NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Effect of Stool Sampling on a Routine Clinical Method for the Quantification of Six Short Chain Fatty Acids in Stool Using Gas Chromatography-Mass Spectrometry.

Author(s): Mahdi T, Desmons A, Krasniqi P, Lacorte JM, Kapel N, Lamazière A, Fourati S, Eguether T

Publication: Microorganisms, 2024, Vol. 12, Page

PubMed ID: 38674773 PubMed Review Paper? No

Purpose of Paper

This paper compared the levels of six short-chain fatty acids (SCFAs) in matched fecal specimens that were stored at room temperature for up to 8 h; diluted in ethanol, methanol, or RNAlater; lyophilized, lyophilized and diluted in glycerol, or analyzed fresh; lyophilized or not and freeze-thaw cycled up to two times; lyophilized and stored at 4°C for up to 130 days; or stored after extraction and derivatization at room temperature for up to 7 days. Levels of the six SCFAs were also compared in feces from healthy volunteers and patients with irritable bowel disease. 

Conclusion of Paper

Storage of feces at room temperature resulted in declines in each of the six SCFAs analyzed during the first 2 h, but levels subsequently increased such that after 6 h they were more similar to specimens that were immediately frozen. Concentrations of each of the six SCFAs were lower when diluted with ethanol, methanol or RNAlater than in matched unpreserved specimens. Compared to fresh feces, lyophilized feces had higher levels of each of the six SCFAs, but levels were comparable to fresh feces when 10% glycerol was added to lyophilized samples. Freeze-thaw cycling affected the levels of all six SCFAs, but the magnitude of effects was larger in non-lyophilized than in lyophilized specimens. Changes in the levels of all six SCFAs were limited to <15% in at least two of the three lyophilized specimens that were stored for ≤60 days at 4°C. 
Levels of the six SCFAs changed by <15% when the extract from lyophilized or fresh feces was stored for 7 days at room temperature. Levels of the six SCFAs in feces from patients the IBD cohort fell within the range of those found in feces from healthy patients.  
 

Studies

  1. Study Purpose

    This study compared levels of six short-chain fatty acids (SCFAs) in matched fecal specimens that were stored at room temperature for up to 8 h; diluted in ethanol, methanol, or RNAlater; lyophilized, lyophilized and diluted in glycerol, or analyzed fresh; lyophilized or not and freeze-thaw cycled up to two times; lyophilized and stored at 4°C for up to 130 days; or stored after extraction and derivatization at room temperature for up to 7 days. Levels of the six SCFAs were also compared in feces from healthy volunteers and patients with irritable bowel disease.  Fecal specimens were collected from twenty-eight healthy volunteers (20-65 years of age) and sent to the laboratory within 2 h.  Fecal specimens were either aliquoted and lyophilized or stored at 4°C or -80°C. Lyophilized specimens were frozen at -80°C for >24 h before lyophilization using a Lyovapor Buchi L-200. SCFA were extracted and derivatized using isobutyl chloroformiate and quantified by GS-MS. To evaluate effects of preservation method, aliquots of a single fecal specimen were left undiluted (fresh) or diluted in ethanol, methanol, or RNAlater. To evaluate SCFA stability at room temperature, a single stool specimen was stored at room temperature, and aliquots were frozen at -80°C after 0, 2, 4, 6, and 8 h. To investigate potential effects of freeze-thaw cycling, a fecal specimen was aliquoted and analyzed fresh and after lyophilization and storage at -80°C or after two or five freeze-thaw cycles. To evaluate effects associated with storage of lyophilized feces at 4°C, three lyophilized fecal specimens were analyzed after 1, 12, 33, 60, and 130 days at 4°C. To analyze effects of including 10% glycerol as a cryoprotectant, three fecal specimens were diluted in 10% glycerol and compared to fresh and lyophilized specimens. To investigate the stability of extracts, specimens were extracted, derivatized, and analyzed after 0 and 7 days at 4°C and in ten specimens after 0 and 24 h at room temperature. Levels of the six SCFAs were compared in feces from the twenty-eight healthy volunteers and twenty-seven patients with irritable bowel diseases (four patients with ulcerative colitis and twenty-three patients with Crohn’s disease) who were undergoing fecal calprotectin analysis.

    Summary of Findings:

    The assay had an inter- and intra- day precision standard deviation of 3-10% and 1-5%, respectively, for all six SCFAs evaluated. Recovery of the six SCFAs from spiked specimens generally ranged from 80-120% but was 129% for isovalerate, which the authors state indicates a risk of overestimation. After correction, concentrations of each of the six SCFAs were less than 20% those in matched fresh specimens when diluted with ethanol or RNAlater. In contrast, when feces were diluted in methanol, concentrations of the six SCFAs were 44-68% of those in matched fresh specimens. Storage of feces at room temperature resulted in declines of 11-29% for each of the six SCFAs after only 2 h, but levels then increased such that after 6 h they were more similar to specimens that were immediately frozen; the authors state that SCFAs levels were also acceptable in feces after 8 h of storage at room temperature. Compared to fresh feces, lyophilized feces had higher levels of each of the six SCFAs, but when 10% glycerol was added to lyophilized feces, the levels were comparable to fresh feces. When lyophilized specimens were freeze-thaw cycled twice, isobutyrate and valerate declined by 32 and 35%, respectively, but the other 4 SCFAs declined by less than 10%. However, after five freeze-thaw cycles, acetate, propionate, and butyrate decreased by 10-30% and isovalerate, isobutyrate, and valerate decreased by 48-50%. Freeze-thaw cycling had a larger effect on non-lyophilized feces, with 15-40% declines in all six SCFAs after only two freeze-thaw cycles. Levels of acetate, propionate, and butyrate in lyophilized feces declined by <15% after 60 days at 4°C, and levels of isobutyrate, isovalerate, and valerate also declined by <15% in two of the three lyophilized specimens evaluated after 60 days of storage. Storage of lyophilized feces at 4°C for 130 days led to declines >15% in five of the six SCFAs; however, changes in butyrate were limited to 2-12%.  Levels of the 6 SCFAs changed by -14 to 3% after the extract from lyophilized or fresh feces was stored for 7 days at room temperature. Levels of the six SCFAs in feces from patients the IBD cohort fell within the range of those found in feces from healthy patients.  

    Biospecimens
    Preservative Types
    • Frozen
    • Other Preservative
    • RNAlater
    • Ethanol
    • None (Fresh)
    Diagnoses:
    • Irritable Bowel Syndrome
    • Normal
    Platform:
    AnalyteTechnology Platform
    Small molecule GC-MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Normal
    IBD
    Biospecimen Preservation Type of fixation/preservation Ethanol
    Frozen
    Glycerol
    Lyophilized
    Methanol
    None (fresh)
    RNAlater
    Storage Storage duration 0 days
    12 days
    33 days
    60 days
    130 days
    Storage Storage conditions Lyophilized
    Not-lyophilized
    Storage Time at room temperature Feces for 0 h
    Feces for 2 h
    Feces for 4 h
    Feces for 6 h
    Feces for 8 h
    Extract for 0 h
    Extract for 24 h
    Storage Freeze/thaw cycling 0 cycles
    2 cycles
    5 cycles

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