Identification and metabolic transformations of carotenoids in ocular tissues of the Japanese quail Coturnix japonica

Abstract

As in humans and monkeys, lutein [(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol] and zeaxanthin [a mixture of (3R,3'R)-beta,beta-carotene-3,3'diol and (3R,3'S-meso)-beta,beta-carotene-3,3'-diol] are found in substantial amounts in the retina of the Japanese quail Coturnix japonica. This makes the quail retina an excellent nonprimate small animal model for studying the metabolic transformations of these important macular carotenoids that are thought to play an integral role in protection against light-induced oxidative damage such as that found in age-related macular degeneration (AMD). In this study, we first identified the array of carotenoids present in the quail retina using C30 HPLC coupled with in-line mass spectral and photodiode array detectors. In addition to dietary lutein (2.1%) and zeaxanthin (11.8%), we identified adonirubin (5.4%), 3'-oxolutein (3.8%), meso-zeaxanthin (3.0%), astaxanthin (28.2%), galloxanthin (12.2%), epsilon,epsilon-carotene (18.5%), and beta-apo-2'-carotenol (9.5%) as major ocular carotenoids. We next used deuterium-labeled lutein and zeaxanthin as dietary supplements to study the pharmacokinetics and metabolic transformations of these two ocular pigments in serum and ocular tissues. We then detected and quantitated labeled carotenoids in ocular tissue using both HPLC-coupled mass spectrometry and noninvasive resonance Raman spectroscopy. Results indicated that dietary zeaxanthin is the precursor of 3'-oxolutein, beta-apo-2'-carotenol, adonirubin, astaxanthin, galloxanthin, and epsilon,epsilon-carotene, whereas dietary lutein is the precursor for meso-zeaxanthin. Studies also revealed that the pharmacokinetic patterns of uptake, carotenoid absorption, and transport from serum into ocular tissues were similar to results observed in most human clinical studies.

Figure 1

HPLC chromatogram of saponified and unsaponified retinal extracts from Japanese quail with detection at 450 nm (A). Major identified carotenoids include 1. Adonirubin, 2. 3ε-Oxolutein, 3. Lutein, 4. Astaxanthin, 5. Zeaxanthin and meso-Zeaxanthin, 6. β-apo-2′-Carotenol, 7. Unidentified peak, 8. ε,ε-Carotene, and 9. Galloxanthin. The circled region in the unsaponified chromatogram indicates the presence of esterified carotenoids.

Figure 2

Structures of major identified carotenoids shown in figure 2: 1. Adonirubin, 2 3′-Oxolutein, 3. Lutein, 4. Astaxanthin, 5A. Zeaxanthin, 5B. meso-Zeaxanthin 6. β-apo-2′-Carotenol, 8. ε,ε-Carotene, and 9. Galloxanthin.

Figure 3

Absorbance and mass spectra of major identified carotenoids from saponified preparations: 1. Adonirubin, 2. 3′-Oxolutein, 3. Lutein, 4. Astaxanthin, 5. Zeaxanthins, 6. β-apo-2′-Carotenol, 7. Unidentified peak, 8.ε,ε-Carotene, and 9. Galloxanthin.

Figure 4

Increase and saturation of retinal and serum carotenoids in the supplemented groups (n=4) from 0 to 16 weeks of feeding. Light gray bars with no shading indicate levels of carotenoids in control birds (n=2) at that time point. The difference between control and experimental groups was statistically significant (P < 0.001) after the second month of feeding for all groups except for retinal levels in lutein fed group, which was statistically significant (P < 0.001) after the third month of feeding. L and Z fed groups represent deuterated lutein and zeaxanthin supplemented groups respectively. Black arrows indicate initial detection of deuterated activity in the serum and/or retina.

Figure 5

Mass spectra of undeuterated and deuterated lutein and zeaxanthin from microbial sources and from retinas of quail fed with deuterated carotenoids for 16 weeks. Deuterated lutein and zeaxanthin display isotopomer distribution between m/z 580–605; major mass peaks are labeled in the graphs.

Figure 6

Normalized resonance Raman spectra recorded from living quail eyes. The upper panel shows a spectrum from a bird fed deuterated zeaxanthin for 12 weeks (1) in comparison to a bird fed an undeuterated control diet (2). The lower panel shows spectra of deuterated (3) and undeuterated (4) standards. Note the 20 cm⁻¹ displacement of the C=C stretch peak with deuteration in the standards and in the living eye. See Table 3 for quantitative analysis of the C=C peak height in comparison to HPLC analysis.