A cell-based assay for retinaldehyde dehydrogenase activity: Retinoid quantification as an alternative to current fluorescence-based approaches.

The biologically active metabolite of Vitamin A, retinoic acid, is essential for regulating immune tolerance, development, and metabolism. A key regulator of retinoic acid signaling is its synthesis by retinaldehyde dehydrogenase, whose expression is tightly regulated and cell-type specific. Current cell-based assays for retinaldehyde dehydrogenase activity rely on fluorescent aldehyde substrates, which lack specificity, limiting their accuracy and interpretability. Here, we developed a sensitive, cell-based assay that directly quantifies retinaldehyde dehydrogenase activity by measuring a panel of retinoids, including all-trans-retinoic acid, using liquid chromatography-mass spectrometry. Employing cultured conventional dendritic cells, we demonstrate that retinoic acid synthesis is time-, substrate-, and enzyme-dependent. Compared to fluorescence-based assays, our assay avoided artifactual signals influenced by cell density and provided a direct, quantitative measure of enzymatic activity in the context of broader retinoid metabolism. This assay offers additional practical advantages, including flexibility in sample processing and compatibility with other downstream metabolite analyses. Together, our protocol provides a robust, specific, and functionally relevant approach that complements existing fluorescence-based approaches to study retinoic acid biosynthesis in immune cells and beyond.