Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

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

Conclusion of Paper

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

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • Other Preservative
   • RNAlater
   • Ethanol
   • None (Fresh)

   Diagnoses:

   • Irritable Bowel Syndrome
   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	GC-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(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

   View more