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. 2023 May 4;299(6):104784. doi: 10.1016/j.jbc.2023.104784

Figure 1.

Figure 1

Quantitation of bisretinoids in the eyes of diet-induced obese (high fat diet) mice and mice fed a standard diet (control). Male black C57BL/6J and male albino C57BL/6Jc2j mice with ages as indicated in months (m). Bisretinoids A2E, iso-A2E, and A2-DHP-PE (A2-dihydropyridine-phosphatidylethanolamine) were quantified by HPLC. A2-GPE (A2-glycerophosphoethanolamine) was quantified using UPLC chromatograms. A, representative reverse phase HPLC chromatograms illustrating the detection of A2E, iso-A2E, and A2-DHP-PE (left). A2-GPE was detected by UPLC (right); age 6 months. Absorbance monitored at 430 nm; retention time in minutes. (Insets) UV/visible absorbance spectra corresponding to indicated peaks. B, chromatographic quantitation of bisretinoids. The value for A2E represents the sum of all-trans and cis-isomers; age 6 months. Individual values and means ± SD are plotted, n = 4. Each value is based on pooling of 1 to 7 eyes/sample. C and D, body weights (g) (C) and blood glucose (mg/dl) (D) in diet-induced obese (high fat diet, HFD) mice and mice fed a standard diet (control). Male albino C57BL/6Jc2j mice, age 12 months. Values are mean ± SD, n = 16. p values were determined by unpaired two-tailed t test. E, chromatographic quantitation of bisretinoids A2E, A2-GPE, and A2-DHP-PE at age 12 months. Individual values and means ± SD are plotted, n = 4. Each plotted value represents pooling of four eyes. F, quantitation of SW-AF (short-wavelength fundus autofluorescence; 488 nm excitation) in HFD-fed and standard diet mice; age 13 months. Individual values and means ± SD are plotted, n = 4. G, photoreceptor cell viability measured as outer nuclear layer (ONL) thickness in albino C57BL/6Jc2j mice fed the HFD from age 6 weeks to 13 months. ONL thicknesses (mean ± SEM) are plotted as distance from the optic nerve head (ONH), n = 8 eyes. H, chromatographic quantitation of bisretinoids (A2E, A2-DHP-PE, A2-GPE, and atRALdi-PE (all trans retinal dimer-phosphatidylethanolamine)); age 36 months (m). Two to ten eyes/each plotted value presented as pmoles/eye. Individual values and means ± SD are plotted; n = 2 to 4. Values for A2E are the sum of all-trans-A2E and cis-isomers of A2E (B, E, and H). p values determined by two-way ANOVA and Sidak’s multiple comparison test (B, E, and H) or unpaired two-tailed t test (C, D, and F). Extraction methods A (B, H) and B (E) were used. PE, phosphatidylethanolamine; UPLC, ultra-performance liquid chromatography.