Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

Gulette, G. A.; Hass, D. T.; Pandey, K.; Zhang, Q.; Han, J. Y. S.; Engel, A. L.; Chao, J. R.; Philp, N. J.; Hurley, J. B.; Miller, J. M. L.

doi:10.1101/2024.06.03.597221

bioRxiv · 10.1101/2024.06.03.597221

Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

Gulette, G. A.·Hass, D. T.·Pandey, K.·Zhang, Q.·Han, J. Y. S.·Engel, A. L.·Chao, J. R.·Philp, N. J.·Hurley, J. B.·Miller, J. M. L.

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Abstract

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 ({beta}-HB). Prior work, based on detecting {beta}-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export {beta}-HB across the apical membrane. Here, we compare the accuracy of measuring {beta}-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of {beta}-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete {beta}-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming {beta}-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 {beta}-HB. Altogether, we substantiate {beta}-HB secretion in RPE but find that the secretion is equal apically and basally, RPE {beta}-HB can derive from ketogenic amino acids or fatty acids, and accurate {beta}-HB assessment requires mass spectrometric analysis.

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Gulette, G. A., Hass, D. T., Pandey, K., Zhang, Q., Han, J. Y. S., Engel, A. L., Chao, J. R., Philp, N. J., Hurley, J. B., Miller, J. M. L.. 2024-06-04. Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results. https://doi.org/10.1101/2024.06.03.597221

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