Production, separation, and characterization of apo-luteinoids by LC-MS/MS.

It has been postulated that chemical or enzymatic catabolism of carotenoids could produce apo­carotenoids which have biological activity. Our objective was to generate and chemically characterize a series of apo­luteinoids (i.e. products resulting from the catabolism of lutein) which could putatively be found in vivo. Lutein was oxidized using potassium permanganate to produce a series of apo­luteinals/luteinone of subsequently shorter chain lengths, from apo­8'­luteinal to apo­11­luteinal. Sodium borohydride reduced this mixture into the corresponding alcohols (i.e. apo­luteinols). Similarly, Tollens' reagent was employed to oxidize the aldehyde series into carboxylic acids (i.e. apo­luteinoic acids). Mixtures of products were separated via HPLC and characterized in-line using photodiode array (PDA) and tandem mass spectrometry (MS-MS). A global HPLC-PDA-MS/MS method was developed to separate the products, and application of the methods to the symmetric xanthophyll zeaxanthin further confirmed the ε- and ß-ring species. These methods can be employed for the study of lutein oxidation products in plants, foods and biological samples.

Carotenoids: Experimental Ionization Energies and Capacity at Inhibiting Lipid Peroxidation in a Chemical Model of Dietary Oxidative Stress.

Carotenoids are important natural pigments and micronutrients contributing to health prevention by several mechanisms, including their electron-donating (antioxidant) activity. In this work, a large series of carotenoids, including 11 carotenes and 14 xanthophylls, have been investigated by wavelength-resolved atmospheric pressure photoionization mass spectrometry (DISCO line of SOLEIL synchrotron), thus allowing the experimental determination of their ionization energy (IE) for the first time. On the other hand, the carotenoids have been also investigated for their ability to inhibit the heme iron-induced peroxidation of linoleic acid in mildly acidic micelles, a simple but relevant chemical model of oxidative stress in the gastric compartment. Thus, the carotenoids can be easily classified from IC50 concentrations deduced from the time dependence of the lipid hydroperoxide concentration. With a selection of two carotenes and three xanthophylls a quantitative analysis is also provided to extract stoichio-kinetic parameters. The influence of the carotenoid structure (number of conjugated carbon-carbon double bonds, presence of terminal six-membered rings, hydroxyl, keto, and/or epoxy groups) on the IE, IC50, and stoichio-kinetic parameters is discussed in details. The data show that the antioxidant activity of carotenes is well correlated to their electron-donating capacity, which itself largely depends on the length of the conjugated polyene chain. By contrast, the IE of xanthophylls is poorly correlated to the polyene chain length because of the strong, and sometimes unexpected, electronic effects of the O-atoms. Although IE remains an approximate predictor of the antioxidant activity of xanthophylls, other factors (interaction with the aqueous phase, competing radical-scavenging mechanisms, the residual activity of the antioxidant's oxidation products) probably play a significant role.

Production of asymmetric oxidative metabolites of [13C]-ß-carotene during digestion in the gastrointestinal lumen of healthy men.

Background: Asymmetric ß-apo-carotenoids (nonvitamin A-active metabolites) of provitamin A carotenoids have been observed in humans, but no study has investigated their formation during digestion. Objective: The aim of this study was to follow the formation and absorption of asymmetric ß-apo-carotenoids during digestion. Design: Healthy men were intragastrically and intraduodenally intubated, and randomly assigned to consume a lipid-rich control meal (n = 3) or a lipid-rich test meal containing 20 mg [13C-10]-ß-carotene (n = 7). Digesta samples were collected over 5 h, and blood collected over 7 h. The triglyceride-rich lipoprotein (TRL) fractions of plasma were also isolated. Lipophilic extracts of digesta, plasma, and TRL were analyzed via a high-performance liquid chromatography-tandem mass spectrometry method developed to identify [13C]-labeled ß-apo-carotenals/carotenone, [13C]-ß-apo-carotenols, and [13C]-ß-apo-carotenoic acids. Results: Relative to [13C]-ß-carotene, [13C]-ß-apo-carotenal levels remained ∼3 orders of magnitude lower throughout digestion (no [13C]-ß-apo-carotenols, or [13C]-ß-apo-carotenoic acids were observed). A mixed model determined relative influence of digesta type and time on digesta metabolite level. Increasing time significantly increased the model levels of digesta [13C]-ß-apo-10',12',14',15-carotenal and [13C]-ß-apo-13-carotenone (P < 0.05) and trended toward decreased [13C]-ß-apo-8'-carotenal (P = 0.0876). Gastric digesta were associated with a significantly higher level of [13C]-ß-apo-8'-carotenal (P = 0.0289), and lower levels of [13C]-ß-apo-12',14',15-carotenal (P < 0.05), relative to duodenal digesta. Anticipated retinoids, but no asymmetric [13C]-ß-apo-carotenals, [13C]-ß-apo-carotenols, or [13C]-ß-apo-carotenoic acids, were observed in the blood or TRL samples. Conclusions: ß-Carotene appears to be robust to digestion, with minor amounts of ß-apo-carotenals/carotenone formed. Absence of asymmetric [13C]-ß-apo-carotenals in plasma and TRL suggests lack of absorption, levels below the limit of detection, lack of stability, or further conversion during the digestive process to as-yet unidentified products. Lack of asymmetric [13C]-ß-apo-carotenals in plasma also suggests a lack of postprandial intestinal BCO2 activity in healthy humans. This trial was registered at clinicaltrials.gov as NCT03492593.

Thermal degradation of antioxidant micronutrients in citrus juice: kinetics and newly formed compounds.

The thermal degradation kinetics of vitamin C, two carotenoids (beta-carotene and beta-cryptoxanthin), and hesperidin, as a function of temperature, were determined for Citrus juice [Citrus sinensis (L.) Osbeck and Citrus clementina Hort. ex Tan]. The influence of dissolved oxygen on the rate of ascorbic acid degradation was also assessed. Analysis of kinetic data suggested a first-order reaction for the degradation of vitamin C and carotenoids. The kinetics parameters Dtheta, z, and Ea have been calculated. Following the Arrhenius relationship, the activation energy of ascorbic acid was 35.9 kJ mol-1 and agreed with the range of literature reported value. The results on vitamin C and carotenoids from citrus juice made it possible to validate the predicting model. Thermal degradation of carotenoids revealed differences in stability among the main provitamin A carotenoids and between these and other carotenoids belonging to the xanthophyll family. The activation energies for the two provitamin A carotenoids were 110 and 156 kJ mol-1 for beta-carotene and beta-cryptoxanthin, respectively. On the other hand, no degradation of hesperidin was observed during thermal treatment. Finally, the vitamin C in citrus juice was not as heat sensitive as expected and the main provitamin A carotenoids present in citrus juice displayed a relative heat stability. The high-performance liquid chromatography-diode array detection-mass spectrometry analysis of degradation products showed that the isomerization of the epoxide function in position 5,6 into a furanoxide function in position 5,8 was a common reaction for several xanthophylls. These findings will help determine optimal processing conditions for minimizing the degradation of important quality factors such as vitamin C and carotenoid in citrus juice.

Effects of high power ultrasound on all-E-ß-carotene, newly formed compounds analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry.

To study effects of high power ultrasound treatment (20 kHz) on ß-carotene degradation, a second-order central composite design (CCD) was performed to investigate maximum ß-carotene loss with three independent factors (ultrasonic intensity, sonication time, and temperature). Results based on variance analysis and Pareto chart have shown that sonication time is the most important factor, followed by ultrasonic intensity level. The evolved degradation products have been tentatively identified using ultra high performance liquid chromatography coupled to both diode array detector and a mass spectrometer (UHPLC-DAD-MS). The main degradation products, tentatively identified, are three Z-isomers of ß-carotene and seven ß-apo-carotenals/ones. Hypothesis on the degradation mechanism of carotenoids are presented.

Cleavage products of lycopene produced by in vitro oxidations: characterization and mechanisms of formation.

The aim of this study was to produce in vitro oxidation products of lycopene, which could be possible in vivo metabolites. An oxidation of lycopene with potassium permanganate gave a range of lycopene degradation compounds resulting from the oxidative cleavage of one or two carbon-carbon double bonds. Eleven apo-lycopenals/ones and six apo-carotendials were obtained and tentatively characterized by HPLC-DAD-MS. Apo-11-lycopenal and apo-8,6'-carotendial were isolated and characterized by (1)H NMR for the first time. Lycopene was submitted to an oxidation by atmospheric oxygen catalyzed by a metalloporphyrin, a model system of the active center of cytochrome P450 enzymes. (Z)-Isomers, monoxides, and cleavage compounds of (E)-lycopene were formed. We propose a mechanism of oxidation of lycopene by this system.

Cashew apple (Anacardium occidentale L.) extract from by-product of juice processing: a focus on carotenoids.

Cashew apple fibrous residue is a by-product of the cashew juice industry. After pressing using a helical type continuous press followed by crossflow microfiltration, an aqueous extract was obtained from these cashew apple fibres. It was characterised by an intense yellow colour due to carotenoid pigments. Carotenoids were identified and quantified in the cashew apple before extraction, in its aqueous extract and in the concentrate obtained by microfiltration. Cashew apple aqueous extract and its concentrate presented a carotenoid profile with 11 carotenoids, most of them were tentatively identified by HPLC-DAD-MS and are xanthophylls present under an esterified form. Auroxanthin and ß-cryptoxanthin represented around 50% of total carotenoids. Concentration of the extract by microfiltration led to epoxy-furanoxy rearrangement of violaxanthin and antheraxanthin. The process allowed an increase of 10 times total carotenoid content compared with initial cashew apple. Total carotenoid content of the final concentrated extract reached 54 mg/kg.

(13Z)- and (9Z)-lycopene isomers are major intermediates in the oxidative degradation of lycopene by cigarette smoke and Sin-1.

The breakdown of lycopene in the presence of reactive oxygen and reactive nitrogen species has been studied in order to identify key in vitro intermediates. These compounds may in turn be produced as metabolites in the body and may have significant physiological properties, such as increased antioxidant capacity. We have studied the in vitro degradation of lycopene in solvent, in plasma and in low density lipoprotein, when challenged with freshly generated gaseous cigarette smoke or free radicals generated in situ by S-morpholinosydonimine at 37°C. The emphasis has been to establish the major intermediates and to compare the data with previous studies using different reactants. We have found that (13Z)-lycopene is the major intermediate in both cigarette smoke and S-morpholinosydonimine reactions (representing ≥60% of the converted (all-E)-lycopene at ∼50% depletion). Additionally, (9Z)-lycopene and various (all-E) and (Z)-lycopene epoxides were predominant. Notably, (5Z)-lycopene appeared to be the most stable form of lycopene under the stated conditions. Previous theoretical studies of isomer thermodynamics and rotational energy barriers for carbon double bonds fully support the pattern of isomer production and stability. In contrast to ß-carotene studies, nitro-derivatives of lycopene could not be detected. In conclusion, (Z)-lycopene production and (5Z)-lycopene stability may help explain elevated (Z)-lycopene in plasma over (Z)-lycopene content in lycopene-containing foods in the diet.

The degradation of (all-E)-beta-carotene by cigarette smoke.

The effects of cigarette smoke in promoting the degradation of (all-E)-beta-carotene have been studied, but some conflicting results promoted a further study. beta-Carotene was solubilized in hexane and challenged with filtered cigarette smoke both at room temperature and at -20 degrees C. The products arising from smoke-induced oxidation were assessed using a combination of HLPC-DAD, LC-MS and GC-MS. At room temperature the degradation of beta-carotene was very rapid, with only a few products being detected using HPLC-DAD. A range of volatile products including beta-ionone, beta-cyclocitral and 5,6-epoxy-beta-ionone were detected using GC-MS. In contrast, when the reaction was slowed (by reducing the reaction temperature), a much wider range of products could be detected by HPLC-DAD, including 4-nitro-beta-carotene and several of its geometric isomers. These degradation products suggest that the C4 position on the beta-carotene end-group plays a key role in initiating free radical attack.