

Nuclear factor-κB mediated lipopolysaccharide-induced mRNA expression of hepcidin in human peripheral blood leukocytes

Zhang

et al. 2011

Innate Immun

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Hepcidin is a known key modulator of iron homeostasis and an innate immune molecule secreted by the liver. The transcriptional mechanism of hepcidin in hepatocytes during inflammation is mediated via the IL-6/STAT3 pathway. Recently, hepcidin demonstrated an anti-inflammatory function in endotoxic mice, and a TLR4-dependent inducible expression of hepcidin was detected in myeloid cells. In this study, we explored the expression and signaling mechanism regulating hepcidin mRNA expression in peripheral blood leukocytes. The mRNA levels of hepcidin in peripheral blood leukocytes from patients with severe sepsis (n = 14) was significantly higher than those in healthy controls (n = 16;0.286 ± 0.065 vs 0.068 ± 0.025; P < 0.05). Ex vivo studies found hepcidin mRNA can be highly induced by challenge of 100 ng/ml LPS or 20 ng/ml TNF-α in peripheral blood leukocytes rather than IL-6, IL-1 and IFN-γ. Anti-TNF-α antibody significantly decreased the levels of hepcidin mRNA induced by LPS. Inhibitor of nuclear factor (NF)-κB rather than that of STAT3 completely abolished the inducibility of hepcidin mRNA in PBMCs and neutrophils. These results indicate that hepcidin mRNA expression in peripheral blood leukocytes induced by LPS depends on NF-κB, and TNF-α may be a key mediator in this procedure.

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Heterogeneous expressions of hepcidin isoforms in hepatoma‐derived cells detected using simultaneous LC‐MS/MS

Cited by 12 publications

References 25 publications

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS)

Nuclear factor-κB mediated lipopolysaccharide-induced mRNA expression of hepcidin in human peripheral blood leukocytes

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