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Use of EDTA to minimize ionic strength dependent frequency shifts in the 1H NMR spectra of urine

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Abstract

The 1H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). 1H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d4, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra.

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Acknowledgements

This work was supported by the NIH Roadmap Initiative on Metabolomics Technology, Grant NIH/NIDDK 3 R21 DK070290-01 and a Collaborative Biomedical Research Grant from Purdue University/Discovery Park.

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  1. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907, USA

    Vincent M. Asiago, G. A. Nagana Gowda, Shucha Zhang, Narasimhamurthy Shanaiah, Jason Clark & Daniel Raftery

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  1. Vincent M. Asiago
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  2. G. A. Nagana Gowda
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Correspondence to Daniel Raftery.

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11306_2008_121_MOESM1_ESM.doc

Figure S1: Histograms showing standard deviation values of the chemical shifts for six metabolites alanine (1.46 ppm), citrate (2.52 ppm), glycine (3.54 ppm), creatinine (3.05 ppm), hippurate (7.84 ppm) and formate (8.40 ppm) for samples with different urine to buffer volumes (3:1, 1:1, and 1:2). (DOCX 16 kb)

11306_2008_121_MOESM2_ESM.doc

Figure S2: PCA loadings plot for PC1 and PC2 of 1H NMR spectra of urine samples from donors with different buffer concentrations without EDTA (a and b), and with 2.5 μmol EDTA added (c and d). See Fig. 5 for the PCA scores plots. (DOCX 448 kb)

11306_2008_121_MOESM3_ESM.doc

Figure S3: PCA scores plot of 1H NMR spectra of urine samples with different urine to buffer volumes ratios 3:1, 1:1, and 1:2 (a) without EDTA and (b) with 2.5 μmol EDTA added. Urea and residual water peaks were removed prior to PCA. (DOCX 21 kb)

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Asiago, V.M., Nagana Gowda, G.A., Zhang, S. et al. Use of EDTA to minimize ionic strength dependent frequency shifts in the 1H NMR spectra of urine. Metabolomics 4, 328–336 (2008). https://doi.org/10.1007/s11306-008-0121-7

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  • DOI: https://doi.org/10.1007/s11306-008-0121-7

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