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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis.

A rapid nondestructive estimation of carotenoid levels in intact fruits and vegetables and their juices could have great value when selecting nutritionally valuable crops for further propagation and commercial use. Carotenoid levels of a variety of agricultural products and juices were measured using resonance Raman spectroscopy and compared to levels determined by extraction and high-pressure liquid chromatography. A strong correlation was observed between the two methods when evaluating juices and when comparing different strains of intact tomatoes at the same stage of ripening.

Photophysical properties of xanthophylls in carotenoproteins from human retinas.

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.

Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum.

Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238. Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm(-1) (C-C stretch) and 1525 cm(-1) (C=C stretch) upon excitation at 488 nm. A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.