The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and β-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells
Introduction
Among the more than 600 carotenoids existing in nature [1], [2], three are of basic importance for the function of the retinal pigment epithelial (RPE) cells in the mammalian eye: β-carotene, lutein and zeaxanthin. β-carotene itself is not abundant but its derivative 11-cis-retinal is part of the visual pigment rhodopsin [3], whereas lutein and zeaxanthin are the only carotenoids of high abundance in RPE cell membranes [4]. Previously we studied the incorporation of various carotenoids into liposomes [5], [6] and pig liver microsomes [7]. Now we extend the investigations to RPE cells. We investigate the incorporation yields of lutein and zeaxanthin, isomeric di-hydroxy compounds of different stereometry, of the di-keto compound canthaxanthin and of the non-polar β-carotene. The structures of these four carotenoids are presented (Fig. 1). The differential comparison of the incorporation yields of these four carotenoids into each of the three membrane types shall allow us to evaluate membrane specific carotenoid incorporation properties.
A main physiological function of the xanthophylls lutein and zeaxanthin consists in their protection of the eye from potentially harmful short-wavelength radiation [8]. The overlapping of the blue light hazard spectrum and the absorption spectrum of the macular pigments has been demonstrated [9]. Antioxidant and radical scavenging effects have as well been reported [8], [10], [11]. A poor absorption of xanthophylls from food matrix or disturbances in their supply to the RPE is supposed to be a risk factor for the age dependent macular degeneration [12], [13].
A basic biophysical question is why only the two xanthophylls, lutein and zeaxanthin out of more than 20 carotenoids present in human plasma are incorporated into the macula [14], [15]. In this paper we look for incorporation features which may favour the exclusive predominance of the macular xanthophylls in RPE cells. We investigate the incorporation rates of the carotenoids into liposomes, microsomes and the RPE cells and its consequences on the membrane fluidity using β-carotene and canthaxanthin as reference pigments. The aim is not to compare quantitatively these three membrane types with regard to their capability to be supplemented with carotenoids, but to compare the carotenoids lutein, zeaxanthin, canthaxanthin and β-carotene in their quantitative relation to each other in which they incorporate differentially into each of these membranes.
The method we apply for the quantification of the carotenoids is UV–Vis absorption spectrometry using the characteristic absorption bands of the carotenoids. To detect the membrane modulating effect of the carotenoids we co-incubate the fluorophor Laurdan and evaluate its fluorescence spectra.
Section snippets
Chemicals
β-carotene, lutein, and zeaxanthin were bought from S.C. Proplanta S.A. Cluj-Napoca, Romania. They were purified from natural sources and checked for purity by HPLC. Canthaxanthin was purchased from Carl Roth (Karlsruhe, Germany). High Purity Egg Yolk phosphatidylcholine (EYPC), MW 750, was purchased from Lipoid KG (Ludwigshafen, Germany). The lipid purity of the preparation was higher than 99% and used without further purification.
The fluorescent probe used was Laurdan
Liposomes
Two sets of incorporation experiments were performed. One was intended to measure the incorporation rates of the carotenoids: the incubation concentration was 1.5 mol% related to liposomal lipids for each carotenoid.
The other set of experiments intended to evaluate the effect of the incorporated carotenoids onto the membranes. To obtain carotenoid specific, and not concentration dependent results, we incubated the liposomes with such carotenoid concentrations (IC) that the carotenoid
Discussion
The xanthophylls lutein and zeaxanthin are the only carotenoids which are located in retinal pigment epithelial (RPE) cell membranes, particularly in the macula lutea. We investigate their incorporation yields into the human RPE cell line D407. We included two reference carotenoids: (i) β-carotene and canthaxanthin as a completely unpolar and less polar one, respectively, and (ii) we compared the incorporation pattern in RPE cells with those in liposomal and in microsomal membranes as physical
Conclusions
The incorporation preference for lutein and zeaxanthin compared to β-carotene and canthaxanthin into RPE cells is significantly higher than into liver microsomes and into liposomes.
As a carrier for carotenoid incorporation into RPE cells fetal calf serum is recommended instead of tetrahydrofuran.
Membrane bound Laurdan fluorescence quenching may reveal a base to determine directly carotenoid concentrations incorporated into membranes.
Acknowledgements
We thank Dr. Adela Pintea and Dr. Svitlana Tykhonova for their helpful discussions, and Christina Kenst, Edda Toma and Rainer Doschke for their valuable technical assistance. M.W.I.S. thanks the Egyptian Government for a scholarship and the DFG for support. C.S. thanks the PHD programme of the faculty and the International Office of the University of Bremen for the travel assistance.
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