Jun Ueyama1, Masaya Oda2, Masaaki Hirayama2, Kuniyo Sugitate3, Norihiro Sakui3, Risa Hamada2, Mikako Ito4, Isao Saito2, Kinji Ohno4. 1. Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 1-1-20 Daikominami, Higashi-ku, Nagoya, 461-8673, Japan. Electronic address: ueyama@met.nagoya-u.ac.jp. 2. Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 1-1-20 Daikominami, Higashi-ku, Nagoya, 461-8673, Japan. 3. Agilent Technologies Japan, Ltd, 9-1 Takakura-cho, Hachioji, Tokyo, 192-8510, Japan. 4. Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Abstract
BACKGROUND: The analysis methods for fecal short-chain fatty acids (SCFAs) have evolved considerably. Recently, the role of SCFAs in gastrointestinal physiology and their association with intestinal microbiota and disease were reported. However, the intra-fecal variability and storage stability of SCFAs have not been extensively investigated. The aim of this study was to understand the limitations of the measurement of SCFAs in crude feces and develop a useful pre-examination procedure using the freeze-drying technique. METHODS: SCFAs in crude feces, obtained from healthy volunteers, and freeze-dried feces were determined by derivatization with isobutyl chloroformate, followed by liquid-liquid extraction with hexane, and separation and analysis using gas chromatography-mass spectrometry. RESULTS: Among the SCFAS, the maximum intra-fecal variability was observed for iso-butyrate (coefficient of variation of 37.7%), but the freeze-drying procedure reduced this variability (coefficient of variation of 7.9%). Similar improvements were also observed for other SCFAs. Furthermore, significant decreases in the SCFA amounts were observed with storage at 4 °C for 24 h. CONCLUSIONS: The freeze-drying procedure affords fecal SCFA stability, even with storage at room temperature for 3 d. The freeze-drying procedure allows reliable SCFA measurements without labour-intensive processes. Therefore, the freeze-drying procedure can be applied in basic, clinical, and epidemiological studies.
BACKGROUND: The analysis methods for fecal short-chain fatty acids (SCFAs) have evolved considerably. Recently, the role of SCFAs in gastrointestinal physiology and their association with intestinal microbiota and disease were reported. However, the intra-fecal variability and storage stability of SCFAs have not been extensively investigated. The aim of this study was to understand the limitations of the measurement of SCFAs in crude feces and develop a useful pre-examination procedure using the freeze-drying technique. METHODS: SCFAs in crude feces, obtained from healthy volunteers, and freeze-dried feces were determined by derivatization with isobutyl chloroformate, followed by liquid-liquid extraction with hexane, and separation and analysis using gas chromatography-mass spectrometry. RESULTS: Among the SCFAS, the maximum intra-fecal variability was observed for iso-butyrate (coefficient of variation of 37.7%), but the freeze-drying procedure reduced this variability (coefficient of variation of 7.9%). Similar improvements were also observed for other SCFAs. Furthermore, significant decreases in the SCFA amounts were observed with storage at 4 °C for 24 h. CONCLUSIONS: The freeze-drying procedure affords fecal SCFA stability, even with storage at room temperature for 3 d. The freeze-drying procedure allows reliable SCFA measurements without labour-intensive processes. Therefore, the freeze-drying procedure can be applied in basic, clinical, and epidemiological studies.
Authors: Lisa F Stinson; Melvin C L Gay; Petya T Koleva; Merete Eggesbø; Christine C Johnson; Ganesa Wegienka; Elloise du Toit; Naoki Shimojo; Daniel Munblit; Dianne E Campbell; Susan L Prescott; Donna T Geddes; Anita L Kozyrskyj Journal: Front Immunol Date: 2020-07-21 Impact factor: 7.561