Mohsen Sharifzadeh

Resonance Raman detection of carotenoid antioxidants in living human tissue

Increasing evidence points to the beneficial effects of carotenoid antioxidants in the human body. Several studies, for example, support the protective role of lutein and zeaxanthin in the prevention of age-related eye diseases. If present in high concentrations in the macular region of the retina, lutein and zeaxanthin provide pigmentation in this most light sensitive retinal spot, and as a result of light filtering and/or antioxidant action, delay the onset of macular degeneration with increasing age. Other carotenoids, such as lycopene and beta-carotene, play an important role as well in the protection of skin from UV and short-wavelength visible radiation. Lutein and lycopene may also have protective function for cardiovascular health, and lycopene may play a role in the prevention of prostate cancer. Motivated by the growing importance of carotenoids in health and disease, and recognizing the lack of any accepted noninvasive technology for the detection of carotenoids in living human tissue, we explore resonance Raman spectroscopy as a novel approach for noninvasive, laser optical carotenoid detection. We review the main results achieved recently with the Raman detection approach. Initially we applied the method to the detection of macular carotenoid pigments, and more recently to the detection of carotenoids in human skin and mucosal tissues. Using skin carotenoid Raman instruments, we measure the carotenoid response from the stratum corneum layer of the palm of the hand for a population of 1375 subjects and develop a portable skin Raman scanner for field studies. These experiments reveal that carotenoids are a good indicator of antioxidant status. They show that people with high oxidative stress, like smokers, and subjects with high sunlight exposure, in general, have reduced skin carotenoid levels, independent of their dietary carotenoid consumption. We find the Raman technique to be precise, specific, sensitive, and well suitable for clinical as well as field studies. The noninvasive laser technique may become a useful method for the correlation between tissue carotenoid levels and risk for malignancies or other degenerative diseases associated with oxidative stress.

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 luteins localization to a region of the cell subject to considerable oxidative stress.

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 MPs 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.

Macular Pigment Imaging in AREDS2 Participants: An Ancillary Study of AREDS2 Subjects Enrolled at the Moran Eye Center

PURPOSE Age-Related Eye Disease Study 2 (AREDS2) is a randomized, placebo-controlled study designed to determine whether supplementation with 10 mg of lutein and 2 mg of zeaxanthin per day can slow the rate of progression of age-related macular degeneration (AMD). Although some biomarkers of response to carotenoid supplementation such as serum concentrations are part of the AREDS2 protocol, measurement of carotenoid concentrations in the eye and other tissues is not. In this approved ancillary study, macular pigment optical density (MPOD), macular pigment distributions, and skin carotenoid levels at enrollment and at each annual visit were measured to assess baseline carotenoid status and to monitor response to assigned interventions. METHODS All subjects enrolled at the Moran Eye Center had MPOD and macular pigment spatial distributions measured by dual-wavelength autofluorescence imaging and total skin carotenoids measured by resonance Raman spectroscopy. Results. Baseline MPOD in enrolled subjects was unusually high relative to an age-matched control group that did not consume carotenoid supplements regularly, consistent with the high rate of habitual lutein and zeaxanthin consumption in Utah AREDS2 subjects prior to enrollment. MPOD did not correlate with serum or skin carotenoid measurements. CONCLUSIONS Useful information is provided through this ancillary study on the ocular carotenoid status of AREDS2 participants in the target tissue of lutein and zeaxanthin supplementation: The macula. When treatment assignments are unmasked at the conclusion of the study, unique tissue-based insights will be provided on the progression of AMD in response to long-term, high-dose carotenoid supplementation versus diet alone. (ClinicalTrials.gov number, NCT00345176.).

Blue-light reflectance imaging of macular pigment in infants and children.

PURPOSE While the role of the macular pigment carotenoids in the prevention of age-related macular degeneration has been extensively studied in adults, comparatively little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital video fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children. METHODS We used blue light reflectance to image the macular pigment in premature babies at the time of retinopathy of prematurity (ROP) screening and in children aged under 7 years who were undergoing examinations under anesthesia for other reasons. We correlated the MPOD with skin carotenoid levels measured by resonance Raman spectroscopy, serum carotenoids measured by HPLC, and dietary carotenoid intake. RESULTS We enrolled 51 infants and children ranging from preterm to age 7 years. MPOD correlated significantly with age (r = 0.36; P = 0.0142), with serum lutein + zeaxanthin (r = 0.44; P = 0.0049) and with skin carotenoid levels (r = 0.42; P = 0.0106), but not with dietary lutein + zeaxanthin intake (r = 0.13; P = 0.50). All premature infants had undetectable macular pigment, and most had unusually low serum and skin carotenoid concentrations. CONCLUSIONS Our most remarkable finding is the undetectable MPOD in premature infants. This may be due in part to foveal immaturity, but the very low levels of serum and skin carotenoids suggest that these infants are carotenoid insufficient as a consequence of low dietary intake and/or severe oxidative stress. The potential value of carotenoid supplementation in the prevention of ROP and other disorders of prematurity should be a fruitful direction for further investigation.

Interrelationships Between Maternal Carotenoid Status and Newborn Infant Macular Pigment Optical Density and Carotenoid Status

PURPOSE Deposition of the macular pigment carotenoids lutein and zeaxanthin in the human retina occurs early in life. In this study, we examined the interrelationships of maternal carotenoid status and newborn infant macular pigment levels and systemic carotenoid status. As a secondary measure, we also evaluated the effects of intrauterine growth restriction (IUGR) on carotenoid status in term newborn infants. METHODS We measured mother and infant skin carotenoids using resonance Raman spectroscopy (RRS), serum carotenoids by HPLC, and mother breast milk carotenoids by HPLC. We measured infant macular pigment levels using noninvasive blue light reflectometry. RESULTS We enrolled 30 healthy term infants, their mothers, and 10 IUGR infants and their mothers. A subset of 16 infants was imaged for macular pigment optical density (MPOD). Infant serum zeaxanthin levels correlated with MPOD (r = 0.68, P = 0.007). Mother serum zeaxanthin levels correlated with infant MPOD (r = 0.59, P = 0.032). Infant and mother serum lutein did not correlate with MPOD. Mother-infant correlations were found for total serum carotenoids (r = 0.42, P = 0.020) and skin carotenoids (r = 0.48, P = 0.001). No difference was seen between IUGR infants and controls in total serum or skin carotenoids. Mothers of IUGR infants had lower total serum carotenoids (P = 0.019) and breast milk carotenoids than controls (P = 0.006). CONCLUSIONS Our findings suggest that maternal zeaxanthin status may play a more important role than lutein status in macular pigment deposition in utero. Controlled trials are needed to determine whether maternal zeaxanthin prenatal supplementation can raise infant macular pigment levels and/or improve ocular function.

MACULAR PIGMENT DISTRIBUTION RESPONSES TO HIGH-DOSE ZEAXANTHIN SUPPLEMENTATION IN PATIENTS WITH MACULAR TELANGIECTASIA TYPE 2

Purpose: To analyze macular pigment (MP) amount and distribution in patients with macular telangiectasia Type 2 receiving oral zeaxanthin supplementation in a randomized, open-label, interventional trial. Methods: Eight macular telangiectasia Type 2 patients were randomized to 10 mg or 20 mg of zeaxanthin per day. At each visit, best-corrected visual acuity, contrast sensitivity, fundus biomicroscopy, color fundus photography, autofluorescence imaging, optical coherence tomography, and serum carotenoid levels were tested. Patients were assessed at baseline and after 6, 12, 18, and 24 months of zeaxanthin supplementation. Concentration of MP was analyzed and calculated from autofluorescence imaging obtained at 488-nm excitation wavelength. Serum carotenoid levels were obtained using high-performance liquid chromatography. Results: The majority of patients had definite increases in the intensity of hypofluorescent ring of MP, but none of them deposited MP centrally at the fovea. Although some patients noted subjective improvements in vision, no objective improvements could be documented, and there were no changes in foveal optical coherence tomographic features. Yellowish, hypofluorescent crystals appeared in one patients macular region with no change in visual acuity. These inner retinal crystals disappeared several months after discontinuing her 20-mg zeaxanthin supplement. Conclusion: Based on the current study, zeaxanthin supplementation does not result in any visual benefit in patients with macular telangiectasia Type 2 and does not reestablish a normal peaked distribution of MP in the fovea. One patient developed a novel, reversible, crystalline maculopathy in response to zeaxanthin supplementation that was reminiscent of canthaxanthin crystalline maculopathy.

Autofluorescence imaging of macular pigment: influence and correction of ocular media opacities.

Abstract. The healthy adult human retina contains in its macular region a high concentration of blue-light absorbing carotenoid compounds, known as macular pigment (MP). Consisting of the carotenoids lutein, zeaxanthin, and meso-zeaxanthin, the MP is thought to shield the vulnerable tissue layers in the retina from light-induced damage through its function as an optical attenuator and to protect the tissue cells within its immediate vicinity through its function as a potent antioxidant. Autofluorescence imaging (AFI) is emerging as a viable optical method for MP screening of large subject populations, for tracking of MP changes over time, and for monitoring MP uptake in response to dietary supplementation. To investigate the influence of ocular media opacities on AFI-based MP measurements, in particular, the influence of lens cataracts, we conducted a clinical trial with a large subject population (93 subjects) measured before and after cataract surgery. General AFI image contrast, retinal blood vessel contrast, and presurgery lens opacity scores [Lens Opacities Classification System III (LOCS III)] were investigated as potential predictors for image degradation. These clinical results show that lens cataracts can severely degrade the achievable pixel contrasts in the AFI images, which results in nominal MP optical density levels that are artifactually reduced. While LOCS III scores and blood vessel contrast are found to be only a weak predictor for this effect, a strong correlation exists between the reduction factor and the image contrast, which can be quantified via pixel intensity histogram parameters. Choosing the base width of the histogram, the presence or absence of ocular media opacities can be determined and, if needed, the nominal MP levels can be corrected with factors depending on the strength of the opacity.

Correlations Between Macular, Skin, and Serum Carotenoids

Purpose Ocular and systemic measurement and imaging of the macular carotenoids lutein and zeaxanthin have been employed extensively as potential biomarkers of AMD risk. In this study, we systematically compare dual wavelength retinal autofluorescence imaging (AFI) of macular pigment with skin resonance Raman spectroscopy (RRS) and serum carotenoid levels in a clinic-based population. Methods Eighty-eight patients were recruited from retina and general ophthalmology practices from a tertiary referral center and excluded only if they did not have all three modalities tested, had a diagnosis of macular telangiectasia (MacTel) or Stargardt disease, or had poor AFI image quality. Skin, macular, and serum carotenoid levels were measured by RRS, AFI, and HPLC, respectively. Results Skin RRS measurements and serum zeaxanthin concentrations correlated most strongly with AFI macular pigment volume under the curve (MPVUC) measurements up to 9° eccentricity relative to MPVUC or rotationally averaged macular pigment optical density (MPOD) measurements at smaller eccentricities. These measurements were reproducible and not significantly affected by cataracts. We also found that these techniques could readily identify subjects taking oral carotenoid-containing supplements. Conclusions Larger macular pigment volume AFI and skin RRS measurements are noninvasive, objective, and reliable methods to assess ocular and systemic carotenoid levels. They are an attractive alternative to psychophysical and optical methods that measure MPOD at a limited number of eccentricities. Consequently, skin RRS and MPVUC at 9° are both reasonable biomarkers of macular carotenoid status that could be readily adapted to research and clinical settings.

Reflection-based imaging of macular pigment distributions in infants and children

Abstract. We have developed a reflection-based capability of the RetCam® platform, an FDA-cleared pediatric retinal-imaging instrument, for the purpose of measuring macular pigment levels as well as their spatial distributions in infants and children. Our modifications include narrow-band blue-wavelength excitation of the macular pigment absorption in combination with spectrally selective blue-wavelength readout of the reflection signals received by the instrument’s CCD detector array. Furthermore, an algorithm is developed that allows the computation of optical density maps for the macular pigment relative to peripheral retinal areas. This made it possible for the first time to directly measure macular pigment levels and their spatial features in the developing human retina. In contrast to adults, infants with measurable pigment levels had almost exclusively a narrow, circularly symmetric, pigment distribution. The described methodology holds promise for future investigations into the role of macular pigment in the developing human retina and the effect of dietary interventions in diseases resulting from a lack of normal carotenoid levels.