ライフサイエンスコーパス: A2E

1 A2E accumulation and retinal degeneration were prevented

2 A2E free cells were also irradiated and analyzed.

3 A2E has been shown to undergo light-dependent oxidation

4 A2E in PBS, with and without an oxygen-depleter or singl

5 A2E localized to lysosomes in cultured RPE as well as in

6 A2E that had accumulated in ARPE-19 cells exhibited irra

7 A2E that was not cell-associated underwent irradiation-i

8 A2E, a major lipofuscin fluorophore that accumulated dur

9 A2E, the major fluorophore of lipofuscin, and its precur

10 A2E-loaded RPE cells in culture bound and internalized i

11 A2E-loaded RPE degraded outer segment proteins efficient

12 scence spectroscopy revealed that A2PE-H(2), A2E, and N-retinylidene-N-retinyl-phosphatidylethanolami

13 with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H(2), which form from retinaldehyde, were

14 Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over contro

15 Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media,

16 ARPE-19 cells accumulated A2E before exposure to blue light.

17 inal pigment epithelium that had accumulated A2E and were irradiated (430 nm).

18 overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation.

19 man RPE (ARPE-19) cells that had accumulated A2E were exposed to blue light in the presence and absen

20 man RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light.

21 In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of b

22 ng to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cross-reactivity with various

23 Also, A2E levels in abcr(-/-) mice reared under cyclic light a

24 Accumulation of A2PE-H(2) and A2E in abcr+/- retina and RPE, respectively, was strongl

25 ot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but no cros

26 e mice resulted in the formation of A2PE and A2E.

27 light and electron microscopic analysis, and A2E quantification.

28 epithelium, photoreceptor degeneration, and A2E accumulation.

29 correlate the distribution of lipofuscin and A2E across the human RPE.

30 tween the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the

31 Lipofuscin and A2E levels in the RPE increased with age and more so in

32 and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic li

33 posure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors fr

34 A2E was not found in any of these mice, and A2E oxidation was not induced by blue light and UV illum

35 ygen-depleter or singlet-oxygen quencher and A2E-laden RPE, were exposed to 430-nm light and examined

36 gy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in copin

37 effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to AB

38 NF-alpha, IL-1beta, and TLR4 transcripts and A2E were significantly lower in naloxone-treated DKO ani

39 fluorescence spectroscopy studies, antibody-A2E binding was evidenced by a fluorescence increase and

40 compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer.

41 of the final product A2E and was consumed as A2E accumulated.

42 Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of

43 of cytotoxic lipofuscin fluorophores such as A2E within the retinal pigment epithelium.

44 ion and to the formation of pigments such as A2E, and is believed to play a key role in the formation

45 indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment ep

46 Using cell-associated A2E as a model of RPE bisretinoid behavior, photobleachi

47 ughter ions that were identical to authentic A2E and its daughter ions.

48 Because A2E accumulation in the RPE is associated with pathogene

49 the RPE to be characterized as yet have been A2E and its isomers.

50 rans-retinal and not, as generally believed, A2E or other retinoid condensation products.

51 When the pyridinium bisretinoid A2E, an age-related fluorophore in the retinal pigment e

52 f RPE lipofuscin, the pyridinium bisretinoid A2E, is a diretinal conjugate that forms in photorecepto

53 The bisretinoid A2E was measured by quantitative high performance liquid

54 were allowed to accumulate the bisretinoid, A2E, and were irradiated at 430 nm.

55 light and reduced levels of the bisretinoids A2E and A2-glycero-phosphoethanolamine (A2-GPE).

56 Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown tha

57 Analysis of the bisretinoids A2E, A2-GPE, A2-dihydropyridine-phosphatidylethanolamine

58 s dot-blot analysis revealed binding to both A2E and A2E-rabbit serum albumin (A2E-RSA) conjugate but

59 plays a role in coping with stress caused by A2E accumulation.

60 onstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic

61 vidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular w

62 Comparable A2E concentrations were detected in the eyes of 6-month-

63 loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis ei

64 l cells accumulate the lipofuscin component, A2E.

65 and analyzed for the bis-retinoid compounds A2E and iso-A2E.

66 One of these compounds, A2E, is generated by phosphate hydrolysis of a phosphati

67 ight- exposed RPE cells that did not contain A2E remained viable.

68 lculation, based on the structure of dihydro-A2E, produced a simulated UV-visible absorbance spectrum

69 proximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes th

70 to investigate the role of autophagy during A2E accumulation.

71 compound diretinoid-pyridinium-ethanolamine (A2E) were increased in Rdh12-deficient mice of various g

72 ation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a

73 cluding di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primari

74 ARPE19 cells were examined to evaluate A2E and RALdi oxidation and toxicity induced by exposure

75 on and intersystem crossing of photo-excited A2E.

76 Light-exposed A2E-laden retinal pigment epithelium exhibits a propensi

77 C measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold

78 The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage

79 ed by measurements of lipofuscin fluorophore A2E in the RPE using liquid chromatography/mass spectrom

80 The lipofuscin fluorophore A2E is known to be an initiator of blue-light-induced ap

81 The major constituent is the fluorophore A2E, a pyridinium bisretinoid.

82 RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cy

83 A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in th

84 The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid.

85 tigated the role of lipofuscin fluorophores (A2E-lipofuscin) on oxidative stress and complement activ

86 The binding constant for A2E and purified RPE65 was calculated to be 250 nM.

87 vitro, suggesting a potential mechanism for A2E cytotoxicity.

88 lyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry.

89 iously hypothesized biosynthetic pathway for A2E and implicate A2PE-H(2) as a precursor in this pathw

90 intracellular lipofuscin resembles that for A2E.

91 Also, once formed, A2E was not eliminated by the RPE.

92 st in part, for cellular damage ensuing from A2E photooxidation.

93 cies of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated c

94 that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-y

95 epithelium that were irradiated to generate A2E photooxidation products.

96 A2-PE; (iii) hydrolysis of A2-PE to generate A2E.

97 ction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatograp

98 l illuminance was not correlated with higher A2E.

99 all-trans-retinal clearance, and the highest A2E amounts were found in Rdh8(-/-)Rdh12(-/-)Abca4(-/-)

100 The death of blue-light-illuminated A2E-laden RPE was blocked in oxygen-depleted media.

101 These studies implicate A2E as an initiator of blue light-induced apoptosis of R

102 n the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected wi

103 emission maximum consistent with a change in A2E milieu upon antibody binding.

104 es oxygen-dependent photochemical changes in A2E, indicates that the effects of singlet oxygen may be

105 The decline in A2E levels in the Abca4(-/-) mice corresponded to reduce

106 dant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both

107 DNA damage was induced in A2E-containing RPE exposed to 430-nm illumination.

108 the cell death program that is initiated in A2E-containing cells in response to blue light.

109 ther autophagy-related molecules involved in A2E accumulation, we performed gene expression array ana

110 yde, which constitutes the first reactant in A2E biosynthesis.

111 noparticles induced 36% and 29% reduction in A2E accumulation respectively.

112 l mechanism for cholesterol sequestration in A2E-loaded cells.

113 phane and N-acetylcysteine; samples included A2E-free cells.

114 ccumulated A2E and were irradiated to induce A2E photooxidation.

115 ell damage mediated by aging/disease-induced A2E accumulation.

116 jugate indicated the presence of five intact A2E molecules covalently linked to BSA, thus deeming it

117 rations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as as

118 ation of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a

119 e oxo-aldehydes are released from irradiated A2E-containing RPE cells.

120 best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side

121 est characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid vis

122 orescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.

123 d for the bis-retinoid compounds A2E and iso-A2E.

124 Likewise, A2E also inhibited isomerohydrolase activity in cells co

125 ts of retinal pigment epithelium lipofuscin, A2E isomers with cis olefins at positions other than the

126 However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE

127 found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibit

128 measured after exposure to 50 and 100 microM A2E were attributable to membrane damage in a subpopulat

129 ting in cells incubated with 10 to 25 microM A2E were comparable to the amounts of A2E present in equ

130 ometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups

131 o detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr-/- m

132 Moreover, A2E levels declined after 8 months of age, a change not

133 e revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxyge

134 Ocular A2E was measured using HPLC.

135 cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE.

136 se data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fl

137 No significant ocular accumulation of A2E occurred under these conditions.

138 hy, which is associated with accumulation of A2E, a diretinoid adduct within the retinal pigment epit

139 The amount of A2E, a major lipofuscin component, increased 10- to 12-f

140 Amounts of A2E in the RPE correlated with diminished all-trans-reti

141 microM A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from

142 sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing cha

143 iments suggest a direct, specific binding of A2E to RPE65.

144 The role of A2PE-H(2) in the biogenesis of A2E and its relationship to other retinal fluorophores h

145 e previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff

146 ations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by li

147 ned questions related to the biosynthesis of A2E, a fluorophore that accumulates in retinal pigment e

148 Dillon & J. Sparrow, for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-tr

149 stigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a

150 on and as a function of the concentration of A2E used to load the cells before illumination.

151 oducts was accompanied by the consumption of A2E, the latter being diminished, however, when illumina

152 ed in the mechanisms leading to the death of A2E-containing RPE cells after blue light illumination.

153 ries of higher molecular mass derivatives of A2E.

154 s no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in

155 t description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry.

156 The spatial distributions of A2E and its oxides were determined using matrix-assisted

157 rum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products b

158 Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative

159 Given the adverse effects of A2E, there is considerable interest in combating its dep

160 Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40%

161 Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pig

162 Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr

163 The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent

164 As predicted by this model, formation of A2E was completely inhibited when abcr-/- mice were rais

165 RPE65 antagonists abolishes the formation of A2E.

166 Whereas the generation of A2E from A2-PE by acid hydrolysis was found to occur ver

167 The identity of A2E was confirmed by tandem mass spectrometry.

168 induced by blue light and UV illumination of A2E-loaded ARPE19 cells.

169 Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase ac

170 ts that can be damaged by the interaction of A2E and blue light.

171 To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were

172 Internalization of A2E by the RPE cells was evidenced by the acquisition of

173 metry detection revealed that irradiation of A2E was associated with A2E photoisomerization, photooxi

174 The levels of A2E accumulated by the cultured cells were quantified by

175 The levels of A2E accumulating in cells incubated with 10 to 25 microM

176 ased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofusc

177 tudied the effect of physiological levels of A2E in RPE cultures on their ability to phagocytose oute

178 tion of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased towa

179 Loading of A2E or exposure to blue light alone had little effect on

180 Loading of A2E, with or without exposure to blue light, upregulated

181 esults corroborate the proposed mechanism of A2E biogenesis.

182 This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20

183 emonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approa

184 retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and pho

185 -MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyl

186 inoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to sing

187 of products resulting from photooxidation of A2E might include a range of fragments that could be rec

188 glet oxygen production and photooxidation of A2E.

189 dylethanolamine (A2PE), a known precursor of A2E, share common electronic and resonant structures.

190 ations and was proposed to be a precursor of A2E.

191 The precursors of A2E identified in this study may represent pharmacologic

192 Here, we identify three precursors of A2E in ocular tissues from abcr-/- mice and humans with

193 eptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine)

194 generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, r

195 sly reported that photooxidation products of A2E can activate complement.

196 ay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the

197 the generation of reactive photo-products of A2E.

198 products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation

199 However, the precise role of A2E in vision loss is unclear.

200 An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines ide

201 ght-dependent oxidation to yield a series of A2E epoxides or oxiranes.

202 lyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on

203 ules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies sign

204 were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside

205 performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of f

206 to define the effect of increasing light on A2E accumulation.

207 Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold t

208 exposed to continuously generated H2O(2) or A2E-mediated photooxidation.

209 osure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expres

210 Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <1

211 However oxidized A2E was not found in any of these mice, and A2E oxidatio

212 crotiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation

213 he production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage e

214 By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or tw

215 tes with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum,

216 rum incubated in wells precoated with peroxy-A2E, the lipofuscin pigment all-trans-retinal dimer, and

217 on at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE.

218 different chain lengths, and the phosphoryl-A2E moiety, m/z 673.

219 ning moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monopero

220 The autofluorescent lipofuscin pigment A2E accumulates in retinal pigment epithelial cells with

221 f free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death.

222 Although endogenously produced A2E in the RPE has been associated with macular degenera

223 ce it formed in advance of the final product A2E and was consumed as A2E accumulated.

224 s accumulation of its condensation products, A2E, and all-trans-retinal dimer (RALdi), both associate

225 xen-1- yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) is followed by RPE atrophy.

226 gical concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis.

227 ments in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (

228 autofluorescence, decreased HPLC-quantified A2E, outer nuclear layer thinning, and increased methylg

229 Rather, A2E prevents cholesterol efflux from these organelles, w

230 g, the rabbit polyclonal antibody recognized A2E that had accumulated in cultured cells, whereas dot-

231 and Abca4 all protect the retina and reduce A2E production by facilitating all-trans-retinal clearan

232 e (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis

233 l, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen.

234 f 11-cis-retinal, is associated with reduced A2E accumulation.

235 Funduscopy, histopathology, retinal A2E quantification, proteomics, RT-PCR gene expression a

236 Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates

237 cludes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component.

238 uch as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium.

239 ion of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal d

240 sed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine.

241 inoid, N-retinylidene-N-retinylethanolamine (A2E).

242 inoid, N-retinylidene-N-retinylethanolamine (A2E).

243 s like N-retinylidene-N-retinylethanolamine (A2E).

244 uch as N-retinylidene-N-retinylethanolamine (A2E).

245 tinoid N-retinylidene-N-retinylethanolamine (A2E).

246 bsorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in m

247 etic defects in vitamin A processing, slowed A2E biosynthesis.

248 ) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but n

249 tive to wild type and was more abundant than A2E.

250 rans-retinal clearance contributes more than A2E oxidation to light-induced cellular toxicity.

251 e efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more rea

252 merohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in

253 Our results demonstrate that A2E accumulation exacerbates the effects of moderate mit

254 ss spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation an

255 These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chr

256 Our data also demonstrate that A2E, which is a cone-shaped lipid, increases the chemica

257 Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit rea

258 Our results provide direct evidence that A2E causes aberrant cholesterol metabolism in RPE cells

259 Taken together, these findings indicate that A2E biosynthesis involves the oxidation of a dihydropyri

260 uter segments as control RPE indicating that A2E does not alter early steps of phagocytosis.

261 Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key e

262 Here, we show that A2E, a quaternary amine and retinoid by-product of the v

263 These results suggest that A2E accumulation causes oxidative stress, complement act

264 versed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes.

265 chromatography, thus confirming A2-PE as the A2E precursor.

266 -IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts.

267 bdamax 490 and 330 nm) was identified in the A2E biomimetic reaction mixture.

268 similar to A2E, but represented <10% of the A2E levels.

269 to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nucle

270 ight mass spectrometry (MALDI-TOF MS) of the A2E-BSA conjugate indicated the presence of five intact

271 evealed that detectable levels of A2-PE, the A2E precursor, are formed within photoreceptor outer seg

272 ator-activated receptor pathway relieves the A2E-induced block on cholesterol efflux and restores cho

273 gates in which the protein was linked to the A2E molecule via its pyridinium ethanolamine moiety.

274 dverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates

275 In addition to A2E and related cis isomers, we previously showed that c

276 ether acute exposure of healthy RPE cells to A2E-lipofuscin affects oxidative stress and expression o

277 PE65 is also on the rate-limiting pathway to A2E formation.

278 iesterase activity that can convert A2-PE to A2E may indicate that some portion of the A2-PE that for

279 ly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels.

280 ells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of

281 LISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation.

282 Unexpectedly, A2E levels were not higher in the albino mice.

283 Unlike A2E, the oxiranes were more abundant in albino vs. pigme

284 s more reactive with singlet oxygen than was A2E.

285 When A2E, in either acellular or cellular environments, was i

286 When A2E-laden RPE were transferred to D(2)O-based media and

287 When A2E-loaded cells are exposed to blue light, the model pa

288 ence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in t

289 determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorp

290 which represents a unique mechanism by which A2E may impair vision in STGD.

291 Studies in which A2E was incubated with a singlet oxygen generator yielde

292 that irradiation of A2E was associated with A2E photoisomerization, photooxidation, and photodegrada

293 ontribute to the pathologies associated with A2E.

294 were elevated in NHS placed in contact with A2E-laden retinal pigment epithelium that were irradiate

295 ence of RPE65 decreased upon incubation with A2E.

296 ated FAF intensities correlate linearly with A2E contents.

297 ith the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds.

298 ic titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence.

299 2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Re

300 ) hydrolysis of the phosphate ester to yield A2E.