Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

Our lab investigates two critical vitamin A transporters, STRA6 and Rbpr2. These membrane proteins facilitate the intake of vitamin A in the blood into tissues such as the eye and liver. Through HPLC we can investigate the physiological functions of these transporters by making a systemic vitamin A profile in transgenic mice.

Another technique used in our field is Surface Plasmon Resonance, SPR which examinees the binding affinities and kinetics of a protein to its liking and we have applied SPR to investigate the binding between STRA6 or Rbpr2, the RBP4 retinol binding complex. One current challenge of this method is sample throughput. The solvent evaporation step is a major bottleneck within this protocol, and future investigators should experiment with drying methods such as nitrogen blow down evaporation, which have a far greater throughput when compared to vacuum evaporation.

We have determined the role of Rbpr2 as an important facilitator in maintaining the systemic vitamin A homeostasis through its function in the liver. And disruption of the Rbpr2 expression in transgenic mice has led to the significant disruption in vitamin A levels across all the examine systemic tissues as quantified through HPLC. Most other protocols in HPLC based vitamin A detection utilize reverse face methods and typically do not allow for retinoid isomers to resolve separately.

Our normal face protocol allows for the resolution of both retinaldehyde and retinal isomers, allowing for us to create a detailed vitamin A profile, which is critical given the importance of vitamin A photoisomerization in the phototransduction cascade.