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 procedure that efficiently recovers 11-cis retinal 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-trans retinol 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-cis retinal. The i/h procedure utilized isopropanol and hexane and, unlike the HCHO method, efficiently recovered all-trans retinol. Illumination (onset at time zero) that produced an approximately exponential decline of 11-cis retinal (time constant of 24 s) led to an increase and then a gradual decline in all-trans retinal. The normalized peak level of all-trans retinal, 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-trans retinol reached approximately 0.3 in retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approximately exponential further growth to approximately 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-trans retinal to all-trans retinol, a reaction thought to be important for both operation of the retinoid visual cycle and shut-off of the phototransduction cascade.
Okajima
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TL
Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Pepperberg
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DR
D000818:Animals; D002025:Bufonidae; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003623:Dark Adaptation; D008027:Light; D008954:Models, Biological; D012160:Retina / Q000737:chemistry* / Q000378:metabolism; D012172:Retinaldehyde / Q000032:analysis / Q000302:isolation & purification; D012176:Retinoids / Q000032:analysis / Q000302:isolation & purification; D014801:Vitamin A / Q000032:analysis* / Q000378:metabolism
D012176:Retinoids; D014801:Vitamin A; D012172:Retinaldehyde