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Separation of carotenol fatty acid esters by high-performance liquid chromatography.

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.

Carotenoids↗

Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach.

An experimental design procedure was used to investigate the effects of some operating parameters on the supercritical fluid extraction of carotenoids beta-carotene, beta-cryptoxanthin and zeaxanthin from Spirulina Pacifica algae, a carotenoid-rich dietary product. Variables tested were temperature and pressure of the supercritical fluid, dynamic extraction time and percentage of ethanol added as the modifier. Each variable was tested at three levels; 31 experiments were performed in random order. Analyses of the extracts were performed by high-performance liquid chromatography with UV-Vis photodiode array detection. Analytical responses (chromatographic peak areas) were processed by using a stepwise multiple regression analysis, in order to find polynomial functions describing the relationships between variables and responses. For all the analytes the experimental conditions providing the highest extraction yield inside the experimental domain considered were found. Supercritical fluid extraction results obtained in these conditions were compared with those obtained by performing solvent extraction in order to evaluate the effectiveness of the supercritical fluid extraction procedure.

Carotenoids↗

Separation and quantitative analysis of some carotenoid fatty acid esters of fruits by liquid chromatography.

The adsorption and partition properties of several fatty acid esters of capsanthin, capsorubin, beta-cryptoxanthin, lutein, violaxanthin and beta-citraurin isolated from fruits were studied by normal-phase (silica) and reversed-phase (octadecylsilane) liquid chromatography using Sudan 1 as internal standard. The separation on a normal phase was based on the functional group of the carotenoids and individual esters of the same carotenoid did not resolve. The separation on a reversed phase was more dependent on the number of acyl carbons than the functional group, and individual esters of the same carotenoid differing only two acyl carbons were separated with a resolution of 3. There was a linear relationship between number of acyl carbons and retention times of the same carotenoid on reversed phase. The separation on a normal phase was the reverse of that on a reversed phase, and a combination of normal-phase followed by reversed-phase chromatography was used for the separation of esters with the same or close retention times.

Carotenoids↗

Separation of serum carotenoids and vitamin A on chromsil-amino and -cyano phases by a bi-directional gradient elution technique.

Vitamin A, beta-carotene, lycopene, beta-cryptoxanthin, lutein and zeaxanthin can be simultaneously separated in human serum extracts by normal-phase liquid chromatography. Two stationary phases (containing amino and cyano groups, respectively) were investigated. With a bi-directional (up-down) gradient-elution technique both packings are useful for the determination of serum levels of carotenoids and retinol in serum extracts. The small sample size, the simplicity of extraction, and a good reproducibility render these procedures ideal for clinical use.

Carotenoids↗

Measurements of the major isoforms of vitamins A and E and carotenoids in the blood of people with spinal-cord injuries.

We used reversed-phase HPLC with diode array detection to simultaneously measure the major isoforms of vitamins A, E, and the carotenoids in serum from 55 healthy people with spinal cord injuries. Typically, the method measured retinol (vitamin A), alpha-tocopherol (vitamin E) and beta-carotene, alpha-carotene, lutein, lycopene, and cryptoxanthin (carotenoids). gamma-Tocopherol (vitamin E), 25-hydroxycalciferol (vitamin D), and the carotenoid zeaxanthin could also be measured when they were present in high concentrations. Healthy people with spinal cord injuries were more likely than similar people without injuries to have low concentrations of alpha-tocopherol, and to a lesser extent retinol and beta-carotene.

Adult↗

Liquid chromatography-electrospray mass spectrometry of beta-carotene and xanthophylls. Validation of the analytical method.

The investigation of beta-carotene and the xanthophylls beta-cryptoxanthin, lutein, zeaxanthin, canthaxanthin and astaxanthin using reversed-phase liquid chromatography-electrospray mass spectrometry interfaced with TurboIonspray (LC-TurboISP-MS) is described. Two narrow-bore C18 columns connected in series and an isocratic solvent system containing acetonitrile-methanol (0.1 M ammonium acetate)-dichloromethane at a flow-rate of 300 microl/min (without splitting) were used. Operating in the positive-ion mode over m/z 500-650, the effects on the formation of the molecular ion species or adduct ions and the MS detector response were investigated for carotenoids, varying the orifice plate voltage, the ring voltage and the ISP voltage. Both conventional ISP and TurboISP were performed; using the TurboISP-MS system, ionization efficiency increased with respect to ISP-MS, particularly at the highest temperature (500 degrees C). Good results were particularly obtained for beta-carotene, which was detectable at the low ng level, without the use of solution-phase oxidants. Using LC columns and acquiring in single-ion monitoring mode, detection limits were estimated to be in the 0.1-1 ng range; dynamic range was established between one- and two-orders of magnitude. Better sensitivity under positive-ion than negative-ion conditions was demonstrated.

Chromatography, High Pressure Liquid↗

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

Analysis of Variance↗

Purification and characterization of cellular carotenoid-binding protein from mammalian liver.

A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. beta-[14C]Carotene copurified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites; a high-affinity site with an apparent Kd of 56 x 10(-9) M and a low-affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high-affinity site was 1 mol/mol whereas that for the low-affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambda max with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa size has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.

Animals↗

Effects of fruit and vegetable consumption on plasma antioxidant concentrations and blood pressure: a randomised controlled trial.

BACKGROUND: High dietary intakes of fruit and vegetables are associated with reduced risks of cancer and cardiovascular disease. Short-term intensive dietary interventions in selected populations increase fruit and vegetable intake, raise plasma antioxidant concentrations, and lower blood pressure, but long-term effects of interventions in the general population are not certain. We assessed the effect of an intervention to increase fruit and vegetable consumption on plasma concentrations of antioxidant vitamins, daily fruit and vegetable intake, and blood pressure. METHODS: We undertook a 6-month, randomised, controlled trial of a brief negotiation method to encourage an increase in consumption of fruit and vegetables to at least five daily portions. We included 690 healthy participants aged 25-64 years recruited from a primary-care health centre. FINDINGS: Plasma concentrations of alpha-carotene, beta-carotene, lutein, beta-cryptoxanthin, and ascorbic acid increased by more in the intervention group than in controls (significance of between-group differences ranged from p=0.032 to 0.0002). Groups did not differ for changes in lycopene, retinol, alpha-tocopherol, gamma-tocopherol, or total cholesterol concentrations. Self-reported fruit and vegetable intake increased by a mean 1.4 (SD 1.7) portions in the intervention group and by 0.1 (1.3) portion in the control group (between-group difference=1.4, 95% CI 1.2-1.6; p<0.0001). Systolic blood pressure fell more in the intervention group than in controls (difference=4.0 mm Hg, 2.0-6.0; p<0.0001), as did diastolic blood pressure (1.5 mm Hg, 0.2-2.7; p=0.02). INTERPRETATION: The effects of the intervention on fruit and vegetable consumption, plasma antioxidants, and blood pressure would be expected to reduce cardiovascular disease in the general population.

Adult↗

Dietary antioxidant intake and incidence of early age-related maculopathy: the Blue Mountains Eye Study.

OBJECTIVE: To investigate associations between dietary intake, including modest supplement intake, of antioxidant vitamins and zinc at baseline and the 5-year incidence of early age-related maculopathy (ARM). DESIGN: Population-based cohort study. PARTICIPANTS: From 1992 through 1994, 3654 persons aged 49 years or more (82% of those eligible) living in two postcode areas west of Sydney, Australia, were examined for the Blue Mountains Eye Study baseline. Five years later, 2335 persons (75% of known survivors) were reexamined. METHODS: A 145-item Food Frequency Questionnaire (FFQ) was used to assess nutrient intakes. Of the 2335 people who attended a follow-up visit, 1989 (85%) had completed a FFQ at baseline. The nutrients examined in this study included: alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein and zeaxanthin, lycopene, retinol, vitamin A, vitamin C, and zinc. MAIN OUTCOME MEASURES: Early ARM was assessed by masked grading of stereo retinal photographs. Definitions for incidence closely followed those used in the Beaver Dam Eye Study. RESULTS: Early ARM developed in 192 persons (8.7% 5-year incidence) who did not have either late or early ARM at baseline. Of these, 159 persons completed the FFQ at baseline. After adjusting for age, gender, family history of ARM, and smoking status at baseline, no associations, or any trends suggesting possible association, were found between baseline intake of the nutrients examined, apart from vitamin C, and the 5-year incidence of early ARM. Compared with the lowest quintile, increasing baseline intakes of vitamin C, from diet and supplements, was associated with an increased risk of incident early ARM (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.0-3.0; and OR, 2.3; 95% CI, 1.3-4.0 for the fourth and fifth quintiles, respectively). CONCLUSIONS: Our cohort study of an older population could not find evidence of protection associated with usual dietary antioxidant or zinc intakes (including use of supplements) on the 5-year incidence of early ARM.

Aged↗

Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer's disease.

In order to assess peripheral levels and activities of a broad spectrum of non-enzymatic and enzymatic antioxidants in elderly subjects with mild cognitive impairment (MCI) and Alzheimer's disease (AD), plasma levels of water-soluble (Vitamin C and uric acid) and of lipophilic (Vitamin A, Vitamin E and carotenoids including lutein, zeaxanthin, beta-cryptoxanthin, lycopene, alpha- and beta-carotene) antioxidant micronutrients as well as activities of plasma and red blood cell (RBC) superoxide dismutase (SOD) and of plasma glutathione peroxidase (GPx) were measured in 25 patients with MCI, 63 AD patients and 53 controls. Peripheral levels and activities of antioxidants were similarly lower in MCI and AD patients as compared to controls. As MCI may represent a prodromal stage of AD, and oxidative damage appears to occur as one of the earliest pathophysiological events in AD, an increased intake of antioxidants in patients with MCI could be helpful in lowering the risk of conversion to dementia.

Aged↗

DNA damage and antioxidants; fluctuations through the year in a central European population group.

Dietary antioxidant levels in the blood depend on intake of fruits and vegetables and therefore might be expected to show seasonal variation. A group of healthy male subjects in Bratislava, Slovakia gave blood samples each month for 1 year. Vitamin C, alpha- and gamma-tocopherol and several carotenoids were measured in plasma, and concentrations of essential metals zinc, copper and selenium in serum. Oxidative DNA damage was assessed in lymphocytes using the comet assay. Seasonal variations in antioxidant levels did not follow a common pattern. beta-Cryptoxanthin was highest in the spring. Lycopene peaked in late summer. Lutein/zeaxanthin was higher in summer than in winter. The concentration of zinc in serum was higher in winter than in summer. DNA damage was lower in summer than in winter. Selenium as well as several antioxidants correlated negatively with indices of DNA damage, while zinc levels showed a positive correlation with DNA damage. These results provide some support for a link between consumption of antioxidants and protection against DNA oxidation.

Antioxidants↗

Simultaneous measurement of seven carotenoids, retinol and alpha-tocopherol in serum by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative measurement in serum of seven carotenoids (lutein, zeaxanthin, canthaxanthin, beta-cryptoxanthin, lycopenes, alpha-carotene and beta-carotene), retinol, alpha-tocopherol and two internal standards (tocol and echinenone) has been developed. The geometric isomers, lutein and zeaxanthin, were completely separated as well as at least nine unidentified carotenoids. All compounds were resolved on an Adsorbosphere HS C18 (3 microm) column (250x4.6 mm I.D.) with a step gradient, 7.1 min after injection, from acetonitrile-methanol (60:40, v/v) containing 0.05% acetic acid to acetonitrile-methanol-dichloromethane (45.6:30.4:24, v/v) containing 0.04% acetic acid, in a total run time of 23 min. Chromatograms at four different wavelengths (292, 325, 450 and 473 nm) and spectra were monitored with a diode array detector. Because of its specificity and sensitivity for a large number of carotenoids, this procedure may be of interest for nutritional and epidemiological studies. Its speed and robustness make it suitable for analyses on large numbers of subjects.

Adult↗

Stability of individual carotenoids, retinol and tocopherols in human plasma during exposure to light and after extraction.

We have modified gradient HPLC procedures for simultaneous quantification of retinol, gamma-tocopherol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, trans-lycopene, cis-lycopene, alpha-carotene and beta-carotene in 200-microl aliquots of human plasma. The photosensitivity of these analytes in plasma exposed to fluorescent lighting for up to 72 h was investigated and most were stable under these conditions. The stability of these analytes held in darkness at -20 degrees C, 4 degrees C or room temperature for up to 48 h after extraction from plasma was also investigated. Variability in measurement of most analytes was greater at room temperature than at 4 degrees C or -20 degrees C. There were statistically significant variations in the measured concentrations of some analytes in samples kept cold. However, the magnitude of these variations was small and of little biological significance, particularly over the first 24 h.

Carotenoids↗

Plasma lipophilic antioxidants and malondialdehyde in congestive heart failure patients: relationship to disease severity.

Plasma levels of malondialdehyde (MDA), vitamin A, and of antioxidant micronutrients including vitamin E, lutein, zeaxanthin, beta-cryptoxanthin, lycopene, and alpha- and beta-carotene were measured in 30 patients with class II and III congestive heart failure (CHF) according to the New York Heart Association (NYHA) classification and in 55 controls. Ejection fraction was evaluated by echocardiography in all patients as a measure of the emptying capacity of the heart. Plasma levels of all measured compounds were significantly lower and MDA significantly higher in patients compared to controls (p <.001). Class II NYHA patients showed significantly lower MDA levels and significantly higher levels of vitamin A, vitamin E, lutein, and lycopene than class III patients. Ejection fraction was inversely correlated with MDA levels and directly correlated with vitamin A, vitamin E, lutein, and lycopene levels in patients. The present study supports the concept that an increased consumption of vitamin-rich fruits and vegetables might help in achieving cardiovascular health.

Aged↗

Relation of vitamin A and carotenoid status to growth failure and mortality among Ugandan infants with human immunodeficiency virus.

Although growth failure is common during pediatric infection with human immunodeficiency virus (HIV) and associated with increased mortality, the relation of specific nutrition factors with growth and mortality has not been well characterized. A longitudinal study was conducted with 194 HIV-infected infants in Kampala, Uganda. Plasma vitamin A, carotenoids (alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, and lutein/zeaxanthin), and vitamin E were measured at age 14 wk, and weight and height were followed up to age 12 mo. Vitamin A and low plasma carotenoid concentrations were predictive of decreased weight and height velocity. Between ages 14 wk and 12 mo, 32% of infants died. Underweight, stunting, and low concentrations of plasma carotenoids were associated with increased risk of death in univariate analyses. Plasma vitamin A concentrations were not associated with risk of death. In a final multivariate model adjusting for weight-for-age, plasma beta-carotene was significantly associated with increased mortality (odds ratio: 3.16, 95% confidence interval: 1.38 to 7.21, P < 0.006). These data suggest that low concentrations of plasma carotenoids are associated with increased risk of death during HIV infection among infants in Uganda.

Body Height↗

Plasma status of retinol, alpha- and gamma-tocopherols, and main carotenoids to first myocardial infarction: case control and follow-up study.

OBJECTIVE: Epidemiologic studies have suggested that dietary intake and plasma concentrations of antioxidants have an inverse relation with coronary heart disease. To test whether fat-soluble antioxidants can play a role against the occurrence of myocardial infarction (MI), we measured plasma levels of retinol, tocopherols, and individual carotenoids in MI patients. METHODS: A case-control and follow-up study of patients in the Móstoles area (Madrid, Spain). One hundred six patients (62 after 1 y) and 104 control subjects participated in the study. Blood samples were collected after overnight fast or during the first 24 h of MI onset for biochemical profiles of retinol, alpha- and gamma-tocopherols, and carotenoid by means of a quality-controlled high-performance liquid chromatography. RESULTS: During the acute phase after MI onset, plasma levels of retinol, gamma-tocopherol, and xanthophylls (lutein/zeaxanthin and beta-cryptoxanthin) decreased, whereas alpha-tocopherol, alpha-carotene, beta-carotene, and lycopene showed levels similar to those of control subjects. Logistic regression analysis showed low concentrations of gamma-tocopherol (and retinol) in plasma as the only statistically significant factor associated with MI, after adjusting for traditional risk factors. However, 1 y later, the MI patients showed a general improvement in plasma lipids and fat-soluble antioxidant status, and none of the analytes was associated with MI. CONCLUSIONS: The decreased plasma status of retinol, gamma-tocopherol, and xanthophylls during the acute phase of MI normalized the year after the MI event, suggesting that most subjects had followed an overall healthier lifestyle and dietary pattern. The results also raise concerns on the usefulness of these plasma compounds as specific, relevant, and predictive markers in relation to coronary heart disease.

Acute-Phase Reaction↗

Decrease of serum carotenoids in Crohn's disease.

Crohn's disease (CD) is frequently complicated by various nutritional disturbances. Although it is important to correct these disturbances, the nutritional status of CD patients has been poorly documented, especially concerning vitamin status. The aims of this study were (a) to measure the serum concentrations of vitamin A and six other carotenoids (lutein, zeaxanthin, alpha-, beta-carotene, alpha-, beta-cryptoxanthin) in patients with CD and to compare them with those in healthy controls and (b) to follow the changes of serum carotenoid levels in CD patients during treatment. Twenty-eight patients with CD and 23 healthy persons were included in this study. The results of twelve patients were followed up through one year. The patients were free of any nutritional treatment. The serum concentrations of carotenoids were measured with high-pressure liquid chromatography (HPLC). The serum concentrations of five carotenoids were significantly lower in the patients than in the controls (vitamin A, zeaxanthin: P < 0.001; alpha-, beta-carotene: P < 0.01; lutein: P < 0.05). The carotenoid status of the followed patients advanced to the normal range, but this increase was not significant. These findings suggest that there is a deficiency of vitamin A and its provitamins in Crohn' s disease prior to treatment. However, because we did not evaluate the vitamin intake in this study, we could not conclude which of the factors--poor intake, increased requirement, or malabsorption--was more important in decreasing of carotenoid levels.

Adolescent↗