RETINOL KINETICS IN THE ISOLATED RETINA DETERMINED BY RETINOID EXTRACTION AND HPLC

Suzuki et al. [Vis, Res. 26, 425-9 (1986); Vis. Res. 28, 1061-70 (1988 )] have described a formaldehyde-based (HCHO-based) extraction procedu re that efficiently recovers 11-cis retinal initially present as rhodo psin 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 perform ance liquid chromatography (HPLC), can account quantitatively for ligh t-induced changes in retinoid levels and thus serve as an alternative to spectrophotometry for tracking the formation of all-trans retinol i n this intact rod preparation. Initially dark-adapted retinas were inc ubated in bright light or in darkness and then analysed by homogenizat ion and extraction using the HCHO and i/h methods. Combined data obtai ned using the two extraction procedures indicated a near-conservation of total retinoid recovered from dark-incubated and illuminated retina s, and thus accounted for light-induced changes in retinoid levels. Th e HCHO procedure, employing formaldehyde, isopropanol and hexane, was similar to that described by Suzuki et al. and recovered retinaldehyde s including chromophoric Il-cis retinal. The i/h procedure utilized is opropanol and hexane and, unlike the HCHO method, efficiently recovere d all-trans retinol. Illumination (onset at time zero) that produced a n approximately exponential decline of Il-cis retinal (time constant o f 24 s) led to an increase and then a gradual decline in all-trans ret inal. The normalized peak level of all-trans retinal, representing abo ut 0.54 of the total molar quantity of recovered retinoid, developed w ith illumination periods of 10-80 s. The normalized level of all-trans retinol reached similar or equal to 0.3 in retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approx imately exponential further growth to similar or equal to 0.9 with a t ime constant of 9.2 min. The results indicate the workability of the H CHO and i/h extraction procedures for tracking the in situ conversion of all-trans retinal to all-trans retinol, a reaction thought to be im portant for both operation of the retinoid Visual cycle and shutoff of the phototransduction cascade. (C) 1997 Academic Press Limited.