No abstract

Imaging of the human fundus of the eye with excitation wavelengths in the visible spectrum reveals a natural autofluorescence, that in a healthy retina originates primarily from the bisretinoids that constitute the lipofuscin of retinal pigment epithelial (RPE) cells. Since the intensity and distribution of fundus autofluorescence is altered in the presence of retinal disease, we have examined the fluorescence properties of the retinal bisretinoids with a view to aiding clinical interpretations. As is also observed for fundus autofluorescence, fluorescence emission from RPE lipofuscin was generated with a wide range of exciting wavelengths; with increasing excitation wavelength, the emission maximum shifted towards longer wavelengths and spectral width was decreased. These features are consistent with fluorescence generation from a mixture of compounds. While the bisretinoids that constitute RPE lipofuscin all fluoresced with maxima that were centered around 600 nm, fluorescence intensities varied when excited at 488 nm, the excitation wavelength utilized for fundus autofuorescence imaging. For instance the fluorescence efficiency of the bisretinoid A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE) was greater than A2E and relative to both of the latter, all-trans-retinal dimer-phosphatidylethanolamine was weakly fluorescent. On the other hand, certain photooxidized forms of the bisretinoids present in both RPE and photoreceptor cells were more strongly fluorescent than the parent compound. We also sought to evaluate whether diffuse puncta of autofluorescence observed in some retinal disorders of monogenic origin are attributable to retinoid accumulation. However, two retinoids of the visual cycle, all-trans-retinyl ester and all-trans-retinal, did not exhibit fluorescence at 488 nm excitation.

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.

To date, several bisretinoids of RPE lipofuscin have been isolated and characterized, and for all of these, formation involves the membrane phospholipid phosphatidylethanolamine. Conversely, the bisretinoid A2-GPE is detected as sn-glycero-3-phosphoethanolamine (GPE) derivatized by two all-trans-retinal. The pathways by which A2-GPE may form under conditions of increased availability of all-trans-retinal, for instance in the Abca4(-/-) mouse, are discussed.

Three new flavonoid glycosides ( 1- 3), 11-hydroxyhainanolidol ( 4), and a new dibenzylbutyrolactone lignan glycoside ( 5) were isolated from the aerial parts of Cephalotaxus koreana Nakai, along with 19 known flavonoids. The structures of the new compounds were elucidated using spectroscopic evidence, primarily NMR and MS. Twenty-four compounds were isolated, and among these isoscutellarein 5-O-beta-D-glucopyranoside ( 3), apigenin ( 6), kaempferol 3-O-alpha-L-rhamnopyranosyl(1'''-->6'')-beta-D-glucopyranoside ( 7), tamarixetin 3-O-alpha-L-rhamnopyranosyl(1'''-->6'')-beta-D-glucopyranoside ( 8), quercetin 3-O-[6''-O-acetyl]-beta-D-glucopyranoside ( 9), and quercetin 3-O-alpha-L-rhamnopyranoside ( 10) showed significant inhibitory activities against osteoclast differentiation at concentrations of 0.1 and 1.0 microg/mL.