Abstract
Purpose :
Production and secretion of ketone bodies, specifically β-hydroxybutyrate (BHB), by the retinal pigment epithelium (RPE) has been postulated to provide overlying photoreceptors with an alternative metabolic fuel source. Previous publications have quantified BHB via enzymatic assays. Here, by comparing results from these enzymatic assays with quantification of BHB via mass spectrometry (MS), we show that commercially available enzymatic assays are too insensitive to measure BHB secretion in RPE culture.
Methods :
Primary human fetal RPE were cultured on Transwells under various cell culture media compositions. BHB secretions from apical and basal supernatants were collected at multiple timepoints and purified using deproteination schemes. BHB levels were assessed enzymatically using the Amplite® Fluorimetric BHB Assay Kit or Stanbio Laboratory BHB Liquicolor Procedure No. 2440 Kit and compared to BHB levels as measured by MS analysis. MS analysis included internal deuterated standards for both BHB and structurally similar but functionally different isomer, α-hydroxybutyrate.
Results :
BHB measured through MS confirmed that RPE cultures are producing BHB in a time dependent manner, but unlike previous reports, it is secreted equally to the apical and basolateral sides. A false BHB signal using enzymatic assay kits was caused by a component secreted by RPE cultures, particularly apically, that is >10kDa; taking media in which molecular components >10kDa were subtracted out revealed barely recordable and highly variable BHB signals. Other media components had less prominent, but still significant effects on accuracy of BHB measurement. Once confounding factors were eliminated, assay kits were not sensitive enough to measure β-hydroxybutyrate in the range that is secreted by the culture system.
Conclusions :
We demonstrate that existing enzymatic assays for BHB quantification in RPE cultures are inadequate. We suggest future research on RPE ketogenesis may require MS methods. By MS analysis, we confirm that the RPE secretes BHB. In contrast to previous reports of an apical-dominant secretion, however, the secretion appears to be both apical and basolateral. While apical secretion may play a role in providing an alternative fuel source to photoreceptors, we speculate that basolateral secretion may play a role in regulating choroid-derived monocyte activation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.