Deuterium Labeling as a Tool for Analyzing Cholesterol Biosynthesis and Efflux in Pediatric Metabolic Disorders — 65a — Reagan E. Fisher^1,2, Jazmine D. W. Yaeger², Amelia G. Lawver ^2,3, Kevin R. Francis^2,4

¹ South Dakota Biomedical Research Infrastructure Network (SD BRIN) Scholar from Augustana University, Sioux Falls, SD, USA

² Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA

³ Sanford Program for Undergraduate Research (SPUR) Scholar from Bethel University, Arden Hills, MN, USA

⁴ Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA

Disruption of cholesterol biosynthesis detrimentally impacts cellular health and function.  Smith-Lemli-Opitz Syndrome (SLOS) is a rare disease resulting from mutations in DHCR7 which codes for the enzyme 7-dehydrocholesterol reductase that catalyzes the last step in cholesterol biosynthesis.  In SLOS, cholesterol production is inhibited and precursor sterols accumulate, namely 7-dehydrocholesterol (7DHC).  Despite the known genetics, the cellular mechanisms that lead to physical abnormalities and neurological dysfunction in SLOS are poorly understood.  Transport of cholesterol between cells and intracellular compartments is important for cell signaling. How altered membrane sterol biochemistry affects sterol transport is not well understood. To determine how cholesterol biosynthesis impacts sterol efflux, we established a deuterium labeling method to analyze cellular cholesterol production and release. Using immortalized human embryonic kidney cells (HEK293T) and microglia (HMC3), we subjected cells to a cholesterol-depleted environment (lipoprotein deficient serum; LPDS) to induce cholesterol biosynthesis and supplemented the media with deuterated acetate (acetate-D₃). To measure sterol content, we used gas chromatography with mass spectrometry (GC-MS). We observed both production of deuterated cellular cholesterol and deuterated cholesterol within the media, suggesting cells utilized acetate-D₃ to both produce cholesterol and efflux synthesized sterols. Importantly, acetate-D₃ labeling was both dose and time dependent. Next, we assessed how impaired cholesterol biosynthesis affected sterol labeling and efflux. When HEK293T cells were treated with AY9944, an inhibitor of DHCR7, cells produced less deuterated cholesterol but instead accumulated deuterated 7DHC. However, no deuterated 7DHC was observed in the media, suggesting 7DHC efflux is inhibited. Lastly, we performed deuterium-labeling on fibroblasts from a SLOS patient.  As observed with AY9944, SLOS fibroblasts produced less deuterated cholesterol and exhibited accumulation of deuterium-labeled 7DHC. This work demonstrates proof-of-concept measurement of deuterated sterols, suggests cellular secretion of sterols may be structurally specific, and provides insight into SLOS mechanisms.

Sanford Research
Kevin Francis