Miniaturized 1H-NMR method for analyzing limited-quantity samples applied to a mouse model of Leigh disease.

INTRODUCTION: The analysis of limited-quantity samples remains a challenge associated with mouse models, especially for multi-platform metabolomics studies. Although inherently insensitive, the highly specific characteristics of nuclear magnetic resonance (NMR) spectroscopy make it an advantageous platform for global metabolite profiling, particularly in mitochondrial disease research. OBJECTIVES: Show method equivalency between a well-established standard operating protocol (SOP) and our novel miniaturized 1H-NMR method. METHOD: The miniaturized method was performed in a 2 mm NMR tube on a standard 500 MHz NMR spectrometer with a 5 mm triple-resonance inverse TXI probe at room temperature. RESULTS: Firstly, using synthetic urine spiked with low (50 µM), medium (250 µM) and high (500 µM) levels (n = 10) of nine standards, both the SOP and miniaturized method were shown to have acceptable precision (CV < 15%), relative accuracy (80-120%), and linearity (R2 > 0.95), except for taurine. Furthermore, statistical equivalence was shown using the two one-sided test. Secondly, pooled mouse quadriceps muscle extract was used to further confirm method equivalence (n = 3), as well as explore the analytical dynamics of this novel approach by analyzing more-concentrated versions of samples (up to 10× concentration) to expand identification of metabolites qualitatively, with quantitative linearity. Lastly, we demonstrate the new technique's application in a pilot metabolomics study using minute soleus muscle tissue from a mouse model of Leigh syndrome using Ndufs4 KO mice. CONCLUSION: We demonstrate method equivalency, supporting our novel miniaturized 1H-NMR method as a financially feasible alternative to cryoprobe technology-for limited-quantity biological samples in metabolomics studies that requires a volume one-tenth of the SOP.