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. 1998 Dec 8;95(25):14609-13.
doi: 10.1073/pnas.95.25.14609.

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

C A Parish  1 , M Hashimoto, K Nakanishi, J Dillon, J Sparrow
Affiliations

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

C A Parish et al. Proc Natl Acad Sci U S A. .

Abstract

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms VSports手机版. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures. .

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Figure 1
Figure 1
Structures of A2E and iso-A2E. Molecular modeling on each compound was performed by optimizing (MOPAC 93, AM1) initial conformers, which were constructed according to the NOE data.
Figure 2
Figure 2
Biogenesis of A2E from retinal and (phosphatidyl)ethanolamine.
Figure 3
Figure 3
HPLC of aged and fetal eye extracts. Samples extracted from human RPE/choroid tissue were injected onto a reversed phase (C18) column (Cosmosil 5C18, Nacalai Tesque, 150 mm × 4.6 mm), eluting with a gradient of methanol in water (85–96% methanol + 0.1% TFA, 1 ml/min). The displayed chromatograms correspond to absorbances at 320 nm. Upper trace: The HPLC of extracted pigments (10 μl of a 200 μl extract) from an 80-year-old eye contained a mixture of pigments, which have been partially identified: peak 1, A2E; peak 2, iso-A2E; peak 3, all-trans-retinol; peak 4, 13-cis-retinol; and ∗, unidentified pigment. All-trans-retinal has a retention time of 14.5 min under these conditions. Lower trace: The HPLC of fetal eye extracts (entire extract) did not contain any detectable amount of these materials.
Figure 4
Figure 4
UV spectra of synthetic and extracted pigments. (A) UV spectra of A2E (solid line) and iso-A2E (dashed line) in methanol. A2E: λmax 439 nm (ɛM 36, 900), 336 (ɛM 25, 600); iso-A2E: λmax 426 nm (ɛM 31,000), 335 (ɛM 27,000); (B) UV spectra of peak 1 (A2E) and peak 2 (iso-A2E) from eye extracts. The weak iso-A2E spectrum is due to the limited quantity of iso-A2E present in the extracts. (C) UV spectra of peak 3 (all-trans-retinol) and peak 4 (13-cis-retinol) from eye extracts. The λmax of retinol is ≈325 nm. The spectra provided in B and C were obtained by PDA detection in the HPLC eluent, ≈10% methanol in water containing 0.1% TFA.
Figure 5
Figure 5
1H-NMR spectra of A2E and iso-A2E. Region of 1H-NMR (CD3OD, 500 MHz) spectra of A2E (A) and iso-A2E (B) corresponding to aromatic and olefinic protons. The critical ROESY correlation peak between Ha and Hb of iso-A2E is identified. This correlation peak is absent in A2E (14). The assignment of all 1H-NMR chemical shifts as well as the COSY and ROESY correlation peaks, which were important for assigning the configuration of each double bond, are indicated.
Figure 6
Figure 6
Photoisomerization of A2E and iso-A2E. Methanolic solutions of purified (A) A2E and (C) iso-A2E were exposed to room light for 30 min. The samples were reanalyzed by HPLC and resulted in similar product mixtures containing approximately 4:1 A2E:iso-A2E: (B) A2E + light; (D) iso-A2E + light. A small impurity (∗) is present in the synthetic iso-A2E sample.
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V体育ios版 - References

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