Retinol Kinetics in the Isolated Retina Determined by Retinoid Extraction and HPLC

Abstract

Suzuki et al. [Vis. Res.26, 425–9 (1986);Vis. Res.28, 1061–70 (1988)] have described a formaldehyde-based (HCHO-based) extraction procedure that efficiently recovers 11-cisretinal initially present as rhodopsin chromophore in photoreceptor membranes. Using the isolated retina of the toad (Bufo marinus), we tested whether this procedure (‘HCHO’ method), in combination with a formaldehyde-free extraction procedure (‘i/h’ method) and the analysis of extracted retinoids by high performance liquid chromatography (HPLC), can account quantitatively for light-induced changes in retinoid levels and thus serve as an alternative to spectrophotometry for tracking the formation of all-transretinol in this intact rod preparation. Initially dark-adapted retinas were incubated in bright light or in darkness and then analysed by homogenization and extraction using the HCHO and i/h methods. Combined data obtained using the two extraction procedures indicated a near-conservation of total retinoid recovered from dark-incubated and illuminated retinas, and thus accounted for light-induced changes in retinoid levels. The HCHO procedure, employing formaldehyde, isopropanol and hexane, was similar to that described by Suzuki et al. and recovered retinaldehydes including chromophoric 11-cisretinal. The i/h procedure utilized isopropanol and hexane and, unlike the HCHO method, efficiently recovered all-transretinol. Illumination (onset at time zero) that produced an approximately exponential decline of 11-cisretinal (time constant of 24 s) led to an increase and then a gradual decline in all-transretinal. The normalized peak level of all-transretinal, representing about 0.54 of the total molar quantity of recovered retinoid, developed with illumination periods of 10–80 s. The normalized level of all-transretinol reached ≃0.3 in retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approximately exponential further growth to ≃0.9 with a time constant of 9.2 min. The results indicate the workability of the HCHO and i/h extraction procedures for tracking the in situ conversion of all-transretinal to all-transretinol, a reaction thought to be important for both operation of the retinoid visual cycle and shut-off of the phototransduction cascade.