Cholesterol is synthesized endogenously in the brain in a complex pathway that starts with the condensation of acetate and acetoacetate. The last step in this pathway is the conversion of 7-dehydrocholesterol (7-DHC) into cholesterol, a step catalyzed by the enzyme dehydrocholesterol reductase 7 (DHCR7). Mutations in this enzyme lead to the accumulation of 7-DHC, a hallmark of the disorder known as Smith-Lemli-Opitz Syndrome (SLOS). The sterol profile in SLOS cells is characterized by lower levels of desmosterol and cholesterol and higher than normal levels of 7-DHC, the most oxidizable lipid known to date. As a tentative approach to improve this phenotype, we tested in this study the effects of ketone bodies using well-established cell culture models of SLOS. WT and Dhcr7-deficient Neuro2a cells were supplemented with different concentrations of acetoacetate (AcAc) and β-hydroxybutyrate and the sterol profile was analyzed. The levels of five sterols in the post-lanosterol pathway, including 7-DHC and cholesterol, were quantified using UHPLC-MS/MS and several isotopically labeled internal standards. Supplementing Dhcr7-deficient Neuro2a cells with AcAc for 96 hours led to a decrease in 7-DHC and an increase in desmosterol and cholesterol when compared to the control conditions, suggesting an improvement in the sterol profile. On the other hand, the same treatment in WT Neuro2a cells did not affect 7-DHC levels, indicating that the ketone bodies – especially AcAc – could have beneficial effects in SLOS cells. These observations are now being confirmed in primary human fibroblasts taken from healthy and SLOS patients. In conclusion, these results suggest that ketone bodies can affect positively the cholesterol biosynthesis in cultured SLOS cells, where they improve the sterol profile.NIH, NICHD.