Prakash Bhosale

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.

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.

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.

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.

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.

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.

Photophysical properties of xanthophylls in carotenoproteins from human retina

Abstract The macula of the human retina contains high amounts of the xanthophyll carotenoids lutein and zeaxanthin [a mixture of (3R,3′R)-zeaxanthin and (3R,3′S-meso)-zeaxanthin]. Recently, it was shown that the uptake and the stabilization of zeaxanthin and lutein into the retina are likely to be mediated by specific xanthophyll-binding proteins (XBP). Here, we have used femtosecond pump–probe spectroscopy to study the dynamics of the S1 state of these xanthophylls in xanthophyll-enriched and native XBP. The results from the native XBP and the enriched XBP were then compared with those for carotenoids in organic solvents and in detergent micelles. Steady-state and transient absorption spectra show that the incorporation of xanthophylls into the protein causes a redshift of the spectra, which is stronger for lutein than for zeaxanthin. The transient absorption spectra further indicate that a part of the xanthophylls remains unbound in the xanthophyll-enriched XBP. The transient absorption spectra of the native XBP prove the presence of both xanthophylls in native XBP. Although the S1 lifetime of lutein does not exhibit any changes when measured in solution, micelles or XBP, we have observed the influence of the environment on the S1 lifetime of meso-zeaxanthin, which has a longer (12 ps) lifetime in XBP than in solution (9 ps). The most pronounced effect was found for vibrational relaxation in the S1 state, which is significantly slower for xanthophylls in XBP compared with micelles and solution. This effect is more pronounced for meso-zeaxanthin, suggesting a specific site of binding of this carotenoid to XBP.

Factorial analysis of tricarboxylic acid cycle intermediates for optimization of zeaxanthin production from Flavobacterium multivorum

Aims:  To study the effect of intermediates of the tricarboxylic acid (TCA) cycle on the production of zeaxanthin from Flavobacterium multivorum in order to optimize production of this xanthophyll carotenoid.

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 mothers 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 babys 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 mothers 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 groups total serum and skin carotenoid decreased significantly during the study. Conclusions: RRS of infants 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.

Production of Deuterated Lutein by Chlorella protothecoides and its Detection by Mass Spectrometric Methods

Chlorellaprotothecoides, 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 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.