Semi-Automatic Enrichment with High Resolution/Selected Reaction Monitoring (HR/SRM) Scan for the Detection of Urinary Hepcidin in Patients with Sickle Cell Disease
Authors: Lin Xionghao , Adjei Elena Afia , Kumari Namita , Diaz Sharmin , Jerebtsova Marina , Oneal Patricia A. , Nekhai Sergei 
Published: October 2019

Abstract
Background
Urinary hepcidin is a potential biomarker of renal inflammation and acute kidney injury (AKI) which is elevated in sickle cell disease (SCD). Hepcidin in circulation is filtered through glomeruli filtration barrier and reabsorbed by the renal tubules. Hepcidin can also be synthesized by the kidney tubular cells. Thus, increased urinary levels of hepcidin may reflect either a reduction in tubular uptake or an increase in renal production. Recent studies suggested that urinary hepcidin may protect against AKI by attenuating heme-mediated injury. Thus decreased hepcidin levels in SCD patients may contribute to AKI and serve as potentially informative marker of SCD-associated kidney injury. Previously, hepcidin was measured by ELISA and mass spectrometry. Immunoassays are limited due to the cross-reactivity of antibodies to prohepcidin and truncated hepcidin-20, -22, and -24 isoforms of active hepcidin-25. Mass spectrometric assays are specific for hepcidin-25 but sample preparation remains a challenge.
Objective
To develop a sensitive, reliable and reproducible nanoLC/FT-MS method with simplified sample preparation for measuring of hepcidin in urine samples. Also to correlate urinary hepcidin with urinary albumin and urinary protein to access the degree of kidney dysfunction.
Methods
Samples were enriched and purified semi-automaticaly on 10-uL ZipTip and online trap column. Stable isotope-labeled hepcidin was used as internal standard. The standard concentration range was 1.56-800 nM and quality control samples were 5 nM, 20 nM, 80 nM and 400 nM. Samples were subjected to an LC-20AD nano HPLC system coupled to an LTQ XL™ Orbitrap mass spectrometer with an in-house made nano-HPLC column. High resolution/selected reaction monitoring (HR/SRM) scan was carried out and the narrow mass range ([M+H]+ ±0.01 Da) was used to extract ion chromatograms (EICs) for quantification. Urinary samples were collected from 20 SCD patients and 13 controls. Urinary albumin, protein and creatinine were detected by ELISA. The urine hepcidin concentrations were normalized to urine creatinine (Cr) values.
Results
Semi-automatic approach simplified sample preparation and accelerated the analysis. At least 24 samples could be prepared and processed at the same time. Online column trapping further purified and enriched hepcidin and improved the sensitivity and specificity of this method by eliminating interferences from urine. Hepcidin showed a good linearity within the concentration range of 1.56-800 nM with an r2 value of 0.9994. The precision intraday (n = 5) and interday (n = 5) and the repeatability (n=5) of the method were good with relative standard deviations (RSDs) lower than 5%. The analyzed samples were stable for 3 days at +4°C (RSDs<5%). The percent mean recoveries of hepcidin was within the acceptable range of 89.65-104.79%. We found that SCD patients had significantly lower (about 2-fold) urinary hepcidin levels compared to controls, and urinary hepcidin levels in 2 SCD patients were below the lower limit of detection (<0.5 nM). We found that there was no difference in urine albumin between SCD and control subjects, total urine protein was significantly increased in SCD patients. There was no positive correlation between urine hepcidin and urine albumin or total protein.
Conclusion
We developed an LC-MS based method for measuring levels of urinary hepcidin. This method is promising in terms of recovery, sensitivity, selectivity, repeatability and simplicity of sample preparation. SCD patients showed significantly decreased hepcidin levels in urine suggesting a potentially novel mechanism of AKI in SCD.
Acknowledgments
This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005 and 5G12MD007597). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Disclosures
No relevant conflicts of interest to declare.