A2-E was synthesized by coupling all-
trans retinaldehyde to ethanolamine (2:1), exactly according to the protocol
of Eldred and Lasky.
24 The reaction product was
extensively purified by sequential thin-layer chromatography, again
following established protocols of Eldred and Lasky
24 and
Reinboth et al.
29 Although the yield of the whole
procedure is relatively low, the final product was found to be of
excellent purity. In analytical high-performance thin-layer
chromatography on silica plates using an established 11-component
primary developing system
30 a single band was detected
under white light, and a single autofluorescent band was observed under
366-nm illumination. High-performance liquid chromatography analysis of
the product using the procedure of Parish et al.
26 with
photometric detection at 320 nm and 434 nm and fluorescence detection
(excitation 420 nm; emission 605 nm) confirmed the absence of
contaminations in our A2-E preparation. The UV spectra and fluorescent
properties of our synthetic A2-E corresponded to those reported by
other investigators.
26 30 A2-E was complexed with
low-density lipoprotein (LDL),
31 by the addition of 7
nanomoles A2-E to 5 mg LDL particles (Sigma, Munich, Germany) in 1 ml
culture medium, and incubated at 37°C for 2 hours. Approximately 90%
of the A2-E-associated fluorescence was found in the lipoprotein
fraction when the A2-E–LDL complex was analyzed by
ultracentrifugation.
32