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

BackgroundThe 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.MethodsIn 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.ResultsThe 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.ConclusionsWe 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.     

Prostaglandin levels: Predictors of indomethacin responsiveness

Summary Pretreatment plasma dilator prostaglandin levels were measured in 16 premature infants with patent ductus arteriosus in an attempt to correlate abnormally elevated levels with clinical responsiveness to indomethacin therapy. Nine of the 16 infants responded well to indomethacin, with complete disappearance of their murmurs by 48 h. Eight of these nine infants had elevated baseline 6 keto PGF₁ α levels (>500 pg/ml). In contrast, seven of the 16 infants did not respond to indomethacin, and six of these had 6 keto PGF₁ α within the normal range (<500 pg/ml). PGE₂ levels varied in the same general direction, but lacked the specificity and sensitivity of the 6 keto PGF₁ α levels. Thus, 6 keto PGF₁ α levels seem to correlate with, and may eventually be helpful in predicting, clinical indomethacin responsiveness in the premature neonate with patency of the ductus arteriosus.