Metabolite Identification Using Infrared Ion Spectroscopy─Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Abstract

Untargeted liquid chromatography−mass spectrometry (LC−MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identificationconfidently determining the chemical structures of m/z-detected unknowns.Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC−MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for massisolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics−IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

Identification of Δ-1-pyrroline-5-carboxylate derived biomarkers for hyperprolinemia type II

“…A similar fragmentation pattern was observed for the P6C analogs we previously annotated 9 , 10 , namely the neutral loss of C 3 H 6 O for feature A and B and C 2 H 4 O 2 for feature C. We, therefore, hypothesized a similar structure and connectivity of these fragments to the P5C-core leading to the proposed structures of 2-OPC ( 4 ) and CPC ( 5 ) (Fig. 1d ).…”

“…In contrast, the calculated frequency belonging to the NH 2 -vibration does not match well with the experimentally obtained spectra for features A and B. This discrepancy can be attributed to the anharmonic behavior of the NH bending mode, resulting in a redshift in spectra predicted within the harmonic approximation 14 , 15 , as was noted before for the corresponding P6C biomarker 9 . Overall the spectral region between 1200 and 1500 cm −1 is adequately predicted by the computed spectra.…”

“…Using our previously established approach, combining untargeted metabolomics with infrared ion spectroscopy (IRIS) and synthesis of reference standards, we show here that P5C reacts with acetoacetic acid or malonic acid in an analogous manner as we previously elucidated for P6C 9 , leading to the formation of biomarkers 2-OPC ( 4 ) and CPC ( 5 ), respectively (Fig. 1d ).…”

“…Through an untargeted metabolomics approach (Next Generation Metabolic Screening, NGMS) 8 , our team has recently identified biomarkers for PDE-ALDH7A1. These biomarkers are the result of P6C reacting with acetoacetic acid or malonic acid 9 , 10 . Due to the structural similarity between the cyclic imines P6C and P5C, we hypothesized that this mechanism of adduct formation would also occur for the reaction of P5C, leading to the formation of (2 R )-5-( 2 - o xopropyl) p yrrolidine-2- c arboxylic acid (2-OPC, 4 ) and (2 R )-5-( c arboxymethyl) p yrrolidine-2- c arboxylic acid (CPC, 5 ) (Fig.…”

Harnessing Metabolomics to Advance Epilepsy Research

“…Thus, far, only a small number of studies have used metabolomics to investigate epileptogenesis. 21 - 24 Heischmann et al 22 explored the hippocampal and plasma metabolome in the rat kainic acid model of mesial temporal lobe epilepsy (MTLE) at different time points during epileptogenesis. The authors found that disease development was associated with perturbations in vitamin D3 metabolism, which is important for mitochondrial function, red-ox processes, and immune modulation.…”