Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy

Abstract

A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (∼1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10–67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry.

Massive Glutamine Cyclization to Pyroglutamic Acid in Human Serum Discovered Using NMR Spectroscopy

“…8,17 The assignment of glutamine and pyroglutamic acid peaks was further confirmed by spiking with standard compounds. Chenomx NMR Suite Professional Software package (version 5.1; Chenomx Inc., Edmonton, Alberta, Canada) was used for absolute quantitation of glutamine and pyroglutamic acid.…”

“…17–20 In fact, recognizing the deleterious effects of metabolite binding to proteins, we recently showed that protein precipitation by methanol is a far superior approach for the quantitative analysis of serum/plasma by NMR. 8,17 Because of the protein binding of pyroglutamic acid, the nonobservance of its NMR peaks neither confirms its absence in serum nor the absence of glutamine cyclization. However, the close association of the glutamine levels in intact serum with the same value for the sum of apparent glutamine and pyroglutamic acid levels for ultrafiltered and protein precipitated samples support the near absence of glutamine cyclization in intact serum.…”

“…8 The 1D NOESY and CPMG (Carr–Purcell– Meiboom–Gill) pulse sequences with water suppression using presaturation were used for 1 H 1D NMR experiments. Additional CPMG experiments for intact serum were performed after spiking with standard pyroglutamic acid.…”

“…5–7 Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) enable reliable identification and quantitation of a large number of blood metabolites (including glutamine) on a routine basis. 8,9 However, numerous investigations using standard compounds have shown that under abnormal conditions, the glutamine molecule is not stable. Under extreme acidic, alkaline or high-temperature conditions, glutamine undergoes spontaneous cyclization to form pyroglutamic acid with the loss of ammonia.…”

Recent Advances in NMR-Based Metabolomics

“…This is due to the poor solubility of many metabolites in acetonitrile, and hence it is clearly unsuitable for quantitative analysis of, specifically, hydrophilic blood metabolites. 68 Using methanol precipitation and combining with an array of 1D/2D NMR experiments, database searches, and spiking with authentic compounds, quantitation of nearly 70 blood metabolites, a large number (nearly 1/3) of which had not been previously identified in the NMR spectra of blood was reported. Further, experimental protocols and comprehensive peak annotations were provided to take advantage of the high reproducibility and quantitative nature of NMR and to mitigate against the sensitivity of NMR chemical shifts to altered sample conditions.…”