FIGURE 2.

Incorporation of ¹⁸O into at-RAL liberated from photoactivated Rho. A, exchange of ¹⁸O in synthetic at-[¹⁸O]RAL upon incubation in an aqueous solution containing 50% methanol (●), 1% BSA (○), or liposomes (▾). The 5-min time frame used for these experiments is indicated by the gray background. B, representative mass spectra indicate changes in the isotopic composition of at-[¹⁸O]RAL upon incubation in 10 mm Bistris propane buffer, pH 7.0, in 70% H₂¹⁸O with 100 mm NaCl and 1% BSA. The peak at m/z 285.3 corresponds to at-RAL ([M + H]⁺). The 2-Da shift in mass of at-[¹⁸O]RAL is indicated by the ion at m/z 287.3 ([M + H]⁺). C, incorporation of ¹⁸O into at-RAL released from photobleached Rho. Purified bovine ROS washed and resuspended in buffers containing various ratios of H₂¹⁸O were extracted with hexane, and the retinoid composition was examined by MS. The pool of analyzed at-RAL was significantly enriched in ¹⁸O. The ratio between at-RAL and at-[¹⁸O]RAL closely reflects the water composition present in the experimental samples (gray bars). The theoretical percentage of at-[¹⁸O]RAL that could be produced from unlabeled at-RAL due to oxygen back-exchange into buffers present in the samples is shown (white bars). D, retinal composition and oxygen back-exchange in isolated mouse retina. at-RAL or at-[¹⁸O]RAL (300 pmol) was added to homogenized retinas isolated from Lrat^−/− mice and washed with buffers containing H₂¹⁸O or H₂O. Samples were incubated on ice for 5 min and extracted with hexane. Oxygen back-exchange did not exceed 15% in the examined samples (light gray and white bars). WT mouse retinas washed with 90% H₂¹⁸O-containing buffer and exposed to light under similar experimental conditions revealed the presence of at-RAL highly enriched in ¹⁸O (dark gray bar). All data represent averaged values of three independent experiments, each performed in duplicate.