Analytical tools for the analysis of β-carotene and its degradation products

Abstract

β-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on β-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with β-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of β-carotene. Their generation originates in the polyene structure of β-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs).

Due to their instability and hydrophobicity, the handling of standards and real samples containing β-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.     

Reaction pH and protein affect the oxidation products of β-pentagalloyl glucose

To better understand the biochemical consequences when polymeric polyphenols serve as biological antioxidants, we studied how reaction pH (pH 2.1–7.4) and protein affected the oxidation of pentagalloyl glucose (PGG) by NaIO₄ in aqueous solution. PGG oxidation produced an o-semiquinone radical intermediate, which tended to form polymeric products at pH values below 5, and o-quinones at higher pH. The model protein bovine serum albumin promoted the formation of quinone even at low pH. Two other polyphenols, procyanidin (epicatechin₁₆-(4→8)-catechin) and epigallocatechin gallate, had similar pH-dependent oxidation patterns.     

n-alkane oxidation by apseudomonas formation and β-oxidation of intermediate fatty acids

Summary From a culture broth ofPseudomonas aeruginosa (KSLA strain 473) grown on heptane as the sole source of carbon, fatty acids could be isolated after a period of decreased oxygen supply. The corresponding methyl esters—obtained by treatment with diazomethane—were separated by gas-liquid chromatography and identified by mass spectrometry. Heptylic, valeric and propionic acids were shown to be present in the original culture broth. Using the same techniques the formation of caproic acid from hexane was shown to occur, whereas the amount of butyric acid formed was extremely small and inconsistent. These results show conclusively that this microbiological oxidation of heptane and hexane proceeds by way of the corresponding fatty acids, which are further degraded by β-oxidation. The absence of caproic and valeric acids in heptane and hexane oxidation, respectively, shows that decarboxylation of fatty acids does not occur.     

POSS-based fluorescence sensor for rapid analysis of β-carotene in health products

In recent years, fluorescent organic–inorganic hybrid nanomaterials have received much interest as potential fluorescent sensor materials. In this study, fluorescent organic–inorganic hybrid nanomaterials (POSS@ANT) were created using polyhedral oligomeric silsesquioxane as the precursor and 9,10-bromoanthracene as the monomer. The morphology and composition of POSS@ANT, as well as its pore characteristics and fluorescence properties were studied. POSS@ANT displayed steady fluorescence emission at an excitation wavelength of 374 nm. Next, a β-carotene fluorescence sensor was developed using the capacity of β-carotene to quench the fluorescence of POSS@ANT. The quenching process is linked to acceptor electron transfer and energy transfer, and the sensor has a high selectivity for β-carotene. This β-carotene fluorescence analysis method that we established has a linear range of 0.2–4.3 mg/L and a detection limit of 0.081 mg/L. Finally, it was used to quantify β-carotene in health products, the recovery rate was 91.1–109.9%, the relative standard deviation (RSD) was 2.2–4.3%, and the results were comparable with the results of high-performance liquid chromatography. The approach is reliable and can be used to determine β-carotene in health products.     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Transcriptome profiling of β-carotene biosynthesis genes and β-carotene accumulation in leaf blades and petioles of celery cv. Jinnanshiqin

β-carotene is a kind of carotenoids and has many biological functions.Proper amount of β-carotene is beneficial to promote the synthesis of vitamin A [1].The unsaturated double bonds in β-carotene structure make it have strong antioxidant capacity.     

Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

Iron-induced oxidation of (all-E)-β-carotene under model gastric conditions: kinetics,products, and mechanism

The stability of (all-E)-β-carotene toward dietary iron was studied in a mildly acidic (pH 4) micellar solution as a simple model of the postprandial gastric conditions. The oxidation was initiated by free iron (Fe^II, Fe^III) or by heme iron (metmyoglobin, MbFe^III). Fe^II and metmyoglobin were much more efficient than Fe^III at initiating β-carotene oxidation. Whatever the initiator, hydrogen peroxide did not accumulate. Moreover, β-carotene markedly inhibited the conversion of Fe^II into Fe^III. β-Carotene oxidation induced by Fe^II or MbFe^III was maximal with 5–10 eq Fe^II or 0.05–0.1 eq MbFe^III and was inhibited at higher iron concentrations, especially with Fe^II. UPLC/DAD/MS and GC/MS analyses revealed a complex distribution of β-carotene-derived products including Z-isomers, epoxides, and cleavage products of various chain lengths. Finally, the mechanism of iron-induced β-carotene oxidation is discussed. Altogether, our results suggest that dietary iron, especially free (loosely bound) Fe^II and heme iron, may efficiently induce β-carotene autoxidation within the upper digestive tract, thereby limiting its supply to tissues (bioavailability) and consequently its biological activity.     

Fenretinide inhibits vitamin A formation from β-carotene and regulates carotenoid levels in mice

N-[4-hydroxyphenyl]retinamide, commonly known as fenretinide, a synthetic retinoid with pleiotropic benefits for human health, is currently utilized in clinical trials for cancer, cystic fibrosis, and COVID-19. However, fenretinide reduces plasma vitamin A levels by interacting with retinol-binding protein 4 (RBP4), which often results in reversible night blindness in patients. Cell culture and in vitro studies show that fenretinide binds and inhibits the activity of β-carotene oxygenase 1 (BCO1), the enzyme responsible for endogenous vitamin A formation. Whether fenretinide inhibits vitamin A synthesis in mammals, however, remains unknown. The goal of this study was to determine if the inhibition of BCO1 by fenretinide affects vitamin A formation in mice fed β-carotene. Our results show that wild-type mice treated with fenretinide for ten days had a reduction in tissue vitamin A stores accompanied by a two-fold increase in β-carotene in plasma (P < 0.01) and several tissues. These effects persisted in RBP4-deficient mice and were independent of changes in intestinal β-carotene absorption, suggesting that fenretinide inhibits vitamin A synthesis in mice. Using Bco1^?/? and Bco2^?/? mice we also show that fenretinide regulates intestinal carotenoid and vitamin E uptake by activating vitamin A signaling during short-term vitamin A deficiency. This study provides a deeper understanding of the impact of fenretinide on vitamin A, carotenoid, and vitamin E homeostasis, which is crucial for the pharmacological utilization of this retinoid.     

Absolute configuration of β,γ-carotene and biosynthetic implications

Racemic γ-ionone, partly resolved via its menthydrazone, was used for total synthesis of β,γ-carotene enriched in the 6′R and 6′S enantiomers. By CD correlation with natural β,γ-carotene isolated from Caloscypha fulgens 6′S-chirality is demonstrated for the natural carotene. Biosynthetic implications regarding the cyclization reaction are discussed.     

Photoreceptor-specific efficiencies of β-carotene,zeaxanthin and lutein for photopigment formation deduced from receptor mutant Drosophila melanogaster

Summary Drosophila rearing media had only -carotene, zeaxanthin or lutein as precursors for photopigment chromophores. Zeaxanthin and lutein are potentially optimum sources of the 3-hydroxylated retinoids of visual and accessory photopigments. Mutants made the electroretinogram in white (w) eyes selective for compound eye photoreceptors R1–6, R7 and R8: R1–6 domiantes w's electroretinogram; R7/8 generates w;ora's (ora = outer rhabdomeres absent); R8 generates w sev;- ora's (sev = sevenless). Microspectrophotometry revealed R1-6's visual pigment. In w, all 3 carotenoids yielded monotonic dose-responses for sensitivity (Fig. 4) or visual pigment (Fig. 7). An ultraviolet sensitivity peak from R1-6's sensitizing pigment was present at high but not low doses (Fig. 1). In w;ora, all 3 carotenoids gave similar spectra dominated by R7's high ultraviolet sensitivity (Fig. 2). For w sev;ora, all spectra were the shape expected for R8, peaking around 510 nm (Fig. 3). The sensitivity dose-response was at its ceiling except for low doses in w;ora (Fig. 5) and zero supplementation in w sev;ora (Fig. 6). Hence, without R1-6, most of our dose range mediated maximal visual pigment formation. In Drosophila, -carotene, zeaxanthin and lutein mediate the formation of all major photopigments in R1-6, R7 and R8.Abbreviations ERG electroretinogram - MSP microspectrophotometry - HPLC high pressure liquid chromatography - n.a. numerical aperture - w, sev, ora Drosophila mutants - y, p, r marg types of R7 and R8     

The synthesis of oligosaccharides by the reversed hydrolysis reaction of β-glucosidase at high substrate concentration and at high temperature

Summary In the presence of -glucosidase from almond, a 90% glucose solution gave four kind of -linked glucose-disaccharides. The yield increased as the concentration of glucose was increased and as the reaction temperature was raised. The maximum yield of disaccharides from 90% glucose solution was 40% at 55°C.