Verification of Meso-Zeaxanthin in Fish

Abstract

Background/objectives: The carotenoids lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) accumulate in the central retina (the macula), where they are collectively known as macular pigment (MP). MP has been shown to enhance visual function in both diseased and non-diseased retinae, and therefore an understanding and confirmation of, the origins of these carotenoids is needed. Studies have shown that L and Z are present in many foodstuffs found in a typical Western diet (e.g. spinach, kale, peppers, yellow corn and eggs). It has been shown that MZ is generated from L in the primate retina and earlier reports suggested that MZ was present in some fish species. Recently, however, one research group reported that MZ is not present in fish and suggested that the earlier reports showing MZ in these marine species were a methodological artefact. The current study was designed to investigate the reason for the contradiction, and test for the presence of MZ in fish and some other foods.

Methods: Raw fruits, vegetables and fish were extracted for carotenoid analysis by high performance liquid chromatography.

Results: MZ was not detected in any of the fruits or vegetables tested in our study. However, using retention time matching, absorption spectrum comparison, and sample spiking, we verified the presence of MZ in salmon skin, sardine skin, trout skin and trout flesh.

Conclusion: This study confirmed the presence MZ in nature, and in the human food chain.

Keywords: Carotenoids; Fish; Food analysis; Food composition; HPLC; Lutein; Macular pigment; Meso-zeaxanthin; Seafood; Zeaxanthin.

Figure 1. Saponification of Lutein under different saponification conditions

MZ = meso-zeaxanthin (highlighted in red); Z = zeaxanthin; L = lutein; Normal phase chiral conditions were used to obtain the above chromatographs. Absorbance spectra are shown for each peak with the exception of MZ in chromatograph C (no absorption spectrum was obtained for this small MZ peak). MZ peak highlighted in red. A. illustrates a standard mixture of MZ, Z and L which was used for retention time matching as part of MZ identification (see retention times and absorption spectra); B. illustrates a non-saponified L standard; C. illustrates the L standard after mild saponification (5% KOH at 45°C overnight); D. illustrates the L standard after intense saponification (10% KOH at 120°C overnight); Note: only following intense saponification of the standard was L converted to MZ, reflected in an altered MZ:L %ratio

Figure 2. Chromatography illustrating the presence of MZ in several fish species tested

MZ = meso-zeaxanthin (highlighted in red); Z = zeaxanthin; L = lutein; Normal phase chiral conditions were used to obtain the above chromatographs. Absorbance spectra are shown for the MZ peak. A, D, G and J = chromatography for non-saponified samples showing ester peaks obtained from salmon skin, sardine skin, trout skin, and trout flesh, respectively; B, E, H and K = chromatography for saponified samples (5% KOH at room temperature overnight) showing MZ peaks obtained from salmon skin, sardine skin, trout skin, and trout flesh, respectively; C, F, I and L = chromatography for saponified samples (5% KOH at room temperature overnight) co-eluted (spiked) with MZ standard showing increased MZ peaks obtained from salmon skin, sardine skin, trout skin, and trout flesh, respectively