Date of Award

Spring 4-28-2025

Embargo Period

12-1-2025

Document Type

Thesis - MUSC Only

Degree Name

Master of Biomedical Science

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Besim Ogretmen

Second Advisor

Katherine Chetta

Third Advisor

David Long

Fourth Advisor

John Baatz

Fifth Advisor

Gangajaru Vamsi

Abstract

ABSTRACT

Healthy gut microbiota establishment in neonates is crucial for infant health. Influential to the establishment of a healthy immune system, a healthy gut is essential for improved long-term health outcomes, and in mitigating the risk for developing diseases later in life. Fecal matter formed in the digestive tract provide valuable data that may be useful in the detection of gut microbiota dysbiosis through the analysis of end metabolic products of digestion in the intestines. However, due to the complex nature of infant fecal material, proper sample collection, preparation and accurate analytical methods are vital in generating precise data in gut microbiome research. Quantitative and qualitative analyses of free fatty acid metabolites in several biological matrices (e.g., serum and urine) have been developed. However, standardized fecal matter sample preparation methodology for targeted free fatty acid analysis was not previously developed prior to this thesis. In this study, the stability and reliability of the fecal matrix in the development of a long-chain free fatty acid profile utilizing high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) was evaluated for future investigations into neonatal diseases and metabolic syndromes. An extraction and purification protocol was optimized using a liquid-liquid extraction assay developed to quantify several polyunsaturated fatty acid species in human stool. We modified the Bligh & Dyer liquid–liquid extraction protocol and compared differences due to internal standard recovery and resolute chromatographic separation. Our results indicate that a minimum fecal (wet) mass of 5.0 mg diluted in buffer solution accurately represents the complex texture of stool with consistent, reproducible results. Our method provides accurate, sensitive, and simultaneous quantification of long to very long chain free fatty acids, specifically, α-linolenic (ALA), eicosapentaenoic (EPA), docosahexaenoic acid (DHA), arachidonic (AA), and linoleic acids (LA).

Rights

Copyright is held by the author. All rights reserved.

Download

Available for download on Monday, December 01, 2025

Share

COinS