Abstract
Purpose :
Zeaxanthin (Z) is a carotenoid that is found in goji berries (Lycium barbarum; LB). Our previous study indicated that LB can attenuate ultraviolet (UV) light-induced cataractogenesis but the amount of Z absorbed by the lens was not measured. This study was undertaken to develop a method to extract Z from lenses incubated with LB solution.
Methods :
Lenses were dissected and cultured 24 - 48 h in medium before being irradiated with UVB light (2.0 J/cm2; 2 hours; n=8). Sham-UV lenses were placed in light-proof boxes before irradiating with UV to account for any heat effects. Six control lenses were never exposed to UV light or heat. Lenses were left in media with no LB (n=3), or with low (0.04% (w/v); n=5), medium (0.14% (w/v); n=6) or high (0.44% (w/v); n=4) levels of LB for 1 month in the dark at 4°C to ensure saturation of LB. Lenses were rinsed then dissolved with isopropyl alchohol before filtering to extract soluble species. Lutein (L) and zeaxanthin (Z) were extracted from a commerically available combined L:Z (20:1 mg) pill for calibration and to confirm the Z extraction method. Z was extracted from each lens then isolated using a differential mobility spectrometer prior to mass spectrometric detection. Culture media were also tested.
Results :
We were able to separately detect L and Z. Z was detected in culture media containing LB but not in media without LB. All lenses showed similar amounts of Z (p = 0.205): 159 ± 46 ng (high LB); 188 ± 43 ng (medium LB); 138 ± 91 ng (low LB); 260 ± 49 ng (no LB). This finding likely reflects detection of endogenous Z. The prior UV condition had no effect on Z amounts in the lens (p=0.413).
Conclusions :
The results from all tests indicated a limit of detection for Z at about 10 ng/ml and a lower limit of quantification at about 100 ng/ml. These limits could be improved through the use of isotopically-labelled L and Z standards. The quantification of Z in the lenses should be interpreted cautiously since the Z levels are close to the lower limit of Z quantification. The observation that (presumably endogenous) levels of Z were present for all lenses suggests that there was no transport of Z across the membranes of the lens, despite the month-long incubation in LB media. This finding might suggest that lenses need to be metabolically active in order to uptake Z. Future work will involve testing metabolically active lenses and expanding the analyses to include lutein and other carotenoids.
This is a 2020 ARVO Annual Meeting abstract.