These results suggest that supplementation with all three macular carotenoids potentially offered advantages over preparations lacking MZ, both in terms of MPOD response and visual performance enhancement.

PURPOSE. The three carotenoids lutein, zeaxanthin, and mesozeaxanthin, are found at the macula and referred to as macular pigment (MP). This study was undertaken to investigate determinants of MP in a large randomly selected sample from the Republic of Ireland (as part of The Irish Longitudinal Study on Ageing [TILDA]).METHODS. MP optical density (MPOD) was measured using customized heterochromatic flicker photometry in 4373 participants. Sociodemographic and self reported health data was obtained using computer assisted personal interview (CAPI). RESULTS. Mean (SD) MPOD

Background/ObjectivesThe 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.MethodsRaw fruits, vegetables and fish were extracted for carotenoid analysis by high performance liquid chromatography.ResultsMZ 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.ConclusionThis study confirmed the presence MZ in nature, and in the human food chain.

Sir, Response to Bernstein et alWe welcome the letter by Bernstein et al 1 in response to our publication 'What is meso-zeaxanthin, and where does it come from?' in Eye 2013. 2 In their letter, Bernstein and colleagues argue that our review article contains 'several critical errors that need to be considered. ' Bernstein and colleagues endeavour to make their points under the following headings:1. Quantitation of xanthophylls using reverse-and normal-phase HPLC. 2. The role of saponification in the quantitation of xanthophylls in food and supplements. 3. Meso-zeaxanthin in lutein supplements.4. Additional evidence supporting lutein as the precursor of meso-zeaxanthin.In our letter below, we reply directly to these points in normal font. Statements made by Bernstein and colleagues are presented in bold font for clarity.1. Quantitation of xanthophylls using reverse-and normal-phase HPLC. 'Nolan et al argue that the two-step HPLC method used for MZ quantitation by Johnson et al is limited because of the labor involved in the manual collection of the total Z þ MZ fraction in the first step. The authors suggest that this process is prone to human error, that only a portion of the Z þ MZ fraction would be collected, and that this fraction typically is contaminated with L carryover. ' We thank Bernstein et al for summarising the two-step method in their correspondence, commonly used for quantifying MZ. We are very familiar with this method, as we have used it in several of our recently published studies. [3][4][5][6] In our review article, we point out the limitations of the standard 'two-step method' commonly used by many laboratories to quantify MZ. These limitations include the following: its labour intensive nature due to manual collection; operator dependency and potential for human error; and a very long sample run time, rendering it difficult to perform bulk analysis (eg, for clinical trials). Our concerns with respect to the traditional 'two-step method' remain, and we believe that it is important to recognise these limitations when discussing published methodology and findings from papers, and that is why we included these points in our review.Bernstein et al premise their defence of the methodology of carotenoid quantification in the paper by Johnson et al 7 on the basis that:'The fact that L, MZ and Z appear on the subsequent normal-phase, chiral column chromatogram verifies that the desired peaks were collected, and this was also confirmed by absorption spectra.'Bernstein et al attempt to address our concerns with respect to the unknown peak that was found to co-elute with the Z fractions of retinal samples in the report by Johnson et al 7 by stating that 'ythe peak also appeared in the reverse phase HPLC of retinal samples from the carotenoid-free monkeys. ' We agree that identifying the peaks and confirming their presence by assessing their absorbance spectra are important. However, it is clear from the Johnson et al 7 paper that the already challenging method used to analyse MZ was made more diffic...

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