Guidelines for the Use of Deuterium Oxide (D2O) in 1H NMR Metabolomics.

In metabolomics, nuclear magnetic resonance (NMR) spectroscopy allows to identify and quantify compounds in biological samples. The sample preparation generally requires only few steps; however, an indispensable factor is the addition of a locking substance into the biofluid sample, such as deuterium oxide (D2O). While creatinine loss in pure D2O is well-described, the effects of different D2O concentrations on the signal profile of biological samples are unknown. In this work, we investigated the effect of D2O levels in the NMR buffer system in urine samples, in dependence on dwell time and temperature exposition. We reveal a decrease of the urinary creatinine peak area up to 35% after 24 h of dwell time at room temperature (RT) using 25% (v/v) D2O, but only 4% loss using 2.5% D2O. 1H, inverse-gated (IG) 13C, DEPT-HSQC NMR, and mass spectrometry (MS) experiments confirmed a proton-deuterium (H/D) exchange at the CH2. This leads to underestimation of creatinine levels and has an extensive effect when creatinine is used for normalization. This work offers a sample stability examination, depending on the D2O concentration, dwell time, and temperature and enables a method to correct for the successive loss. We propose an equation to correct the creatinine loss for samples prepared with various D2O concentrations and storage temperatures for dwell times up to 24 h. The correction function was validated against an external data set with n = 26 samples. To ensure sufficient creatinine stability in future studies, we suggest that a maximum of 10% D2O should be used at 4 °C or 2.5% D2O at RT, respectively.

Data Processing Optimization in Untargeted Metabolomics of Urine Using Voigt Lineshape Model Non-Linear Regression Analysis.

Nuclear magnetic resonance (NMR) spectroscopy is well-established to address questions in large-scale untargeted metabolomics. Although several approaches in data processing and analysis are available, significant issues remain. NMR spectroscopy of urine generates information-rich but complex spectra in which signals often overlap. Furthermore, slight changes in pH and salt concentrations cause peak shifting, which introduces, in combination with baseline irregularities, un-informative noise in statistical analysis. Within this work, a straight-forward data processing tool addresses these problems by applying a non-linear curve fitting model based on Voigt function line shape and integration of the underlying peak areas. This method allows a rapid untargeted analysis of urine metabolomics datasets without relying on time-consuming 2D-spectra based deconvolution or information from spectral libraries. The approach is validated with spiking experiments and tested on a human urine 1H dataset compared to conventionally used methods and aims to facilitate metabolomics data analysis.

Qualitative and quantitative assessment of the presence of ciguatoxin, P-CTX-1B, in Spanish Mackerel (Scomberomorus commerson) from waters in New South Wales (Australia).

Ciguatera Fish Poisoning (CFP) is a tropical disease caused by the consumption of fish contaminated with ciguatoxins (CTXs). Currently, the only feasible prevention methods for CFP are to avoid the consumption of fish of certain species from some regions, avoid larger fish of certain species, or avoid all fish caught from specific regions. Here, we quantified levels of P-CTX-1B in Spanish Mackerel (Scomberomorus commerson), which is the main fish species that causes CFP in New South Wales and Queensland, Australia, using LC-MS detection against a toxin standard. We found detectable P-CTX-1B in both flesh and liver tissues in fish from New South Wales (n = 71, 1.4% prevalence rate, with a confidence interval of 1%-4%, and 7% prevalence, 1%-12%, in flesh and liver, respectively). In the small sample of fish from Queensland, there was a 46% prevalence (19-73%, n = 13). Toxin levels found were 0.13 µg kg-1 to <0.1 µg kg-1 in flesh, and 1.39 µg kg-1 to <0.4 µg kg-1 in liver, indicating that liver tissue had a significantly higher concentration (∼5 fold) of P-CTX-1B. No apparent relationship was observed between the length or weight of S. commerson and the detection of P-CTX-1B in this study. Footnote.