Authorship:
Christopher M. Benton (1), Chang Kee Lim (2)
(1) Agilent Technologies, (2) Kings College Hospital
| Short Abstract Studying the modification of porphobilinogen is important when trying to understand the pathogenesis of the peripheral neuropathy, chronic renal failure and hepatocellular carcinoma observed in the AHP. Using LC/HRMS we demonstrate how PBG can form various oxygenated species, derived from reactive epoxide, hydroperoxide and endoperoxide intermediates. Furthermore, these intermediates formed adducts with amino acids and peptides, highlighting their reactivity. The mechanisms of formation of these conjugates are postulated to be via epoxyporphobilinogen and PBG endoperoxide intermediates. If formed in vivo, such compounds could covalently bind to cellular macromolecules, such as proteins and nucleic acids, altering their biological function. |
Long Abstract
Introduction
5-Aminolaevulinic acid (ALA) and porphobilinogen (PBG) are precursors of the haem biosynthetic pathway and important for identifying acute hepatic porphyrias (AHP). Various studies have shown that late complications of AHP are peripheral neuropathy, chronic renal failure and hepatocellular carcinoma [1]. The mechanisms of such complications are unknown, and in this respect the AHP are poorly understood. It has been hypothesised that reactive metabolites/derivatives of ALA and/or PBG are potentially mediating such complications [2], however such compounds are yet to be reported. Using liquid chromatography-accurate mass tandem mass spectrometry we demonstrate how PBG can form various oxygenated species, derived from reactive epoxide, hydroperoxide and endoperoxide intermediates. Furthermore, these intermediates formed adducts with amino acids and peptides, highlighting their reactivity.
Methods
The UHPLC system consisted of an Agilent 1290 Infinity LC system (Agilent Technologies, Waldbronn, Germany). The mass spectrometer used was an Agilent 6530 Accurate Mass Q-TOF (Agilent Technologies, Santa Clara, USA) equipped with a Dual Agilent Jet Stream electrospray ionisation source, operated in positive ionisation mode (capillary voltage, 3.00 kV; nozzle voltage, 0 V; fragmentor, 100 V; capillary gas temperature, 200 °C; capillary gas flow, 8 L/min; sheath gas temperature, 400 °C, sheath gas flow, 12 L/min; nebulizer, 35 psi). The data was processed using Agilent MassHunter Qualitative analysis software (version B 07.00), Agilent MassHunter Profinder (version B 06.00) and Mass Profiler Professional (version 12.6.1).
Results
Studying the modification of PBG is important when trying to understand the pathogenesis of the peripheral neuropathy, chronic renal failure and hepatocellular carcinoma observed in the AHP. The results in this study show that PBG reacts with reactive oxygen species (ROS) to give endoperoxide, hydroperoxide and epoxide intermediates, leading to the formation of oxygenated PBG derivatives. To investigate the reactivity of these oxygenated PBG intermediates an aqueous solution of PBG was exposed to ROS in the presence of cysteine and glutathione (GSH). Analysis of the reaction products by LC-accurate mass MS/MS revealed the presence of two sets of oxygenated PBG-cysteine conjugate isomers. The first set of PBG-cysteine conjugate isomers displayed a measured exact m/z 362.1013 corresponding to a molecular formula of C13H20N3O7S, with an error of 0.96 ppm from the calculated exact m/z value of 362.1016. The second set of PBG-cysteine conjugate isomers had a measured exact m/z 333.0748 corresponds to a molecular formula of C12H17N2O7S and error of 0.9 ppm from the calculated exact m/z 333.0751.
GSH similarly reacted with oxygenated PBG to give two main partially resolved isomeric peaks with an exact measured m/z 548.1646, which corresponds to a molecular formula of C20H30N5O11S, with an error of 2.02 ppm from the calculated exact m/z 548.1657. An additional oxygenated PBG-GSH adduct was also observed with a measured exact m/z 519.1385 corresponding to a molecular formula of C19H27N4O11S, with an error of 1.26 ppm from the calculated exact m/z value of 519.1392. The mechanisms of formation of these conjugates are postulated to be via epoxyporphobilinogen and PBG endoperoxide intermediates.
Conclusions
If formed in vivo, such compounds could covalently bind to cellular macromolecules, such as proteins and nucleic acids, altering their biological function and potentially contribute to the late stage complications observed in the AHP.
Research use only. Not for use in diagnostic procedures.
[1] Stewart, M.F., 2012. Journal of Clinical Pathology. 65, 976-980
[2] Marsden, J.T. & Rees, D.C., 2014. Journal of Clinical Pathology. 67, 60-65.