Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (β-HB). Prior work, based on detecting β-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export β-HB across the apical membrane. Here, we compare the accuracy of measuring β-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of β-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete β-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming β-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce β-HB. Altogether, we substantiate β-HB secretion in RPE but find that the secretion is equal apically and basally, RPE β-HB can derive from ketogenic amino acids or fatty acids, and accurate β-HB assessment requires mass spectrometric analysis.