Signal Intensities Derived from Different NMR Probes and Parameters Contribute to Variations in Quantification of Metabolites

Abstract

We discovered that serious issues could arise that may complicate interpretation of metabolomic data when identical samples are analyzed at more than one NMR facility, or using slightly different NMR parameters on the same instrument. This is important because cross-center validation metabolomics studies are essential for the reliable application of metabolomics to clinical biomarker discovery. To test the reproducibility of quantified metabolite data at multiple sites, technical replicates of urine samples were assayed by 1D-1H-NMR at the University of Alberta and the University of Michigan. Urine samples were obtained from healthy controls under a standard operating procedure for collection and processing. Subsequent analysis using standard statistical techniques revealed that quantitative data across sites can be achieved, but also that previously unrecognized NMR parameter differences can dramatically and widely perturb results. We present here a confirmed validation of NMR analysis at two sites, and report the range and magnitude that common NMR parameters involved in solvent suppression can have on quantitated metabolomics data. Specifically, saturation power levels greatly influenced peak height intensities in a frequency-dependent manner for a number of metabolites, which markedly impacted the quantification of metabolites. We also investigated other NMR parameters to determine their effects on further quantitative accuracy and precision. Collectively, these findings highlight the importance of and need for consistent use of NMR parameter settings within and across centers in order to generate reliable, reproducible quantified NMR metabolomics data.

Whole Blood Reveals More Metabolic Detail of the Human Metabolome than Serum as Measured by 1H-NMR Spectroscopy

“…Metabolite data were acquired for 19 WB and 20 serum samples; one WB sample did not properly extract and the NMR spectrum was uninterruptable. The urine metabolomics data from this cohort has been previously published (16). …”

“…Normal, healthy volunteers were identified and recruited for study participation via the Claude D. Pepper Older Americans Independence Center (OAIC) Research Participant Program at the University of Michigan's Geriatric Center and the Michigan Institute of Clinical and Health Research (MICHR) clinical studies website (UMClinicalStudies.org) (16). The study and its associated informed consent form were approved by the University of Michigan's Institutional Review Board approved study (UM IRB; protocol number, HUM00038122).…”

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism

“…In advance of the study, the urine collection containers were coated with 10% NaN 3 to help retard bacterial growth. 40 The containers were air-dried in a biosafety cabinet before being attached to the cage. The water bottle and the food dispenser were filled and the volume and weight, respectively, were recorded before assembly to the cage.…”

“…40,41 The supernatants were carefully transferred to new 15 mL conical tubes so as not to disturb the pellet. The initial pH was recorded and the volume of each urine sample was measured again.…”

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data

“…NMR analysis of metabolites takes advantage of pulse sequences [42,45] and suppression of the intense 1 H water resonance. [50,51] There are several features of NMR that have led to its early and continued use in metabolomics -(1) the observed resonances are quantitative once calibrated with a known amount of a single internal standard such as 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), which has nine identical methyl protons that results in a single high-intensity proton peak that can be set to 0 ppm [29] ; (2) it is a non-destructive technique, so the sample can subsequently be analyzed by GC-or LC-MS; (3) it is highly reproducible and is less susceptible to instrument variability like LC-MS that makes it ideal for inter-and crosscenter studies [50] ; and (4) if observable (particularly if an unknown metabolite is first isolated), the fine structure of the resonances of a metabolite can provide information about its chemical nature. In addition, magic angle NMR allows for the observation of highresolution metabolite signals directly in tissues without preliminary extraction.…”