A2E, the major fluorophore of lipofuscin in RPE cells,
20 is elevated approximately 20-fold in
abca4 −/− versus wild-type mice
10 (Fig. 2A) . The wavelength of maximal absorbance (λ
max) for A2E is 434 nm
20 (
Fig. 2A , inset). A second constituent of lipofuscin, with a λ
max of approximately 500 nm, is 32-fold elevated in
abca4 −/− versus wild-type eyecups
11 (Fig. 2B) . We tentatively identified the molecule responsible for this 500-nm absorption as the A2E-precursor, A2PE-H
2.
11 44 Alternatively, it has been suggested that the major 500-nm absorbing species in
abca4 −/− mouse eyes is the protonated Schiff-base conjugate of all-
trans-RAL-dimer with ethanolamine or a phosphatidylethanolamine,
45 or di-hydro-A2E (A2E-H
2),
46 which lacks the phosphatidic acid moiety of A2PE-H
2. To distinguish between these possibilities, we collected the 9.5-minute, 500-nm peak fraction during normal-phase liquid chromatography of
abca4 −/− eyecup extracts
(Fig. 2B)and submitted it to reverse phase chromatography and electrospray-ionization (ESI)-MS. The 500-nm absorbing material eluted in a major peak between 16.5 and 19.5 minutes
(Fig. 2C) . The 10-nm difference in λ
max of the 500-nm absorbing peak between normal- and reverse-phase chromatography (
Figs. 2B 2C , insets) is a solvent effect of the two mobile phases. ESI-MS analysis showed that this 500-nm peak coeluted with a dominant, singly charged ion of
m/z 1014.81
(Figs. 2D 2E) . The mass of this major ion within the 500-nm peak fraction corresponded to the mass of mono-stearoyl-A2PE-H
2 (1014.73 amu)
(Fig. 2G)and the mono-stearyl-phosphatidylethanolamine Schiff-base of all-
trans-RAL-dimer, but not to A2E-H
2 (594.5 amu). Collision-induced fragmentation of this parent ion yielded no additional structural information. To distinguish between A2PE-H
2 and all-
trans-RAL-dimer protonated Schiff-base, we performed UV-spectral analysis during NaOH-titration of the 9.5-minute normal-phase peak fraction
(Fig. 2B) . If a protonated Schiff-base were responsible for the 500-nm absorption, the addition of NaOH in molar excess would deprotonate it, causing a significant hypsochromic shift in the spectrum. We observed no detectable reduction of 500-nm absorbance during base titration
(Fig. 2F)despite the addition of an estimated 20-fold molar excess of NaOH. These results suggest that the 500-nm absorbing compound, which is dramatically elevated in
abca4 −/− eyecups
(Fig. 2B)and postmortem RPE samples from patients with Stargardt disease,
11 is mono-stearyl A2PE-H
2 or its open-ring precursor, mono-stearyl-PE-
bis-all-
trans-RAL Schiff-base
(Fig. 2G) .