A Novel Source of Methylglyoxal and Glyoxal in Retina: Implications for Age-Related Macular Degeneration

Abstract

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.

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

“…The end products of photodegradation of lipofuscin bisretinoids are a variety of reactive aldehydes and 3-carbon bifunctional fragments carrying two aldehydes (GO) or an aldehyde and a ketone (MG) (7,8). The configuration of these fragments is consistent with the known polyene structures of bisretinoids (63).…”

“…By employing liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), we identified MG and GO along with other aldehyde-bearing fragments within the photo-fragments released by the irradiation of the bisretinoids A2E and all-transretinal dimer. We trapped MG by derivatization with 4-nitrophenylhydrazine, and we showed that bisretinoid photo-cleavage would occur at sites of singlet molecular oxygen addition within the polyenic structures of the bisretinoid molecules A2E and all-trans-retinal dimer (7,8). We also demonstrated that these photofragments can be released from cultured cells into the extracellular milieu.…”

“…We have demonstrated that a previously unknown source of MG and GO is the photooxidation-induced photodegradation of the vitamin A aldehyde-derived molecules, including A2E (adduct of two vitamin A aldehyde and ethanolamine) and alltrans-retinal dimer, that accumulate in retinal pigment epithelial (RPE) cells as bisretinoid constituents of RPE lipofuscin (7,8). By employing liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), we identified MG and GO along with other aldehyde-bearing fragments within the photo-fragments released by the irradiation of the bisretinoids A2E and all-transretinal dimer.…”

Lipofuscin Accumulation into and Clearance from Retinal Pigment Epithelium Lysosomes: Physiopathology and Emerging Therapeutics

“…Cellular photosensitivity is proportional to the amount of LBs accumulated [42] and the wavelength, with a maximum at 430 nm (blue light), which coincides with the excitation spectrum of the LBs [43]. The absorption of blue photons by the LBs' extended double bond conjugated system, in the presence of oxygen, leads to the formation of oxidized LB species [44,45] that after repetitive oxidative atacks become fragmented into far reaching, highly reactive, carbonyl bearing small molecules [43,44,[46][47][48]. These fragments promote cell damage by forming Schif base adducts with free amine groups in lysosomal hydrolases, nucleotides, phospholipids, lipids, proteins [49], DNA [50], proteasomes [51], and molecules in extracellular retinal deposits (drusen), which could trigger local innate and adaptive immune responses [52,53].…”

Vitamin A-aldehyde adducts: AMD risk and targeted therapeutics

“…The toxicity of these vitamin A-aldehyde adducts is likely attributable to their propensity to photogenerate reactive oxygen species and to photodecompose into aldehyde-and dicarbonyl-(glyoxal and methylglyoxal) bearing fragments (23,24). Evidence that proteins modified by the same dicarbonyls are detected in drusen, is indicative of a link between photodegradation of RPE lipofuscin and sub-RPE aging changes that confer risk of AMD.…”