Abstract
Background
In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities.
Aims
To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies.
Methods
Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated.
Results
Integral changes were generally small, but serum citrate signals decreased significantly in some samples.
Conclusions
Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.
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Data availability
The metabolite integrals analysed in the current study are available from the corresponding author on reasonable request.
References
Barrilero, R., Ramírez, N., Vallvé, J. C., Taverner, D., Fuertes, R., Amigó, N., et al. (2017). Unravelling and quantifying the “NMR-Invisible” metabolites interacting with human serum albumin by binding competition and T2 relaxation-based decomposition analysis. Journal of Proteome Research, 16(5), 1847–1856. https://doi.org/10.1021/acs.jproteome.6b00814.
Beckonert, O., Keun, H. C., Ebbels, T. M. D., Bundy, J., Holmes, E., Lindon, J. C., et al. (2007). Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nature Protocols, 2(11), 2692–2703. https://doi.org/10.1038/nprot.2007.376.
Bell, J. D., Brown, J. C. C., Kubal, G., & Sadler, P. J. (1988). NMR-invisible lactate in blood plasma. FEBS Letters, 235(1–2), 81–86. https://doi.org/10.1016/0014-5793(88)81238-9.
Bernini, P., Bertini, I., Luchinat, C., Nincheri, P., Staderini, S., & Turano, P. (2011). Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks. Journal of Biomolecular NMR, 49(3–4), 231–243. https://doi.org/10.1007/s10858-011-9489-1.
Buggeskov, K., Maltesen, R., Rasmussen, B., Hanifa, M., Lund, M., Wimmer, R., et al. (2018). Lung protection strategies during cardiopulmonary bypass affect the composition of blood electrolytes and metabolites—a randomized controlled trial. Journal of Clinical Medicine, 7(11), 462. https://doi.org/10.3390/jcm7110462.
Carr, H. Y., & Purcell, E. M. (1954). Effects of diffusion on free precession in nuclear magnetic resonance experiments. Physical Review, 94(3), 630–638. https://doi.org/10.1103/PhysRev.94.630.
Deprez, S., Sweatman, B. C., Connor, S. C., Haselden, J. N., & Waterfield, C. J. (2002). Optimisation of collection, storage and preparation of rat plasma for 1H NMR spectroscopic analysis in toxicology studies to determine inherent variation in biochemical profiles. Journal of Pharmaceutical and Biomedical Analysis, 30(4), 1297–1310. https://doi.org/10.1016/S0731-7085(02)00455-7.
Dona, A. C., Jiménez, B., Schäfer, H., Humpfer, E., Spraul, M., Lewis, M. R., et al. (2014). Precision high-throughput proton NMR spectroscopy of human urine, serum, and plasma for large-scale metabolic phenotyping. Analytical Chemistry, 86(19), 9887–9894. https://doi.org/10.1021/ac5025039.
Hanifa, M. A., Skott, M., Maltesen, R. G., Rasmussen, B. S., Nielsen, S., Frøkiær, J., et al. (2019). Tissue, urine and blood metabolite signatures of chronic kidney disease in the 5/6 nephrectomy rat model. Metabolomics : Official Journal of the Metabolomic Society, 15(8), 112. https://doi.org/10.1007/s11306-019-1569-3.
Jupin, M., Michiels, P. J., Girard, F. C., Spraul, M., & Wijmenga, S. S. (2013). NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction. Journal of Magnetic Resonance, 228, 81–94. https://doi.org/10.1016/j.jmr.2012.12.010.
Jupin, M., Michiels, P. J., Girard, F. C., Spraul, M., & Wijmenga, S. S. (2014). NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin. Journal of Magnetic Resonance, 239, 34–43. https://doi.org/10.1016/j.jmr.2013.11.019.
Maltesen, R. G., Hanifa, M. A., Kucheryavskiy, S., Pedersen, S., Kristensen, S. R., Rasmussen, B. S., et al. (2016). Predictive biomarkers and metabolic hallmark of postoperative hypoxaemia. Metabolomics, 12(5), 87. https://doi.org/10.1007/s11306-016-1018-5.
Maltesen, R. G., Rasmussen, B. S., Pedersen, S., Hanifa, M. A., Kucheryavskiy, S., Kristensen, S. R., et al. (2017). Metabotyping patients’ journeys reveals early predisposition to lung injury after cardiac surgery. Scientific Reports, 7(1), 40275. https://doi.org/10.1038/srep40275.
Meiboom, S., & Gill, D. (1958). Modified spin-echo method for measuring nuclear relaxation times. Review of Scientific Instruments, 29(8), 688–691. https://doi.org/10.1063/1.1716296.
Nicholson, J. K., & Gartland, K. P. (1989). 1H NMR studies on protein binding of histidine, tyrosine and phenylalanine in blood plasma. NMR in Biomedicine, 2(2), 77–82. https://doi.org/10.1002/nbm.1940020207.
Nicholson, J. K., Lindon, J. C., & Holmes, E. (1999). “Metabonomics”: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica; the Fate of Foreign Compounds in Biological Systems, 29(11), 1181–1189. https://doi.org/10.1080/004982599238047.
Oliver, S. G., Winson, M. K., Kell, D. B., & Baganz, F. (1998). Systematic functional analysis of the yeast genome. Trends in Biotechnology, 16(9), 373–378. https://doi.org/10.1016/S0167-7799(98)01214-1.
Pinto, J., Domingues, M. R. M., Galhano, E., Pita, C., Almeida, M. do C., Carreira, I. M., & Gil, A. M. (2014). Human plasma stability during handling and storage: impact on NMR metabolomics. The Analyst, 139(5), 1168–77. https://doi.org/10.1039/c3an02188b
Rist, M. J., Muhle-Goll, C., Görling, B., Bub, A., Heissler, S., Watzl, B., et al. (2013). Influence of freezing and storage procedure on human urine samples in NMR-based metabolomics. Metabolites, 3(2), 243–258. https://doi.org/10.3390/metabo3020243.
Tweeddale, H., Notley-McRobb, L., & Ferenci, T. (1998). Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool (“metabolome”) analysis. Journal of Bacteriology, 180(19), 5109–16. https://www.ncbi.nlm.nih.gov/pubmed/9748443
Wider, G., & Dreier, L. (2006). Measuring protein concentrations by NMR spectroscopy. Journal of the American Chemical Society, 128(8), 2571–2576. https://doi.org/10.1021/ja055336t.
Disclosure
Raluca G Maltesen, Bodil S. Rasmussen, Munsoor A. Hanifa and Reinhard Wimmer hold a patent for metabolic biomarkers of hypoxaemia.
Funding
The NMR laboratory at Aalborg University is supported by the Obel Family, SparNord and Carlsberg Foundations. Human experiments were funded by Aalborg University Hospital, the Mica, Lundbeck, Obel Family, Ehrenreich, Aase & Ejnar Danielsen and Moller Foundations, Pharmanovia A/S and the Heart Centre Research Committee at Rigshospitalet, Copenhagen University Hospital. Animal experiments were carried out at the Water and Salt Research Centre, Aarhus University, which was established and supported by the Danish National Research Foundation.
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MAH devised the concept based upon experiments designed by KBB, RGM, BSR, HBR, TR, SN, JF, MAH, MS and RW. Experiments were performed by BSR, KBB, RGM, MAH and MS. RGM and MAH acquired the NMR data and MAH analysed the data. MAH drafted the original manuscript and all authors were involved in revising the manuscript and approving the final version.
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Animal studies were approved by the Danish Ministry of Justice, and conducted in accordance with the National Institute of Health “Guide for the Care and Use of Laboratory Animals” (8th edition). The human studies are both registered at ClinicalTrials.gov (NCT02475694 and NCT01614951) and were conducted in accordance with the Declaration of Helsinki and approved by the local ethics committees.
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Hanifa, M.A., Maltesen, R.G., Rasmussen, B.S. et al. Citrate NMR peak irreproducibility in blood samples after reacquisition of spectra. Metabolomics 16, 7 (2020). https://doi.org/10.1007/s11306-019-1629-8
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DOI: https://doi.org/10.1007/s11306-019-1629-8