Topical application of L-arginine blocks advanced glycation by ascorbic acid in the lens of hSVCT2 transgenic mice

Abstract

Purpose: Previous experiments from our laboratory showed that the oral intake of selected guanidino compounds could block the formation of crystallin-bound advanced ascorbylation products. Here we tested whether these were also active when applied as eye drops.

Methods: Two month old hSVCT2 transgenic mice (n=10) were treated twice daily with one drop of 0.1% L-arginine, γ-guanidinobutyric acid (GBA), penicillamine (PA) or N-acetylcysteine (NAC) in one eye and vehicle only in the other eye. After seven months, lens crystallins were isolated, dialyzed, and proteolytically digested to determine the protein-bound fluorescence at 335/385 and 370/440 nm excitation/emission and the advanced glycation/ascorbylation endproducts carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), glucosepane, glyoxal, and methylglyoxal hydroimidazolones G-H1 and MG-H1. The topical uptake of L-arginine and NAC was also evaluated in vitro and in vivo in rabbit lens.

Results: In hSVCT2 mice, L-arginine decreased 335/385 and 370/440 nm fluorescence by 40% (p<0.001), CML, CEL, and glucosepane crystallin crosslinks by 35% (p<0.05), 30% (p<0.05), and 37% (p<0.05), respectively, without affecting MG-H1 and G-H1. NAC decreased 335/385 nm fluorescence by 50% (p<0.001) but, like PA and GBA, had no effect on other modifications. L-Arginine uptake into rabbit eyes treated topically reached identical lenticular plateau levels (~400 nmol/g wet weight) at 0.5% and 2.0% but levels remained three times higher at 5 h at 2% versus 0.5% concentration, respectively. In vitro studies showed a 100 fold higher L-arginine level than NAC levels, implicating high affinity uptake of the former.

Conclusions: L-Arginine when applied both orally and topically is a potent and broad suppressor of advanced ascorbylation in the lens. Its uptake in rabbit lens upon topical application suggests transcorneal uptake into the human lens should be feasible for testing its potential anticataract properties in clinical trials.

Figure 1

Structure of AGEs identified in the aging human lens.

Figure 2

Levels of protein-bound fluorescence in transgenic mouse lens protein with and without inhibitor treatment. A: Fluorescence at λ_ex/em 335/385 nm and B: Fluorescence at λ_ex/em 370/440 nm. One-way ANOVA was used followed by post-hoc analysis for all comparisons (n=10 per group). L-Arginine (ARG) significantly reduced fluorescence at 335/385 nm (p<0.001) and 370/440 nm (p<0.001). N-acetylcysteine (NAC) significantly reduced the fluorescence at 335/385 nm (p<0.001). GBA=guanidinobutyric acid, PA=penicillamine.

Figure 3

Additional AGE levels in mouse lens with or without inhibitor eye drop treatment. A: Mouse lens protein CML levels were significantly reduced by L-arginine (p<0.05). B: Mouse lens protein CEL levels were significantly reduced by L-arginine (p<0.05). C: Mouse lens protein GH1 were not affected by inhibitors (p=N.S.) versus vehicle control. D: Mouse lens protein MG-H1 levels were not affected by inhibitors (p=N.S) versus vehicle control. E: Mouse lens protein glucosepane levels were significantly reduced by L-arginine (p<0.05). One-way ANOVA was used followed by post-hoc analysis for all comparisons (n=10 per group). For abbreviations, see Figure 1.

Figure 4

Comparative uptake of L-arginine and N-acetyl-L-cysteine in rabbit lenses (n=2) incubated with 5 mM L-arginine and 5 mM NAC with and without presence of 25 mM D-glucose or 5 mM ascorbic acid and 0.1 mM dehydroascorbic acid for 4 h. The lenses were washed with cold PBS and homogenized in water for L-arginine and NAC determination in supernatant by LC/MS.

Figure 5

Uptake kinetics of L-Arginine and NAC in rabbit lens. Two rabbits in each group were topically applied with 0.5% inhibitor on right eye and 2% inhibitor on the left eye. The inhibitors were quantified by LC-MS in total lens extract. A: 0.5% L-arginine eye drop; B: 2% L-arginine eye drop; C: 0.5% NAC eye drop; D: 2% NAC eye drop.