Resonance Raman spectroscopy and the preterm infant carotenoid status.

OBJECTIVE: The aim of the study was to validate the noninvasive resonance Raman spectroscopy (RRS) method in infants in comparison with the high-performance liquid chromatography (HPLC) method, and to evaluate the carotenoid status in preterm infants fed with mother's milk or formula. METHODS: In the first phase of the study, resonance Raman measurements were made on male term infants' skin and correlated with tissue harvested at the time of circumcision. Each baby's foreskin was weighed, enzymatically digested, and the total carotenoids were extracted and quantitated by the HPLC. Next, to evaluate the carotenoid status of preterm infants (BW <1500 g), the skin and serum carotenoids in infants fed with either human milk or preterm formula were studied from the start of feedings and every 2 weeks until hospital discharge. Skin carotenoids were measured by RRS and the serum total carotenoids by HPLC. RESULTS: Foreskin carotenoid levels measured by RRS correlated with HPLC measurements of total serum carotenoids (R = 0.52, P < 0.01, n = 16). Forty preterm infants were studied for their carotenoid status. Thirty-two infants were fed mother's milk, whereas 8 were fed a preterm infant formula that was not enriched with carotenoids. The gestation and birth weight of the 2 feeding groups were similar. The infants fed human milk had a higher serum total carotenoid concentration and skin Raman counts than formula-fed infants. The skin Raman counts and total serum carotenoid correlated (R = 0.44, P = 0.01). The human milk-fed infants' serum total carotenoid concentrations and Raman values did not change during the study period; however, the formula-fed group's total serum and skin carotenoid decreased significantly during the study. CONCLUSIONS: RRS of infant's skin reliably assesses total carotenoid status noninvasively. Human milk-fed preterm infants have higher serum and skin carotenoids than formula-fed infants suggesting that formula-fed infants may benefit from carotenoid supplementation.

Microbial carotenoids.

Carotenoids are among the most widely distributed pigments in nature, and they are exclusively synthesized by plants and microorganisms. These compounds may serve a protective role against many chronic diseases such as cancers, age-related macular degeneration, and cardiovascular diseases and also act as an excellent antioxidant system within cells. Recent advances in the microbial genome sequences and increased understanding about the genes involved in the carotenoid biosynthetic pathways will assist industrial microbiologists in their exploration of novel microbial carotenoid production strategies. Here we present an overview of microbial carotenogenesis from biochemical, proteomic, and biotechnological points of view.

Noninvasive assessment of dermal carotenoids as a biomarker of fruit and vegetable intake.

BACKGROUND: Resonance Raman spectroscopy (RRS) has been suggested as a feasible method for noninvasive carotenoid measurement of human skin. However, before RRS measures of dermal carotenoids can be used as a biomarker, data on intra- and intersubject variability and validity are needed. OBJECTIVE: The purpose of this study was to evaluate the reproducibility and validity of RRS measures of dermal total carotenoids and lycopene in humans. DESIGN: In study 1, 74 men and women with diverse skin pigmentation were recruited. RRS measures of the palm, inner arm, and outer arm were obtained at baseline, 1 wk, 2 wk, 1 mo, 3 mo, and 6 mo (to maximize seasonal variation). The RRS device used visible light at 488 nm to estimate total carotenoids and at 514 nm to estimate lycopene. Reproducibility was assessed by intraclass correlation coefficients (ICCs). In study 2, we recruited 28 subjects and assessed dietary carotenoid intake, obtained blood for HPLC analyses, performed RRS measures of dermal carotenoid status, and performed dermal biopsies (3-mm punch biopsy) with dermal carotenoids assessed by HPLC. RESULTS: ICCs for total carotenoids across time were 0.97 (palm), 0.95 (inner arm), and 0.93 (outer arm). Total dermal carotenoids assessed by RRS were significantly correlated with total dermal carotenoids assessed by HPLC of dermal biopsies (r = 0.66, P = 0.0001). Similarly, lycopene assessed by RRS was significantly correlated with lycopene assessed by HPLC of dermal biopsies (r = 0.74, P < 0.0001). CONCLUSION: RRS is a feasible and valid method for noninvasively assessing dermal carotenoids as a biomarker for studies of nutrition and health.

Significant correlations of dermal total carotenoids and dermal lycopene with their respective plasma levels in healthy adults.

Carotenoids in skin have been known to play a role in photoprotection against UV radiation. We performed dermal biopsies of healthy humans (N=27) and collected blood samples for pair-wise correlation analyses of total and individual carotenoid content by high performance liquid chromatography (HPLC). The hydrocarbon carotenoids (lycopene and beta-carotene) made up the majority of carotenoids in both skin and plasma, and skin was somewhat enriched in these carotenoids relative to plasma. Beta-cryptoxanthin, a monohydroxycarotenoid, was found in similar proportions in skin as in plasma. In contrast, the dihydroxycarotenoids, lutein and zeaxanthin, were relatively lacking in human skin in absolute and relative levels as compared to plasma. Total carotenoids were significantly correlated in skin and plasma (r=0.53, p<0.01). Our findings suggest that human skin is relatively enriched in lycopene and beta-carotene, compared to lutein and zeaxanthin, possibly reflecting a specific function of hydrocarbon carotenoids in human skin photoprotection.

Purification and partial characterization of a lutein-binding protein from human retina.

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate lutein's localization to a region of the cell subject to considerable oxidative stress.

Retinal carotenoids can attenuate formation of A2E in the retinal pigment epithelium.

A2E, an important constituent of lipofuscin in human retinal pigment epithelium (RPE), is thought to mediate light-induced oxidative damage associated with aging and other ocular disorders. Ocular carotenoids in overlying retinal tissues were measured by HPLC and mass spectrometry and were correlated with levels of RPE A2E. We observed a statistically significant increase in total A2E levels in human RPE/choroid with age, and A2E levels in macular regions were approximately 1/3 lower than in peripheral retinal regions of the same size. There was a statistically significant inverse correlation between peripheral retina carotenoids and peripheral RPE/choroid A2E. Prospective carotenoid supplementation studies in Japanese quail demonstrated nearly complete inhibition of A2E formation and oxidation. These findings support current recommendations to increase dietary intake of xanthophyll carotenoids in individuals at risk for macular degeneration and highlight a new potential mechanism for their protective effects-inhibition of A2E formation and oxidation in the eye.

Transport and retinal capture of lutein and zeaxanthin with reference to age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population in the western world. The etiology and pathogenesis of this disease remain unclear. However, there is an increasing body of evidence supporting the hypothesis that the macular pigment carotenoids, lutein and zeaxanthin, play an important role in protection against AMD, by filtering out blue light at a pre-receptoral level, or by quenching free radicals. Lutein and zeaxanthin are dietary xanthophyll carotenoids, which are delivered to the retina via plasma lipoproteins. The biological mechanisms governing retinal capture and accumulation of lutein and zeaxanthin, to the exclusion of other carotenoids, are still poorly understood. Although these mechanisms remain unclear, it is possible that selective capture of these carotenoids is related to lipoprotein, or apolipoprotein, function and profile. Xanthophyll-binding proteins appear to play an important role in the retinal capture of the xanthophyll carotenoids. The Pi isoform of GSTP1 has been isolated as a specific binding protein for zeaxanthin. The binding protein responsible for retinal uptake of lutein remains elusive. This article reviews the literature germane to the mechanisms involved in the capture, accumulation and stabilization of lutein and zeaxanthin by the retina, and the processes involved in their transport in serum.

Retinol-binding protein and retinol analysis in cerebrospinal fluid and serum of patients with and without idiopathic intracranial hypertension.

BACKGROUND: Several studies have implicated vitamin A-related compounds in the pathogenesis of idiopathic intracranial hypertension (IIH). The goal of this study was to compare cerebrospinal fluid (CSF) and serum concentrations of retinol and retinol-binding protein (RBP) in subjects with and without IIH. METHODS: CSF and serum samples were collected from 87 subjects. The study population was composed of subjects with IIH (IIH group, n = 28), subjects with non-IIH neurologic conditions (neurology controls, n = 42), and subjects undergoing preoperative lumbar puncture but with no known neurologic conditions (anesthesia controls, n = 17). RBP levels (nM) were determined using radial immunodiffusion, and retinol levels (nM) were determined using high-performance liquid chromatography. RESULTS: The retinol/RBP ratio was greater in CSF than in serum, especially in subjects with IIH. CONCLUSIONS: The finding of increased levels of unbound retinol in the CSF of subjects with IIH provides further evidence that vitamin A may be involved in the pathogenesis of IIH. Comparative statistical analyses revealed multivariate relationships that demonstrate the need to further investigate correlations between vitamin A and RBP levels in CSF and serum.

Evaluation of 4-methylpyrazole as a potential therapeutic dark adaptation inhibitor.

PURPOSE: To investigate whether 4-methylpyrazole (4-MP; fomepizole; Antizol), an alcohol dehydrogenase inhibitor that delays dark adaptation in laboratory animals, is a possible pharmaceutical agent for the treatment of Stargardt disease. METHODS: Healthy adults were given intravenous infusions of either 4-MP or placebo during six weekly visits to assess effects on dark adaptation. RESULTS: Each participant exhibited a linear, rod-and cone-mediated, log-based response during the initial phase of dark adaptation during both placebo and 4-MP sessions. There were no statistically significant differences between the linear slopes of the 4-MP and placebo testing sessions (alpha = 0.05). CONCLUSIONS: 4-MP does not appear to be a sufficient inhibitor of the human visual cycle to be considered further as a clinical treatment for Stargardt disease or similar ocular disorders at this time; however, additional testing of 4-MP inhibition of production of lipofuscin's A2E fluorophore in mouse models of Stargardt disease is still warranted.

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

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.

Identification of 3-methoxyzeaxanthin as a novel age-related carotenoid metabolite in the human macula.

PURPOSE: The xanthophyll carotenoids lutein and zeaxanthin, along with their major metabolites, meso-zeaxanthin, and 3'-oxolutein, are highly concentrated in the human macula. In addition to these two metabolites, there are still others that have not yet been identified. A highly sensitive HPLC-mass spectral method was used to identify and quantify a new xanthophyll metabolite that increases with age. METHODS: Maculae (4-mm diameter) from donor eyes free of ocular disease were procured from the local eye bank. The carotenoid extracts from each tissue sample were analyzed by HPLC coupled with an in-line single quadrupole mass spectrometer in a positive ion atmospheric pressure chemical ionization mode. The elution profile, visible absorption spectra and mass spectra were compared to synthetic standards to identify the ocular carotenoids and their metabolites. RESULTS: Along with 3'-oxolutein and meso-zeaxanthin, a relatively nonpolar zeaxanthin derivative was identified, with m/z 582.5 and spectral properties similar to those of dietary zeaxanthin. This compound was identified as 3-methoxyzeaxanthin (3-MZ) based on elution profile, absorption spectra, and mass spectra in comparison to a synthetic standard. 3-MZ increased with age (P < 0.001) and was not detectable in peripheral retina or in nonretinal tissues. CONCLUSIONS: Identification of 3-MZ in the macula of aged donors indicates that O-methylation of carotenoids is a potential biomarker for aging and age-related ocular disorders.

HPLC measurement of ocular carotenoid levels in human donor eyes in the lutein supplementation era.

PURPOSE: A substantial proportion of the population at risk for visual loss from age-related macular degeneration consumes supplements containing high doses of lutein, but clinical studies to date have shown only modest and variable increases in macular carotenoid pigments in response to supplementation. To determine whether lutein supplementation can indeed alter ocular carotenoid levels, the authors chemically measured levels of lutein, zeaxanthin, and their metabolites in the macula, peripheral retina, and lens of 228 eyes from 147 human donors and correlated these results with retrospective supplement histories from families of selected members of the study population. METHODS: Lenses and circular punches of macula (4-mm diameter) and equatorial peripheral retina (8-mm diameter) were dissected from donor eyes free of ocular disease procured from the local eye bank. The amounts of lutein, zeaxanthin, meso-zeaxanthin, and 3'-oxolutein were determined by HPLC with photodiode array and mass spectral detection. RESULTS: Eighteen percent of eyes from donors age 48 and older had unusually high levels (66.3 +/- 15.1 ng) of macular carotenoids that were three times the rest of the older population's mean level (23.0 + 12.1 ng; P < 0.001). Carotenoid levels in these outliers were also unusually high in the lens and in the peripheral retina. Similar outliers were not present in donors younger than 48. Most of these outliers regularly consumed high-dose lutein supplements before death. Lutein supplementation was uncommon in older donors whose macular carotenoids were in the normal range. CONCLUSIONS: The presence of unusually high levels of macular carotenoids in older donors who were regularly consuming high-dose lutein supplements supports the hypothesis that long-term lutein supplementation can raise levels of macular pigment. Elevated carotenoid levels in the peripheral retina and lens in these same donors could have important implications for understanding why some clinical methods of macular pigment measurement have had difficulty detecting robust and consistent responses in carotenoid supplementation trials.

Vertebrate and invertebrate carotenoid-binding proteins.

In invertebrates and vertebrates, carotenoids are ubiquitous colorants, antioxidants, and provitamin A compounds that must be absorbed from dietary sources and transported to target tissues where they are taken up and stabilized to perform their physiological functions. These processes occur in a specific and regulated manner mediated by high-affinity carotenoid-binding proteins. In this mini-review, we examine the published literature on carotenoid-binding proteins in vertebrate and invertebrate systems, and we report our initial purification and characterization of a novel lutein-binding protein isolated from liver of Japanese quail (Coturnix japonica).

Production of deuterated lutein by Chlorella protothecoides and its detection by mass spectrometric methods.

Chlorella protothecoides, a lutein-producing microalga, was grown aerobically in a mineral medium prepared with 70% (v/v) deuterated water. HPLC/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) analysis revealed approximately 58% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 599. The rapidly growing microalga had much higher levels (58%) of deuterium substitution relative to previously reported (9-15%) natural sources of lutein.

Production of deuterated zeaxanthin by Flavobacterium multivorum and its detection by resonance Raman and mass spectrometric methods.

Flavobacterium multivorum, a zeaxanthin-producing organism, was grown aerobically in a medium prepared with deuterated water. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) and resonance Raman spectroscopy (RRS) analysis revealed approximately 75% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 600. Deuterated zeaxanthin upon excitation with a 488 nm laser exhibited characteristic resonance Raman vibrational modes at 1,161 and 1,504 cm(-1) as compared to 1,007, 1,159 and 1,525 cm(-1 )for undeuterated zeaxanthin. HPLC/APCI-MS and HPLC/RRS were specific and sensitive with limits of detection of 2.5 pg and 50 ng, respectively.

Quantitative measurement of 3'-oxolutein from human retina by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.

3-Hydroxy-beta,epsilon-carotene-3'-one (3'-oxolutein) is the major oxidative metabolite of dietary carotenoids in the retina of the human eye. Elucidating the biochemical mechanism of its formation may provide helpful insight into the pathogenesis of age-related macular degeneration; however, it is found in relatively low quantities that require highly sensitive methods for quantitation from individual retinas. Normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry allowed us to do quantitative analysis of 3'-oxolutein from central and peripheral retinas obtained from individual human donors. The limit of quantification for 3'-oxolutein in human retina at a signal-to-noise ratio of 10 was 6 pg. The precision of the assay yielded a coefficient of variation ranging from 4.7 to 7.4% and accuracies of 106-108%. A statistically significant (R = 0.99, p < or = 0.001) linear working range was achieved between 5 and 7200 pg. The 3'-oxolutein contents from 8-mm punches of the central macula and peripheral retina were found to be 375+/-192 and 191+/-95 pg/tissue, respectively.

Microbial xanthophylls.

Xanthophylls are oxygenated carotenoids abundant in the human food supply. Lutein, zeaxanthin, and cryptoxanthin are major xanthophyll carotenoids in human plasma. The consumption of these xanthophylls is directly associated with reduction in the risk of cancers, cardiovascular disease, age-related macular degeneration, and cataract formation. Canthaxanthin and astaxanthin also have considerable importance in aquaculture for salmonid and crustacean pigmentation, and are of commercial interest for the pharmaceutical and food industries. Chemical synthesis is a major source for the heavy demand of xanthophylls in the consumer market; however, microbial producers also have potential as commercial sources. In this review, we discuss the biosynthesis, commercial utility, and major microbial sources of xanthophylls. We also present a critical review of current research and technologies involved in promoting microbes as potential commercial sources for mass production.

Synergistic effects of zeaxanthin and its binding protein in the prevention of lipid membrane oxidation.

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss due to age-related macular degeneration, but the actual mechanisms of their protective effects are still poorly understood. We have recently purified, identified and characterized a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye which specifically and saturably binds to the two forms of zeaxanthin endogenously found in the foveal region. In this report, we studied the synergistic antioxidant role of zeaxanthin and GSTP1 in egg yolk phosphatidylcholine (EYPC) liposomes using hydrophilic 2,2'-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) and lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) as lipid peroxyl radical generators. The two zeaxanthin diastereomers displayed synergistic antioxidant effects against both azo lipid peroxyl radical generators when bound to GSTP1. In the presence of GSTP1, nondietary (3R,3'S-meso)-zeaxanthin was observed to be a better antioxidant than dietary (3R,3'R)-zeaxanthin. This effect was found to be independent of the presence of glutathione. Carotenoid degradation profiles indicated that the zeaxanthin diastereomers in association with GSTP1 were more resistant to degradation which may account for the synergistic antioxidant effects.

Beta-carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea.

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3' R-beta, beta-carotene-3, 3' diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of beta-carotene (beta, beta-carotene), beta-cryptoxanthin (beta, beta-caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on beta-carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and beta-carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l(-1) urea and sodium carbonate, respectively. The maximum beta-carotene level was 7.85 microg ml(-1) culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific beta-carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that beta-carotene was produced maximally during the late exponential phase at 0.41 microg ml(-1) h(-1). It is possible that this organism could be an excellent commercial source of either beta-carotene or zeaxanthin, depending on initial culture conditions.

Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye.

Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.7. Using mass spectral sequencing methods and the public NCBI database, it was identified as a Pi isoform of human glutathione S-transferase (GSTP1). Dietary (3R,3'R)-zeaxanthin displayed the highest affinity with an apparent Kd of 0.33 microm, followed by (3R,3'S-meso)-zeaxanthin with an apparent Kd of 0.52 microm. (3R,3'R,6'R)-Lutein did not display any high-affinity binding to GSTP1. Other human recombinant glutathione S-transferase (GST) proteins, GSTA1 and GSTM1, exhibited only low affinity binding of xanthophylls. (3R,3'S-meso)-Zeaxanthin, an optically inactive nondietary xanthophyll carotenoid present in the human macula, exhibited a strong induced CD spectrum in association with human macular XBP that was nearly identical to the CD spectrum induced by GSTP1. Like-wise, dietary (3R,3'R)-zeaxanthin displayed alterations in its CD spectrum in association with GSTP1 and XBP. Other mammalian xanthophyll carrier proteins such as tubulin, high-density lipoprotein, low-density lipoprotein, albumin, and beta-lactoglobulin did not bind zeaxanthins with high affinity, and they failed to induce or alter xanthophyll CD spectra to any significant extent. Immunocytochemistry with an antibody to GSTP1 on human macula sections showed highest labeling in the outer and inner plexiform layers. These results indicate that GSTP1 is a specific XBP in human macula that interacts with (3R,3'S-meso)-zeaxanthin and dietary (3R,3'R)-zeaxanthin in contrast to apparently weaker interactions with (3R,3'R,6'R)-lutein.