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

Abstract

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 r(2)=0.9512±0.0202 (n=5) for urine and r(2)=0.9709±0.0291 (n=5) for serum [r(2)=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.

Fig. 1

Optimization of mSRM transitions of endogenous hepcidin-25 and isotopic hepcidin-25. (A) MS spectra of endogenous and heavy isotopic hepcidin-25. The most intense parent ions (charge: 4+), 698.18 (endogenous hepcidin-25) and 700.16 (Hepcidin-25H) m/z, were chosen fragmentation to product ions, (B) Representative MS/MS spectra. The product ions, 354.1 (b3) and 501.2 (b4) m/z were chosen for mSRM transition. (C) Extracted ion chromatogram (upper) and selected reaction monitoring of product ions (lower).

Fig. 2

Representative standard curves of the Hepcidin-25H calibration. (A) A linear response of Hepcidin-25H quantitation between 0.5 to 6 picomoles was demonstrated in TSQ Quantum Ultra, (B) The linear response of the Hepcidin-25H quantification between 100 to 500 femtomoles was demonstrated in LTQ-XL linear ion trap instrument.

Fig. 3

Mean hepcidin concentrations in urine and serum of normal subjects. (A, B) Urine hepcidin concentration: Samples were obtained from 10 subjects (women, A, n=5; men, B, n = 5) at 4 time points (0, 2, 4, and 8 h) after 650mg ferrous sulfate treatment at t=0, (C, D) Serum hepcidin concentration: Samples were obtained from 10 subjects (women, C, n=5; men, D, n = 5) at 5 time points (0, 1, 2, 4, and 8 h) after 650mg ferrous sulfate treatment at t=0 (The bars show mean ± SE), n=5 for each gender. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig. 4

Relationship of (A) urine and (B) serum hepcidin to serum ferritin at baseline. The best fit lines for men are defined by the following equations: A. y=0.084x-0.059, r²= 0.7613, p= 0.0536, B. y=0.106x+1.372, r²= 0.7641, p= 0.0526).

Fig. 5

Effects of a single dose of oral iron on (A) serum iron and (B) serum transferrin saturation in subjects studied (mean±SE). The increases are highly significant [p ≤ 0.0378] for both men and women. As expected, levels are lower in women than men at all time points, except for serum iron concentrations at 4 h after oral iron.