Macular and serum carotenoid concentrations in patients with malabsorption syndromes.

The carotenoids lutein and zeaxanthin are believed to protect the human macula by absorbing blue light and quenching free radicals. Intestinal malabsorption syndromes such as celiac and Crohn's disease are known to cause deficiencies of lipid-soluble nutrients. We hypothesized that subjects with nutrient malabsorption syndromes will demonstrate lower carotenoid levels in the macula and blood, and that these lower levels may correlate with early-onset maculopathy. Resonance Raman spectrographic (RRS) measurements of macular carotenoid levels were collected from subjects with and without a history of malabsorption syndromes. Carotenoids were extracted from serum and analyzed by high performance liquid chromatography (HPLC). Subjects with malabsorption (n = 22) had 37% lower levels of macular carotenoids on average versus controls (n = 25, P < 0.001). Malabsorption was not associated with decreased serum carotenoid levels. Convincing signs of early maculopathy were not observed. We conclude that intestinal malabsorption results in lower macular carotenoid levels.

Resonance Raman imaging of macular pigment distributions in the human retina.

We describe resonance Raman imaging (RRI) of macular pigment (MP) distributions in the living human eye. MP consists of the antioxidant carotenoid compounds lutein and zeaxanthin, is typically present in high concentrations in the healthy human macula relative to the peripheral retina, and is thought to protect this important central region from age-related macular degeneration. We demonstrate that RRI is capable of quantifying and imaging the spatially strongly varying MP distribution in the human retina. Using laser excitation of the MP molecules at 488nm, and sequential camera detection of light emitted back from the retina at the MP's strongest Raman peak position and at an off-peak position, RRI maps of MP are obtained at a resolution below 50microm within a fraction of a second per exposure. RRI imaging can be carried out with undilated pupils and provides a highly molecule-specific diagnostic imaging approach for MP distributions in human subjects.

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.

Resonance Raman measurement of macular carotenoids in the living human eye.

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss from age-related macular degeneration. To study this protective effect further, it is important to measure macular carotenoid levels noninvasively in a wide variety of subjects. We have developed and validated resonance Raman spectroscopy as a sensitive and specific objective method to measure macular carotenoid levels in the living human eye. In this minireview, the principles and implementation of ocular carotenoid resonance Raman spectroscopy are reviewed, and the results of observational cross-sectional studies and of prospective supplementation studies on subjects with and without macular pathology are summarized. We have recently extended this technology to an imaging mode which will further enhance our understanding of the roles of lutein and zeaxanthin in normal macular function and in the prevention of age-related visual loss.

Resonance Raman measurement of macular carotenoids in retinal, choroidal, and macular dystrophies.

BACKGROUND: It has been hypothesized that the macular carotenoid pigments lutein and zeaxanthin may protect against macular and retinal degenerations and dystrophies. OBJECTIVE: To test this hypothesis by objectively measuring lutein and zeaxanthin levels in a noninvasive manner in patients who have retinitis pigmentosa (RP), choroideremia (CHM), and Stargardt macular dystrophy and comparing them with an age-matched healthy control population. METHODS: Using resonance Raman spectroscopy, a novel objective noninvasive laser-optical technique, we measured macular carotenoid levels in 30 patients (54 eyes) who have RP, CHM, and Stargardt macular dystrophy and compared them with 76 age-matched subjects (129 eyes) who did not have macular pathologic conditions in a case-control study. RESULTS: As a group, patients with RP and CHM had the same macular carotenoid levels as age-matched healthy control subjects (P =.76, 2-way analysis of variance). Patients with Stargardt macular dystrophy tended to have levels of macular carotenoid pigments that, on average, were about 50% lower than healthy controls (P =.02,unpaired 2-tailed t test). CONCLUSIONS: The patients with RP and CHM had normal levels of macular carotenoids, suggesting that nutritional supplementation with macular carotenoids such as lutein, zeaxanthin, or both will be unlikely to affect the clinical course of RP and CHM. Although the number of patients with Stargardt macular dystrophy examined was limited, their macular carotenoid levels were usually lower than those of subjects of a similar age with no macular pathologic condition.

Chemistry, distribution, and metabolism of tomato carotenoids and their impact on human health.

Recent epidemiological studies have suggested that the consumption of tomatoes and tomato-based food products reduce the risk of prostate cancer in humans. This protective effect has been attributed to carotenoids, which are one of the major classes of phytochemicals in this fruit. The most abundant carotenoid in tomato is lycopene, followed by phytoene, phytofluene, zeta-carotene, gamma-carotene, beta-carotene, neurosporene, and lutein. The distribution of lycopene and related carotenoids in tomatoes and tomato-based food products has been determined by extraction and high-performance liquid chromatography-UV/Visible photodiode array detection. Detailed qualitative and quantitative analysis of human serum, milk, and organs, particularly prostate, have revealed the presence of all the aforementioned carotenoids in biologically significant concentrations. Two oxidative metabolites of lycopene, 2,6-cyclolycopene-1,5-diols A and B, which are only present in tomatoes in extremely low concentrations, have been isolated and identified in human serum, milk, organs (liver, lung, breast, liver, prostate, colon) and skin. Carotenoids may also play an important role in the prevention of age-related macular degeneration, cataracts, and other blinding disorders. Among 25 dietary carotenoids and nine metabolites routinely found in human serum, mainly (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, lycopene, and their metabolites were detected in ocular tissues. In this review we identified and quantified the complete spectrum of carotenoids from pooled human retinal pigment epithelium, ciliary body, iris, lens, and in the uveal tract and in other tissues of the human eye to gain a better insight into the metabolic pathways of ocular carotenoids. Although (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their metabolites constitute the major carotenoids in human ocular tissues, lycopene and a wide range of dietary carotenoids have been detected in high concentrations in ciliary body and retinal pigment epithelium. The possible role of lycopene and other dietary carotenoids in the prevention of age-related macular degeneration and other eye diseases is discussed.

Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models.

PURPOSE: To determine the stereochemistry of carotenoids in human ocular tissues in comparison with plasma and liver and to elucidate the possible transformations of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin in the eye. Similarly, to characterize the carotenoid profiles in the eye tissues, plasma, and liver of quails and frogs to determine whether these can serve as appropriate nonprimate animal models for metabolic studies. METHODS: Configurational isomers of carotenoids and their nondietary by-products from pooled human plasma, liver, retinal pigment epithelium (RPE-choroid), ciliary body, iris, and lens were characterized and quantified by high-performance liquid chromatography (HPLC) on a chiral column. Carotenoids and their nondietary by-products in pooled extracts from quail and frog plasma, liver, retina, RPE-choroid, iris, and lens were similarly characterized and quantified. RESULTS: (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, (3R,3'S; meso)-zeaxanthin, (3R,3'S,6'R)-lutein (3'-epilutein), 3-hydroxy-beta, epsilon -carotene-3'-one, and 5Z- and all-E-lycopene were detected in nearly all human ocular tissues examined. (3R,3'S; meso)-zeaxanthin was not detected in the human plasma and liver but was present in human macula, retina, and RPE-choroid. (3S,3'S)-zeaxanthin was detected in human macula in minute quantities. The carotenoid profiles in quail and frog ocular tissues were somewhat similar to those in humans, with the exception that lycopene was absent. Frog retina, plasma, and liver revealed the presence of (3S,3'S)-zeaxanthin. CONCLUSIONS: The most likely transformations of carotenoids in the human eye involve a series of oxidation-reduction and double-bond isomerization reactions. Quail and frog appear to possess the appropriate enzymes for conversion of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin to the same nondietary by-products observed in humans and thus may serve as excellent nonprimate animal models for metabolic studies.

Resonance Raman measurement of macular carotenoids in normal subjects and in age-related macular degeneration patients.

PURPOSE: Dietary carotenoids lutein and zeaxanthin may play a protective role against visual loss from age-related macular degeneration (AMD) through antioxidant and light screening mechanisms. We used a novel noninvasive objective method to quantify lutein and zeaxanthin in the human macula using resonance Raman spectroscopy and compared macular pigment levels in AMD and normal subjects. DESIGN: Observational study of an ophthalmology clinic-based population. PARTICIPANTS AND CONTROLS: Ninety-three AMD eyes from 63 patients and 220 normal eyes from 138 subjects. METHODS: Macular carotenoid levels were quantified by illuminating the macula with a low-power argon laser spot and measuring Raman backscattered light using a spectrograph. This technique is sensitive, specific, and repeatable even in subjects with significant macular pathologic features. MAIN OUTCOME MEASURE: Raman signal intensity at 1525 cm(-1) generated by the carbon-carbon double-bond vibrations of lutein and zeaxanthin. RESULTS: Carotenoid Raman signal intensity declined with age in normal eyes (P < 0.001). Average levels of lutein and zeaxanthin were 32% lower in AMD eyes versus normal elderly control eyes as long as the subjects were not consuming high-dose lutein supplements (P = 0.001). Patients who had begun to consume supplements containing high doses of lutein (> or =4 mg/day) regularly after their initial diagnosis of AMD had average macular pigment levels that were in the normal range (P = 0.829) and that were significantly higher than in AMD patients not consuming these supplements (P = 0.038). CONCLUSIONS: These findings are consistent with the hypothesis that low levels of lutein and zeaxanthin in the human macula may represent a pathogenic risk factor for the development of AMD. Resonance Raman measurement of macular carotenoid pigments could play an important role in facilitating large-scale prospective clinical studies of lutein and zeaxanthin protection against AMD, and this technology may someday prove useful in the early detection of individuals at risk for visual loss from AMD.

In vivo resonant Raman measurement of macular carotenoid pigments in the young and the aging human retina.

We have used resonant Raman scattering spectroscopy as a novel, noninvasive, in vivo optical technique to measure the concentration of the macular carotenoid pigments lutein and zeaxanthin in the living human retina of young and elderly adults. Using a backscattering geometry and resonant molecular excitation in the visible wavelength range, we measure the Raman signals originating from the single- and double-bond stretch vibrations of the pi-conjugated molecule's carbon backbone. The Raman signals scale linearly with carotenoid content, and the required laser excitation is well below safety limits for macular exposure. Furthermore, the signals decline significantly with increasing age in normal eyes. The Raman technique is objective and quantitative and may lead to a new method for rapid screening of carotenoid pigment levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness in the elderly in the United States.