Abstract
The measurement of absolute metabolite concentrations in small samples remains a significant analytical challenge. This is particularly the case when the sample volume is only a few microliters or less and cannot be determined accurately via direct measurement. We previously developed volume determination with two standards (VDTS) as a method to address this challenge for biofluids. As a proof-of-principle, we applied VDTS to NMR spectra of polar metabolites in the hemolymph (blood) of the tiny yet powerful genetic model Drosophila melanogaster. This showed that VDTS calculation of absolute metabolite concentrations in fed versus starved Drosophila larvae is more accurate than methods utilizing normalization to total spectral signal. Here, we introduce paired VDTS (pVDTS), an improved VDTS method for biofluids and solid tissues that implements the statistical power of paired control and experimental replicates. pVDTS utilizes new equations that also include a correction for dilution errors introduced by the variable surface wetness of solid samples. We then show that metabolite concentrations in Drosophila larvae are more precisely determined and logically consistent using pVDTS than using the original VDTS method. The refined pVDTS workflow described in this study is applicable to a wide range of different tissues and biofluids.
Highlights
Metabolomics is the measurement via nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS) of multiple metabolites in samples of whole organisms, tissues, or biofluids.[1−6] A snapshot of the small molecule profile of a biological sample provides important biological information that is complementary to that obtained from the proteome and the transcriptome
These methods, tend to be ineffective when samples with very different total signal strengths (e.g., >50%) are being compared. Such circumstances can arise either when sample-to-sample differences in volume are large or when a large fraction of the most abundant metabolites in control versus experimental samples differ greatly in concentration. This can lead to failures to provide logically self-consistent sets of peak intensities in PCA loadings plots; for example, we found that different NMR multiplets from the same metabolite could demonstrate opposite sign in probabilistic quotient normalization (PQN)-treated Drosophila larval hemolymph PCA loadings as shown in ref 19
The volume determination using two standards (VDTS) method relied upon the first stage dilution of the target hemolymph sample into a droplet of known volume (Vf) containing a fixed concentration of one standard, followed by extraction of the polar metabolites using a solvent mix containing a known amount of a second standard (DSS, see Figure 2 from ref 19)
Summary
Metabolomics is the measurement via nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS) of multiple metabolites in samples of whole organisms, tissues, or biofluids.[1−6] A snapshot of the small molecule profile (metabolome) of a biological sample provides important biological information that is complementary to that obtained from the proteome and the transcriptome. NMR and MS metabolomics have greatly increased our understanding of many biological and medical processes,[7−11] including physiological changes during development and aging, and metabolic responses to dietary manipulations.[12−17] If the volumes of biofluid analytes (e.g., blood or cerebrospinal fluid) are sufficient for accurate measurement, sample-to-sample differences in volumes can be accounted for in a straightforward manner prior to chemometric analysis In these analytes of known volume, the absolute concentration of metabolites in the starting sample can be determined by reference to a single internal standard.
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