Raman spectroscopic evidence for nuclear disulfide in isolated lenses of hyperbaric oxygen-treated guinea pigs

Abstract

Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O 2 -treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90° geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of -SS-(503 cm −1 ) and -SH (2577 cm −1 ) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS-and -SH regions. -SS-levels in the O 2 -treated nucleus were found to have increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the -SSincrease was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. -SH levels in the O 2 -treated nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in -SS-or loss of -SH was observed in the lens cortex of the O 2 -treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of -SS-and loss of -SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O 2 , disulfidecrosslinking of lens crystallins in the nucleus, and nuclear cataract.

The LEGSKO Mouse: A Mouse Model of Age-Related Nuclear Cataract Based on Genetic Suppression of Lens Glutathione Synthesis

“…gamma-glutamyl transpeptidase deficient mice develop nuclear opacities at 1 week secondary to cysteine deficiency [22], or that treatment with buthionine sulfoxide (BSO) results in lowered GSH levels and cataract in newborn rats [23] and very young mice [24] . Vice-versa, oxidative stress from hyperbaric oxygen duplicates many changes observed in the aging human lens [25]. Knocking out glutathione peroxidase (GPX-1) in mice leads to cataract formation [26], whereby changes in lens epithelial cell morphology and DNA breaks were also found under oxidant stress generated by either hydrogen peroxide (H 2 O 2 ) treatment or GPX-1 null condition [27].…”

Oxidative Damage and the Prevention of Age-Related Cataracts

“…Importantly, these refractive changes regressed after the discontinuation of short-term therapy, suggesting that the increase in oxidation caused by hyperbaric oxygen is initially reversible. Subsequent studies in experimental animals confirmed the effect of high oxygen exposure on nuclear opacification and provided insight into some of the harmful effects of oxygen on the lens, including increased formation of protein-protein disulfide cross-links [57,[67][68][69][70] . These studies provide a direct link between excessive oxygen exposure and nuclear cataract.…”

Surface enhanced Raman spectroscopic direct determination of low molecular weight biothiols in umbilical cord whole blood

“…14,15 Because of that there is growing interest in the use of Raman and SERS for the detection of biothiols and GSH in biological samples. Raman spectroscopy was employed to study the changes in the intensity of S-H and S-S bonds in the lenses of guinea pigs upon treatment 17 as well as for studying oxidative stress in trapped erythrocytes. 18 SERS has been previously employed for improving the sensitivity of Raman detection of GSH by relying on the bonding of the thiol group to Au or Ag nanoparticles (NPs).…”