Single Nucleotide Polymorphisms in ß-Carotene Oxygenase 1 are Associated with Plasma Lycopene Responses to a Tomato-Soy Juice Intervention in Men with Prostate Cancer.

BACKGROUND: Human plasma and tissue lycopene concentrations are heterogeneous even when consuming controlled amounts of tomato or lycopene. OBJECTIVES: Our objective is to determine whether single nucleotide polymorphisms (SNPs) in or near known or putative carotenoid metabolism genes [ß-carotene 15,15' monooxygenase 1 (BCO1), scavenger receptor class B type 1 (SCARB1), ATP-binding cassette transporter subfamily A member 1 (ABCA1), microsomal triglyceride transfer protein (MTTP), apolipoprotein B-48, elongation of very long chain fatty acids protein 2 (ELOVL2), and ATP-binding cassette subfamily B member 1 (ABCB1), and an intergenic superoxide dismutase 2, mitochondrial-associated SNP] are predictive of plasma lycopene responses to steady state tomato juice consumption. METHODS: Secondary linear regression analyses of data from a dose-escalation study of prostate cancer patients [n = 47; mean ± SEM age: 60 ± 1 y; BMI (in kg/m2): 32 ± 1] consuming 0, 1, or 2 cans of tomato-soy juice/d (163 mL/can; 20.6 mg lycopene 1.2 mg ß-carotene/can) for 24 ± 0.7 d before prostatectomy were conducted to explore 11 SNP genotype effects on the change in plasma lycopene and plasma and prostate tissue concentrations of lycopene, ß-carotene, phytoene, and phytofluene. RESULTS: Two BCO1 SNP genotypes were significant predictors of the change in plasma lycopene, with SNP effects differing in magnitude and direction, depending on the level of juice intake (rs12934922 × diet group P = 0.02; rs6564851 × diet group P = 0.046). Further analyses suggested that plasma ß-carotene changes were predicted by BCO1 rs12934922 (P < 0.01), prostate lycopene by trending interaction and main effects of BCO1 SNPs (rs12934922 × diet group P = 0.09; rs12934922 P = 0.02; rs6564851 P = 0.053), and prostate ß-carotene by BCO1 SNP interaction and main effects (rs12934922 × diet group P = 0.01; rs12934922 P < 0.01; rs7501331 P = 0.02). CONCLUSIONS: In conclusion, SNPs in BCO1 and other genes may modulate human plasma and prostate tissue responses to dietary lycopene intake and warrant validation in larger, human controlled feeding intervention and cohort studies. Genetic variants related to carotenoid metabolism may partially explain heterogeneous human blood and tissue responses and may be critical covariates for population studies and clinical trials. This trial was registered at clinicaltrials.gov as NCT01009736.

A Novel Tomato-Soy Juice Induces a Dose-Response Increase in Urinary and Plasma Phytochemical Biomarkers in Men with Prostate Cancer.

Background: Tomato and soy intake is associated with reduced prostate cancer risk or severity in epidemiologic and experimental studies. Objective: On the basis of the principle that multiple bioactives in tomato and soy may act on diverse anticancer pathways, we developed and characterized a tomato-soy juice for clinical trials. In this phase 2 dose-escalating study, we examined plasma, prostate, and urine biomarkers of carotenoid and isoflavone exposure. Methods: Men scheduled for prostatectomy were recruited to consume 0, 1, or 2 cans of tomato-soy juice/d before surgery (mean ± SD duration: 24 ± 4.6 d). The juice provided 20.6 mg lycopene and 66 mg isoflavone aglycone equivalents/177-mL can. Plasma carotenoids and urinary isoflavone metabolites were quantified by HPLC-photometric diode array and prostate carotenoids and isoflavones by HPLC-tandem mass spectrometry. Results: We documented significant dose-response increases (P < 0.05) in plasma concentrations of tomato carotenoids. Plasma concentrations were 1.86-, 1.69-, 1.73-, and 1.69-fold higher for lycopene, ß-carotene, phytoene, and phytofluene, respectively, for the 1-can/d group and 2.34-, 3.43-, 2.54-, and 2.29-fold higher, respectively, for the 2-cans/d group compared with 0 cans/d. Urinary isoflavones daidzein, genistein, and glycitein increased in a dose-dependent manner. Prostate carotenoid and isoflavone concentrations were not dose-dependent in this short intervention; yet, correlations between plasma carotenoid and urinary isoflavones with respective prostate concentrations were documented (R2 = 0.78 for lycopene, P < 0.001; R2 = 0.59 for dihydrodaidzein, P < 0.001). Secondary clustering analyses showed urinary isoflavone metabolite phenotypes. To our knowledge, this is the first demonstration of the phytoene and phytofluene in prostate tissue after a dietary intervention. Secondary analysis showed that the 2-cans/d group experienced a nonsignificant decrease in prostate-specific antigen slope compared with 0 cans/d (P = 0.078). Conclusion: These findings provide the foundation for evaluating a well-characterized tomato-soy juice in human clinical trials to define the impact on human prostate carcinogenesis. This trial is registered at clinicaltrials.gov as NCT01009736.

ß-Carotene-9',10'-oxygenase status modulates the impact of dietary tomato and lycopene on hepatic nuclear receptor-, stress-, and metabolism-related gene expression in mice.

Tomato and lycopene (ψ,ψ-carotene) consumption is hypothesized to protect against nonalcoholic steatohepatitis and hepatocarcinogenesis, processes that may depend upon diet and gene interactions. To investigate the interaction of tomato or lycopene feeding with ß-carotene-9',10'-monooxygenase (Bco2) on hepatic metabolic and signaling pathways, male wild-type (WT) and Bco2(-/-) mice (3-wk-old; n = 36) were fed semi-purified control, 10% tomato powder-containing, or 0.25% lycopene beadlet-containing diets for 3 wk. Serum lycopene concentrations were higher in lycopene- and tomato-fed Bco2(-/-) mice compared with WT (P = 0.03). Tomato- and lycopene-fed mice had detectable hepatic apolipoprotein (apo)-6'-, apo-8'-, and apo-12'-lycopenal concentrations. Hepatic expression of ß-carotene-15,15'-monooxygenase was increased in Bco2(-/-) mice compared with WT (P = 0.02), but not affected by diet. Evaluation of hepatic gene expression by focused quantitative reverse transcriptase-polymerase chain reaction arrays for nuclear receptors and coregulators (84 genes) and stress and metabolism (82 genes) genes indicates that tomato feeding affected 31 genes (≥1.5-fold, P < 0.05) and lycopene feeding affected 19 genes, 16 of which were affected by both diets. Lycopene down-regulation of 7 nuclear receptors and coregulators, estrogen-related receptor-α, histone deacetylase 3, nuclear receptor coactivator 4, RevErbA-ß, glucocorticoid receptor, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ, coactivator 1 ß was dependent upon interaction with Bco2 status. Lycopene and tomato feeding induced gene expression patterns consistent with decreased lipid uptake, decreased cell proliferation and mitosis, down-regulated aryl hydrocarbon receptor signaling, and decreased expression of genes involved in retinoid X receptor heterodimer activation. Tomato feeding also caused expression changes consistent with down-regulation of DNA synthesis and terpenoid metabolism. These data suggest tomato components, particularly lycopene, affect hepatic gene expression, potentially affecting hepatic responses to metabolic, infectious, or chemical stress.

Naturally occurring eccentric cleavage products of provitamin A ß-carotene function as antagonists of retinoic acid receptors.

ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene catalyzed by ß-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of ß-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are ß-apocarotenals and ß-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the ß-apocarotenoids significantly activated RARs. Importantly, however, ß-apo-14'-carotenal, ß-apo-14'-carotenoic acid, and ß-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that ß-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. ß-Apo-13-carotenone and the ß-apo-14'-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nm ß-apo-13-carotenone was present in human plasma. These findings suggest that ß-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary ß-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

Comparison of high-performance liquid chromatography/tandem mass spectrometry and high-performance liquid chromatography/photo-diode array detection for the quantitation of carotenoids, retinyl esters, α-tocopherol and phylloquinone in chylomicron-rich fractions of human plasma.

RATIONALE: Bioavailability of essential lipophilic micronutrients and carotenoids is of utmost interest for human health, as the consumption of these compounds may help alleviate major nutritional deficiencies, cardiovascular disease, and cancer. High-performance liquid chromatography/photo-diode array detection (HPLC-PDA) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) were compared for the quantitative analysis of α- and ß-carotene, ß-cryptoxanthin, lutein, lycopene, α-tocopherol, phylloquinone, and several retinyl esters from chylomicron-containing triglyceride rich lipoprotein (TRL) fractions of human plasma obtained from two clinical trials. METHODS: After selecting an efficient extraction method for the analytes, both the HPLC/PDA and the HPLC/MS/MS methods were developed and several parameters validated using an HP 1200 series HPLC system interfaced with a HP 1200 series diode-array detector (Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 5500 (AB Sciex, Foster City, CA, USA) via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. RESULTS: For lycopene, α- and ß-carotene, HPLC/MS/MS was up to 37 times more sensitive than HPLC-PDA. PDA detection was shown to be up to 8 times more sensitive for lutein. MS/MS signals were enhanced by matrix components for lutein and ß-cryptoxanthin, as determined by referencing to the matrix-independent PDA signal. In contrast, matrix suppression was observed for retinyl palmitate, α-carotene, and ß-carotene. Both detectors showed similar suitability for α-tocopherol, lycopene and retinyl palmitate (representing ~73% of total retinyl esters). MS/MS exclusively allowed the quantitation of minor retinyl esters, phylloquinone, and (Z)-lycopene isomers. CONCLUSIONS: HPLC/MS/MS was more sensitive than HPLC-PDA for six of the eight analytes and represents a powerful tool for the analysis of chylomicron samples and potentially other biological samples of limited sample size. When internal standards are available for the target carotenoid, employing MS/MS detection may reduce the necessary blood sample volume, which is particularly advantageous for minimizing risk and discomfort to human subjects during clinical studies.

An LC/MS method for d8-ß-carotene and d4-retinyl esters: ß-carotene absorption and its conversion to vitamin A in humans.

The intestinal absorption and metabolism of ß-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. MS has provided a better understanding of the absorption of ß-carotene, the most potent provitamin A carotenoid, through the use of stable isotopes of ß-carotene. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-ß-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and ß-carotene were recovered in a single simple extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-ß-carotene and its d4-retinyl ester metabolites was achieved using the same C30 reversed-phase liquid chromatography followed by mass spectrometry in selected ion monitoring and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 min. This HPLC-MS method allowed us to quantify the absorption of intact d8-ß-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-ß-carotene.

Adjustable Intragastric Balloon Leads to Significant Improvement in Obesity-Related Lipidome and Fecal Microbiome Profiles: A Proof-of-Concept Study.

INTRODUCTION: Intragastric balloons (IGBs) are a safe and effective treatment for obesity. However, limited knowledge exists on the underlying biological changes with IGB placement. METHODS: This single-institution study was part of an adjustable IGB randomized controlled trial. Subjects with obesity were randomized in a 2 is to 1 ratio to 32 weeks of IGB with diet/exercise counseling (n = 8) vs counseling alone (controls, n = 4). Diet/exercise counseling was continued for 24 weeks post-IGB removal to assess weight maintenance. We used mass spectrometry for nontargeted plasma lipidomics analysis and 16S rRNA sequencing to profile the fecal microbiome. RESULTS: Subjects with IGBs lost 15.5% of their body weight at 32 weeks vs 2.59% for controls (P < 0.05). Maintenance of a 10.5% weight loss occurred post-IGB explant. IGB placement, followed by weight maintenance, led to a -378.9 µM/L reduction in serum free fatty acids compared with pre-IGB (95% confidence interval: 612.9, -145.0). This reduction was mainly in saturated, mono, and omega-6 fatty acids when compared with pre-IGB. Polyunsaturated phosphatidylcholines also increased after IGB placement (difference of 27 µM/L; 95% confidence interval: 1.1, 52.8). Compared with controls, saturated and omega-6 free fatty acids (linoleic and arachidonic acids) were reduced after IGB placement. The fecal microbiota changed post-IGB placement and weight maintenance vs pre-IGB (P < 0.05). Further analysis showed a possible trend toward reduced Firmicutes and increased Bacteroidetes post-IGB and counseling, a change that was not conclusively different from counseling alone. DISCUSSION: IGB treatment is associated with an altered fecal microbiome profile and may have a better effect on obesity-related lipidome than counseling alone.

Novel methoxy-carotenoids from the burgundy-colored plumage of the Pompadour Cotinga Xipholena punicea.

Recent advances in the fields of chromatography, mass spectrometry, and chemical analysis have greatly improved the efficiency with which carotenoids can be extracted and analyzed from avian plumage. Prior to these technological developments, Brush (1968) concluded that the burgundy-colored plumage of the male pompadour Cotinga Xipholena punicea is produced by a combination of blue structural color and red carotenoids, including astaxanthin, canthaxanthin, isozeaxanthin, and a fourth unidentified, polar carotenoid. However, X. punicea does not in fact exhibit any structural coloration. This work aims to elucidate the carotenoid pigments of the burgundy color of X. punicea plumage using advanced analytical methodology. Feathers were collected from two burgundy male specimens and from a third aberrant orange-colored specimen. Pigments were extracted using a previously published technique (McGraw et al. (2005)), separated by high-performance liquid chromatography (HPLC), and analyzed by UV/Vis absorption spectroscopy, chemical analysis, mass spectrometry, nuclear magnetic resonance (NMR), and comparison with direct synthetic products. Our investigation revealed the presence of eight ketocarotenoids, including astaxanthin and canthaxanthin as reported previously by Brush (1968). Six of the ketocarotenoids contained methoxyl groups, which is rare for naturally-occurring carotenoids and a novel finding in birds. Interestingly, the carotenoid composition was the same in both the burgundy and orange feathers, indicating that feather coloration in X. punicea is determined not only by the presence of carotenoids, but also by interactions between the bound carotenoid pigments and their protein environment in the barb rami and barbules. This paper presents the first evidence of metabolically-derived methoxy-carotenoids in birds.

Hepatic stellate cells are an important cellular site for ß-carotene conversion to retinoid.

Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary ß-carotene, but the ability of HSCs to metabolize ß-carotene in situ has not been explored. To gain understanding of this, we investigated whether ß-carotene-15,15'-monooxygenase (Bcmo1) and ß-carotene-9',10'-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary ß-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12'-carotenal levels of Bcmo1-deficient mice. Although apo-10'-carotenal, like apo-12'-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8'- or apo-14'-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a ß-carotene-containing diet compared to mice receiving no ß-carotene. Collectively, our data establish that HSCs are an important cellular site for ß-carotene accumulation and metabolism within the liver.

Green Tea Extract Treatment in Obese Mice with Nonalcoholic Steatohepatitis Restores the Hepatic Metabolome in Association with Limiting Endotoxemia-TLR4-NFκB-Mediated Inflammation.

SCOPE: Catechin-rich green tea extract (GTE) alleviates nonalcoholic steatohepatitis (NASH) by lowering endotoxin-TLR4 (Toll-like receptor-4)-NFκB (nuclear factor kappa-B) inflammation. This study aimed to define altered MS-metabolomic responses during high-fat (HF)-induced NASH that are restored by GTE utilizing livers from an earlier study in which GTE decreased endotoxin-TLR4-NFκB liver injury. METHODS AND RESULTS: Mice are fed a low-fat (LF) or HF diet for 12 weeks and then randomized to LF or HF diets containing 0% or 2% GTE for an additional 8 weeks. Global MS-based metabolomics and targeted metabolite profiling of catechins/catechin metabolites are evaluated. GTE in HF mice restores hepatic metabolites implicated in dyslipidemia insulin resistance, and inflammation. These include 122 metabolites: amino acids, lipids, nucleotides, vitamins, bile acids, flavonoids, xenobiotics, and carbohydrates. Hepatic amino acids, B-vitamins, and bile acids are inversely correlated with biomarkers of insulin resistance, liver injury, steatosis, and inflammation. Further, phosphatidylcholine metabolites are positively correlated with biomarkers of liver injury and NFκB inflammation. Thirteen catechin metabolites are identified in livers of GTE-treated mice, mostly as phase II conjugates of parental catechins or microbial-derived valerolactones. CONCLUSION: The defined anti-inflammatory/metabolic interactions advance an understanding of the mechanism by which GTE catechins protect against NFκB-mediated liver injury in NASH.

Analysis of Tomato Carotenoids: Comparing Extraction and Chromatographic Methods.

Background: Tomatoes (Solanum lycopersicum) are an economically and nutritionally important crop colored by carotenoids such as lycopene and ß-carotene. Market diversification and interest in the health benefits of carotenoids has created the desire in plant, food, and nutritional scientists for improved extraction and quantification protocols that avoid the analytical bottlenecks caused by current methods. Objective: Our objective was to compare standard and rapid extraction as well as chromatographic separation methods for tomato carotenoids. Method: Comparison was based on accuracy and the ability to discriminate between alleles and genetic backgrounds. Estimates of the contribution to variance in the presence of genetic and environmental effects were further used for comparison. Selections of cherry and processing tomatoes with varying carotenoid profiles were assessed using both established extraction and HPLC-diode array detector (HPLC-DAD) methods and rapid extraction and ultra-HPLC-DAD (UHPLC-DAD) protocols. Results: Discrimination of alleles in samples extracted rapidly (<5 min/sample) was similar to samples extracted using a standard method (10 min/sample), although carotenoid concentrations were lower due to reduced extraction efficiency. Quantification by HPLC-DAD (21.5 min/sample) and UHPLC-DAD (4.2 min/sample) were comparable, but the UHPLC-DAD method could not separate all carotenoids and isomers of tangerine tomatoes. Random effects modeling indicated that extraction and chromatographic methods explained a small proportion of variance compared with genetic and environmental sources. Conclusions: The rapid extraction and UHPLC-DAD methods could enhance throughput for some applications compared with standard protocols.

Cruciferous Vegetables, Isothiocyanates, and Bladder Cancer Prevention.

Bladder cancer is a significant health burden due to its high prevalence, risk of mortality, morbidity, and high cost of medical care. Epidemiologic evidence suggests that diets rich in cruciferous vegetables, particularly broccoli, are associated with lower bladder cancer risk. Phytochemicals in cruciferous vegetables, such as glucosinolates, which are enzymatically hydrolyzed to bioactive isothiocyanates, are possible mediators of an anticancer effect. In vitro studies have shown inhibition of bladder cancer cell lines, cell cycle arrest, and induction of apoptosis by these isothiocyanates, in particular sulforaphane and erucin. Although not yet completely understood, many mechanisms of anticancer activity at the steps of cancer initiation, promotion, and progression have been attributed to these isothiocyanates. They target multiple pathways including the adaptive stress response, phase I/II enzyme modulation, pro-growth, pro-survival, pro-inflammatory signaling, angiogenesis, and even epigenetic modulation. Multiple in vivo studies have shown the bioavailability of isothiocyanates and their antitumoral effects. Although human studies are limited, they support oral bioavailability with reasonable plasma and urine concentrations achieved. Overall, both cell and animal studies support a potential role for isothiocyanates in bladder cancer prevention and treatment. Future studies are necessary to examine clinically relevant outcomes and define guidelines on ameliorating the bladder cancer burden.

Identification of an Epoxide Metabolite of Lycopene in Human Plasma Using 13C-Labeling and QTOF-MS.

The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with 13C-labeling to investigate the post-prandial oxidative metabolism of lycopene in human plasma. Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.

Limited appearance of apocarotenoids is observed in plasma after consumption of tomato juices: a randomized human clinical trial.

Background: Nonvitamin A apocarotenoids occur in foods. Some function as retinoic acid receptor antagonists in vitro, though it is unclear if apocarotenoids are absorbed or accumulate to levels needed to elicit biological function. Objective: The aim of this study was to quantify carotenoids and apocarotenoids (ß-apo-8'-, -10'-, -12'-, and -14'-carotenal, apo-6'-, -8'-, -10'-, -12'-, and -14'-lycopenal, retinal, acycloretinal, ß-apo-13-carotenone, and apo-13-lycopenone) in human plasma after controlled consumption of carotenoid-rich tomato juices. Design: Healthy subjects (n = 35) consumed a low-carotenoid diet for 2 wk, then consumed 360 mL of high-ß-carotene tomato juice (30.4 mg of ß-carotene, 34.5 µg total ß-apocarotenoids/d), high-lycopene tomato juice (42.5 mg of lycopene, 119.2 µg total apolycopenoids/d), or a carotenoid-free control (cucumber juice) per day for 4 wk. Plasma was sampled at baseline (after washout) and after 2 and 4 wk, and analyzed for carotenoids and apocarotenoids using high-pressure liquid chromatography (HPLC) and HPLC-tandem mass spectrometry, respectively. The methods used to analyze the apocarotenoids had limits of detection of ∼ 100 pmol/L. Results: Apocarotenoids are present in tomato juices at 0.1-0.5% of the parent carotenoids. Plasma lycopene and ß-carotene increased (P < 0.001) after consuming high-lycopene and ß-carotene tomato juices, respectively, while retinol remained unchanged. ß-Apo-13-carotenone was found in the blood of all subjects at every visit, although elevated (P < 0.001) after consuming ß-carotene tomato juice for 4 wk (1.01 ± 0.27 nmol/L) compared with both baseline (0.37 ± 0.17 nmol/L) and control (0.46 ± 0.11 nmol/L). Apo-6'-lycopenal was detected or quantifiable in 29 subjects, while ß-apo-10'- and 12'-carotenal were detected in 6 and 2 subjects, respectively. No other apolycopenoids or apocarotenoids were detected. Conclusions: ß-Apo-13-carotenone was the only apocarotenoid that was quantifiable in all subjects, and was elevated in those consuming high-ß-carotene tomato juice. Levels were similar to previous reports of all-trans-retinoic acid. Other apocarotenoids are either poorly absorbed or rapidly metabolized or cleared, and so are absent or limited in blood. ß-Apo-13-carotenone may form from vitamin A and its presence warrants further investigation. This trial was registered at clinicaltrials.gov as NCT02550483.

A metabolomic evaluation of the phytochemical composition of tomato juices being used in human clinical trials.

Juices from the traditional red tomato and a unique tangerine tomato variety are being investigated as health promoting foods in human clinical trials. However, it is unknown how the tangerine and red tomato juices differ in biologically relevant phytochemicals beyond carotenoids. Here liquid-chromatography high-resolution mass spectrometry metabolomics was used to evaluate broadly the similarities and differences in carotenoids and other phytochemicals between red and tangerine tomato juices intended for clinical interventions. This untargeted approach was successful in the rapid detection and extensive characterization of phytochemicals belonging to various compound classes. The tomato juices were found to differ significantly in a number of phytochemicals, including carotenoids, chlorophylls, neutral lipids, and cinnamic acid derivatives. The largest differences were in carotenoids, including lycopene, phytoene, phytofluene, neurosporene, and ζ-carotene. Smaller, but significant, differences were observed in polar phytochemicals, such as chlorogenic acid, hydroxyferulic acid, phloretin-di-C-glycoside, and isopropylmalic acid.

Relative contribution of α-carotene to postprandial vitamin A concentrations in healthy humans after carrot consumption.

Background: Asymmetric α-carotene, a provitamin A carotenoid, is cleaved to produce retinol (vitamin A) and α-retinol (with negligible vitamin A activity). The vitamin A activity of α-carotene-containing foods is likely overestimated because traditional analytic methods do not separate α-retinol derivatives from active retinol.Objective: This study aimed to accurately characterize intestinal α-carotene cleavage and its relative contribution to postprandial vitamin A in humans after consumption of raw carrots.Design: Healthy adults (n = 12) consumed a meal containing 300 g raw carrot (providing 27.3 mg ß-carotene and 18.7 mg α-carotene). Triglyceride-rich lipoprotein fractions of plasma were isolated and extracted, and α-retinyl palmitate (αRP) and retinyl palmitate were measured over 12 h postprandially via high-performance liquid chromatography-tandem mass spectrometry. The complete profile of all α-retinyl esters and retinyl esters was measured at 6 h, and total absorption of α- and ß-carotene was calculated.Results: αRP was identified and quantified in every subject. No difference in preference for absorption of ß- over α-carotene was observed (adjusting for dose, 28% higher, P = 0.103). After absorption, ß-carotene trended toward preferential cleavage compared with α-carotene (22% higher, P = 0.084). A large range of provitamin A carotenoid conversion efficiencies was observed, with α-carotene contributing 12-35% of newly converted vitamin A (predicted contribution = 25.5%). In all subjects, a majority of α-retinol was esterified to palmitic acid (as compared with other fatty acids).Conclusions: α-Retinol is esterified in the enterocyte and transported in the blood analogous to retinol. The percentage of absorption of α-carotene from raw carrots was not significantly different from ß-carotene when adjusting for dose, although a trend toward higher cleavage of ß-carotene was observed. The results demonstrate large interindividual variability in α-carotene conversion. The contribution of newly absorbed α-carotene to postprandial vitamin A should not be estimated but should be measured directly to accurately assess the vitamin A capacity of α-carotene-containing foods. This trial was registered at clinicaltrials.gov as NCT01432210.

Tomatoes protect against development of UV-induced keratinocyte carcinoma via metabolomic alterations.

Prolonged tomato consumption can mitigate ultraviolet (UV) light induced sunburn via unknown mechanisms. Dietary carotenoids distributed to skin are hypothesized to protect skin against UV-induced damage, although other phytochemicals may play a role. We hypothesize that tomato consumption would protect against skin cancer. SKH-1 hairless and immunocompetent mice (n = 180) were fed AIN-93G or AIN-93G + 10% tangerine or red tomato powder for 35 weeks. From weeks 11-20, mice (n = 120) were exposed to 2240 J/m2 UV-B light, 3x/week, and tumors were tracked weekly. Control mice were fed the same diets but not exposed to UV. Tumor number was significantly lower in male mice consuming red tomato diets (1.73 ± 0.50, P = 0.015) or pooled tomato diets (2.03 ± 0.45, P = 0.017) compared to controls (4.04 ± 0.65). Carotenoid levels in plasma and skin were quantitated, with total lycopene higher in skin of tangerine fed animals despite a lower dose. Metabolomic analyses elucidated compounds derived from tomato glycoalkaloids (including tomatidine and hydroxylated-tomatidine) as significantly different metabolites in skin after tomato exposure. Here, we describe that tomato consumption can modulate risk for keratinocyte carcinomas; however, the role of the newly identified specific phytochemicals possibly responsible for this action require further investigation.

Plasma Metabolomics Reveals Steroidal Alkaloids as Novel Biomarkers of Tomato Intake in Mice.

SCOPE: Diets rich in tomato products are associated with a reduced risk of various chronic disease processes. The carotenoid lycopene is most intensely studied as the bioactive mediating health effects, yet tomatoes contain an array of phytochemicals. An untargeted metabolomics study is conducted on blood plasma to identify novel markers of tomato consumption absorbed from the diet and released into the bloodstream in mice. METHODS AND RESULTS: Male mice are fed a control AIN-93G diet or the same diet supplemented with 0.25 % lycopene beadlets, or 10 % freeze-dried red tomato, tangerine tomato, or low-carotenoid tomato for 4 weeks. Untargeted UHPLC-QTOF-MS data acquisition and differential analysis of plasma metabolites reveals several structurally related deglycosylated tomato steroidal alkaloids, including tomatidine and hydroxylated/desaturated derivatives, in plasma after the consumption of all three tomato varieties. Additionally, plasma metabolite profiles reflect glycoalkaloid forms found in the tomato diets. CONCLUSION: Dietary tomato glycoalkaloids are cleaved during digestion to aglycones and further metabolized post-absorption. Steroidal alkaloids in plasma may serve as novel and specific biomarkers of tomato consumption and represent a class of phytochemical metabolites that could potentially have in vivo bioactivity impacting health and disease processes.

Urinary excretion of Citrus flavanones and their major catabolites after consumption of fresh oranges and pasteurized orange juice: A randomized cross-over study.

SCOPE: Orange juice contains flavanones including hesperidin and narirutin, albeit at lower concentrations as compared to orange fruit. Therefore, we compared bioavailability and colonic catabolism of flavanones from orange juice to a 2.4-fold higher dose from fresh oranges. METHODS AND RESULTS: Following a randomized two-way cross-over design, 12 healthy subjects consumed a test meal comprising either fresh oranges or pasteurized orange juice, delivering 1774 and 751 µmol of total Citrus flavanones, respectively. Deglucuronidated and desulfated hesperetin, naringenin, and the flavanone catabolites 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 4-hydroxyhippuric acid, and hippuric acid were quantitated in 24-h urine by UHPLC-MS/MS. Differences in urinary hesperetin excretion were found to be nonsignificant (p = 0.5209) both after consumption of orange fruit (21.6 ± 8.0 µmol) and juice (18.3 ± 7.2 µmol). By analogy, postprandial flavanone catabolite excretions were highly similar between treatments. Excretion of 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid was inversely related to that of hesperetin, illustrating the catabolite/precursor relationship. CONCLUSION: Despite 2.4-fold higher doses, excretion of flavanones from ingested fresh orange fruit did not differ from that following orange juice consumption, possibly due to a saturation of absorption or their entrapment in the fiber-rich matrix of the fruit.

An HPLC-MS/MS method for the separation of α-retinyl esters from retinyl esters.

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene.