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A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

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Abstract

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography–mass spectrometry (GC–MS) analysis. A one-step derivatization using 100 μL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations of all targeted compounds showed good intra- and inter-day (within 7 days) precision (<10 %), and good stability (<20 %) within 4 days at room temperature (23–25 °C), or 7 days when stored at −20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistical analysis of the concentrations of these targeted metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.

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Abbreviations

SCFAs:

Short-chain fatty acids

BCAAs:

Branched-chain amino acids

PCF:

Propyl chloroformate

GC–MS:

Gas chromatography–mass spectrometry

RSDs:

Relative standard deviations

CE:

Capillary electrophoresis

HPLC:

High performance liquid chromatography

GC:

Gas chromatography

HS-SPME:

Headspace solid-phase microextraction

PrOH:

Propanol

Py:

Pyridine

IS:

Internal standard

RT:

Retention time

PLS:

Partial least squares regression

LOD:

Limit of detection

MCFAs:

Medium chain fatty acids

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Acknowledgments

This work was financially supported by the NIH Grants 1R01AA020212-01 and 3P30DK056350-10.

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Authors and Affiliations

  1. Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China

    Xiaojiao Zheng, Aihua Zhao & Wei Jia

  2. School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China

    Xiaojiao Zheng

  3. Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, 28081, USA

    Yunping Qiu, Wei Zhong, Qiong Li, Guoxiang Xie, Shanlei Qiao, Zhanxiang Zhou & Wei Jia

  4. David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC, 28081, USA

    Sarah Baxter, Mingming Su & Brandon M. Ore

  5. UNC Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, NC, 28081, USA

    Melanie D. Spencer & Steven H. Zeisel

Authors
  1. Xiaojiao Zheng
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  2. Yunping Qiu
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  3. Wei Zhong
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  9. Shanlei Qiao
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  12. Zhanxiang Zhou
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  13. Aihua Zhao
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  14. Wei Jia
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Correspondence to Aihua Zhao or Wei Jia.

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Zheng, X., Qiu, Y., Zhong, W. et al. A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids. Metabolomics 9, 818–827 (2013). https://doi.org/10.1007/s11306-013-0500-6

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  • DOI: https://doi.org/10.1007/s11306-013-0500-6

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