Impact of Pre-Analytical Variables and Individual Characteristics on SCFA Profiles in Human Samples

Alshawish, Walaa ; Al-Rawashdeh, Ayah Yazeed Fayez

2. semester

Master of Science (MSc) in Engineering (Bioengineering)

2025

2025-05-26

95 pages

This project aimed to establish a reliable and reproducible GC-MS method for the quantification of short-chain fatty acids (SCFAs) in clinical stool and plasma samples, and to explore how individual factors such as diet, age, and body mass index (BMI), and preparation conditions, raw sample freezing and room Temp. exposure, influence SCFA levels in a small-scale human study. Calibration curves for acetic, propionic, butyric, and valeric acids demonstrated strong linearity, with \( R^2 \) values ranging from 0.9582 to 0.9792, confirming the method’s accuracy and reproducibility. The method was applied to samples collected from eight female participants with varying biological and dietary profiles. SCFA concentrations were consistently higher and more stable in stool than in plasma, with acetic acid being the most abundant compound. Butyric and valeric acids were often undetectable in plasma. Among the influencing factors, diet had the most significant impact on SCFA levels, while no consistent trends were observed for age or BMI. Pre-analytical variables, such as freezing time and room temperature delay, had minimal effect on SCFA stability. The use of iso-valeric acid as an internal standard contributed to improved quantification reliability. Overall, the method proved robust across biological matrices and is well suited for clinical microbiome research. These findings support the use of stool as a preferred matrix and reinforce the relevance of SCFAs as biomarkers of gut health and dietary influence.

SCFA ; Diet type ; Raw samples freezing time ; Extracts room Temp. exposure time ; Age ; BMI

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