Quantification of Urine and Plasma Porphyrin Precursors Using LC-MS in Acute Hepatic Porphyrias: Improvement in Routine Diagnosis and in the Monitoring of Kidney Failure Patients.

BACKGROUND: The quantification of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine are the first-line tests for diagnosis and monitoring of acute hepatic porphyrias (AHP). Ion-exchange chromatography (IEC), which is time- and staff-consuming and limited to urine, is still the preferred method in many specialized laboratories, despite the development of mass spectrometry-based methods. METHODS: We describe a new LC-MS method that allows for rapid and simple quantification of ALA and PBG in urine and plasma with an affordable instrument that was used to analyze 2260 urine samples and 309 blood samples collected in 2 years of routine activity. The results were compared to those obtained with IEC, and urine reference ranges and concentrations in asymptomatic carriers were determined. Plasma concentrations were measured in healthy subjects and subgroups of symptomatic and asymptomatic AHP carriers. RESULTS: In urine, the clinical decision limits were not impacted by the change of method despite discrepancies in low absolute concentrations, leading to lower normal values. Two-thirds of asymptomatic AHP carriers (with the exception of coproporphyria carriers) showed an increased urine PBG concentration. Urine and plasma levels showed a good correlation except in patients with kidney disease in whom the urine/plasma ratio was relatively low. CONCLUSION: We described an LC-MS based method for the routine diagnosis and monitoring of AHP that allows for the detection of more asymptomatic carriers than the historical method. Blood analysis appears to be particularly relevant for patients with kidney disease, where urine measurement underestimates the increase in ALA and PBG levels.

LC-MS/MS method for hepcidin-25 measurement in human and mouse serum: clinical and research implications in iron disorders.

BACKGROUND: The peptide hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. This 25-amino acid hormone is produced and secreted predominantly by hepatocytes. Hepcidin has been suggested as a promising diagnostic marker for iron-related disorders. However, its accurate quantification for clinical use remains so far challenging. In this report we describe a highly specific and quantitative serum hepcidin method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). MATERIAL: The analytical validation included the determination of the limit of detection, of quantification, repeatability, reproducibility and linearity. This assay was developed for human and mouse hepcidin. The human assay was performed on serum patients with unexplained microcytic anemia. We applied our LC-MS/MS method for quantifying hepcidin-1 in mouse in various conditions: inflammation, hemolytic anemia, Hamp-1, Hjv and Hfe KO mice. RESULTS: We show that the LC-MS/MS is suitable for accurate determination of hepcidin-25 in clinical samples, thereby representing a useful tool for the clinical diagnosis and follow-up of iron-related diseases. In mouse, a strong correlation between hepatic Hamp-1 mRNA expression and serum hepcidin-1 levels was found (r=0.88; p=0.0002) and the expected variations in mouse models of iron disorders were observed. CONCLUSIONS: Therefore, we propose this adaptive LC-MS/MS method as a suitable method for accurate determination of hepcidin-25 in clinical samples and as a major tool contributing to the clinical diagnosis, follow-up and management of iron-related disorders. It also opens new avenues to measure hepcidin in animal models without interspecies antigenic limitations.