Determination of N-retinylidene-N-retinylethanolamine (A2E) levels in central and peripheral areas of human retinal pigment epithelium

Abstract

The bis-retinoid N-retinylidene-N-retinylethanolamine (A2E) is one of the major components of lipofuscin, a fluorescent material that accumulates with age in the lysosomes of the retinal pigment epithelium (RPE) of the human eye. Lipofuscin, as well as A2E, exhibit a range of cytotoxic properties, which are thought to contribute to the pathogenesis of degenerative diseases of the retina such as Age-related Macular Degeneration. Consistent with such a pathogenic role, high levels of lipofuscin fluorescence are found in the central area of the human RPE, and decline toward the periphery. Recent reports have however suggested a surprising incongruence between the distributions of lipofuscin and A2E in the human RPE, with A2E levels being lowest in the central area and increasing toward the periphery. To appraise such a possibility, we have quantified the levels of A2E in the central and peripheral RPE areas of 10 eyes from 6 human donors (ages 75-91 years) with HPLC and UV/VIS spectroscopy. The levels of A2E in the central area were on average 3-6 times lower than in peripheral areas of the same eye. Furthermore, continuous accumulation of selected ions (CASI) imaging mass spectrometry showed the presence of A2E in the central RPE, and at lower intensities than in the periphery. We have therefore corroborated that in human RPE the levels of A2E are lower in the central area compared to the periphery. We conclude that the levels of A2E cannot by themselves provide an explanation for the higher lipofuscin fluorescence found in the central area of the human RPE.

Figure 1

Spatially oriented flattened human eyecup after dissection and procurement of RPE samples. Donor age, 78 years; left eye. RPE-choroid samples were obtained with a 7.25 mm trephine blade from the central area (C, central) containing the macula, and 8 peripheral areas. Peripheral areas are denoted in terms of the side (I, inferior; N, nasal; S, superior; T, temporal) and proximity to the center (m, mid-; f, far-). The RPE-choroid tissue remaining in the eyecup following removal of the punches wrinkles up, hence the irregular appearance of the leftover holes, even though they are all of the same shape and size.

Figure 2

HPLC chromatograms of RPE-choroid organic extracts from different areas of a human eye. Donor age, 76 years; left eye. (A) Absorbance traces at 430 nm; the section of the trace used to determine A2E levels has been labeled; see text for details. (B) Absorbance traces at 480 nm; same HPLC runs as in (A). fN, far-nasal; mN, mid-nasal; C, central.

Figure 3

Levels of A2E (A) and of total extracted visible light-absorbing pigments (B) in different areas of a human RPE. Same eye as in Fig. 2 (donor age, 76 years; left eye). Levels of A2E and of total visible light-absorbing pigments were determined from the HPLC chromatograms of organic RPE-choroid extracts. A2E and total pigment levels are lowest in the central area of the RPE. Pigment levels, denoted by A-480, were measured as the Area Under the Curve (AUC) of the absorbance trace at 480 nm, are shown in units of absorbance×time (mOD.min). N, nasal; I, inferior.

Figure 4

Correlation between the levels of A2E in corresponding areas of left and right eyes. Donor ages in years: 75 (◇), 76 (□), 86 (◁), and 91 (○). Open symbols represent peripheral areas, filled symbols central areas. To facilitate comparisons, a line with slope 1 has been drawn through the origin. For the 75 year old set of eyes, no data were obtained from the mid-superior, mS, left eye sample, so data for only 8 pairs of samples (1 central; 7 peripheral) are plotted. For each of the other three sets of eyes, 9 pairs of samples are plotted.

Figure 5

Levels of A2E (A) and total extracted visible light-absorbing pigments (B) are lower in the central compared to peripheral areas of human RPE. The levels of A2E and total pigment were normalized to the levels measured from the central area of each RPE. Data are from 10 eyes from 6 different donors (4 pairs and two single eyes). Donor ages (years): 75, 76, 78, 80, 86, 91. The number of samples for each region was n = 10, with the exception of the mid-superior (mS) and far-inferior (fI) regions, for which n = 9. Error bars represent SEM.

Figure 6

Correlation between the levels of extracted non-A2E visible light-absorbing pigments and A2E in the 88 RPE samples analyzed from the 10 eyes of all 6 donors. Both A2E and non-A2E pigment levels were measured from the Area Under the Curve (AUC) of the chromatogram at 480 nm. Linear regression analysis gave R²= 0.62. The regression line has a slope of 2.3.

Figure 7

Imaging mass spectrometry of A2E in human retina cross section. (A) Optical image of 82 year old human retina section on gold MALDI target plate. ON, optic nerve. (B) Overlay of ion images from A2E (m/z 592.45, green) and an unidentified molecule (m/z 590.32, red) over the optical image of the retina. (C) Ion image of A2E (m/z 592.45). (D) Ion image of m/z 590.32. (E) Overlay of ion images from m/z 592.45 (green) and m/z 590.32 (red) showing their presence in distinct retinal cell layers.