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1.  Genetic Variants Influencing Biomarkers of Nutrition Are Not Associated with Cognitive Capability in Middle-Aged and Older Adults123 
The Journal of Nutrition  2013;143(5):606-612.
Several investigations have observed positive associations between good nutritional status, as indicated by micronutrients, and cognitive measures; however, these associations may not be causal. Genetic polymorphisms that affect nutritional biomarkers may be useful for providing evidence for associations between micronutrients and cognitive measures. As part of the Healthy Ageing across the Life Course (HALCyon) program, men and women aged between 44 and 90 y from 6 UK cohorts were genotyped for polymorphisms associated with circulating concentrations of iron [rs4820268 transmembrane protease, serine 6 (TMPRSS6) and rs1800562 hemochromatosis (HFE)], vitamin B-12 [(rs492602 fucosyltransferase 2 (FUT2)], vitamin D ([rs2282679 group-specific component (GC)] and β-carotene ([rs6564851 beta-carotene 15,15'-monooxygenase 1 (BCMO1)]. Meta-analysis was used to pool within-study effects of the associations between these polymorphisms and the following measures of cognitive capability: word recall, phonemic fluency, semantic fluency, and search speed. Among the several statistical tests conducted, we found little evidence for associations. We found the minor allele of rs1800562 was associated with poorer word recall scores [pooled β on Z-score for carriers vs. noncarriers: −0.05 (95% CI: −0.09, −0.004); P = 0.03, n = 14,105] and poorer word recall scores for the vitamin D–raising allele of rs2282679 [pooled β per T allele: −0.03 (95% CI: −0.05, −0.003); P = 0.03, n = 16,527]. However, there was no evidence for other associations. Our findings provide little evidence to support associations between these genotypes and cognitive capability in older adults. Further investigations are required to elucidate whether the previous positive associations from observational studies between circulating measures of these micronutrients and cognitive performance are due to confounding and reverse causality.
doi:10.3945/jn.112.171520
PMCID: PMC3738233  PMID: 23468552
2.  Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression 
Background
Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression.
Results
We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression.
Conclusion
We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene.
doi:10.1186/1471-2199-7-7
PMCID: PMC1526748  PMID: 16504037
3.  Hepatic stellate cells are an important cellular site for β-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 β-carotene, but the ability of HSCs to metabolize β-carotene in situ has not been explored. To gain understanding of this, we investigated whether β-carotene-15,15′-monooxygensase (Bcmo1) and β-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 β-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 β-carotene-containing diet compared to mice receiving no β-carotene. Collectively, our data establish that HSCs are an important cellular site for β-carotene accumulation and metabolism within the liver.
doi:10.1016/j.abb.2010.05.010
PMCID: PMC3692274  PMID: 20470748
Vitamin A; Carotenoid; Carotene cleavage; Chylomicron; Diet
4.  Beta-carotene affects gene expression in lungs of male and female Bcmo1−/− mice in opposite directions 
Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1−/− mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1−/− mice. Testosterone levels were higher after BC supplementation only in Bcmo1−/− mice, which had, unlike wild-type (Bcmo1+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.
doi:10.1007/s00018-010-0461-0
PMCID: PMC3021199  PMID: 20820853
Sex-hormones; Mouse whole genome microarray gene expression analysis; Beta-carotene 15,15′-monooxygenase 1; Steroids; Retinol; Retinoic acid; Gender effect; Transcriptome
5.  In Vitro and In Vivo Characterization of Retinoid Synthesis from β-carotene 
Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like β-carotene, through the actions β-carotene 15,15′ monooxygenase (BCMO1). As the substrates for retinoid metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact β-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms.
doi:10.1016/j.abb.2008.02.010
PMCID: PMC2587144  PMID: 18295589
β-carotene; cleavage; β-carotene 15; 15′ monooxygenase; retinoid; metabolism; cellular retinol binding protein
6.  Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene 
Cellular and Molecular Life Sciences   2010;67(12):2039-2056.
Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1+/+) mice efficiently cleave BC. Bcmo1−/− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1−/− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1−/− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1−/− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1−/− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.
doi:10.1007/s00018-010-0341-7
PMCID: PMC2877315  PMID: 20372966
ATBC and CARET study; Beta-carotene 15,15′-monooxygenase 1; Whole-mouse genome microarray gene expression; Inflammation; Vitamin A deficiency; ADH7; LRAT
7.  β-Carotene conversion products and their effects on adipose tissue 
Genes & Nutrition  2009;4(3):179-187.
Recent epidemiological data suggest that β-carotene may be protective against metabolic diseases in which adipose tissue plays a key role. Adipose tissue constitutes the major β-carotene storage tissue and its functions have been shown to be modulated in response to β-carotene breakdown products, especially retinal produced after cleavage by β-carotene 15,15′-monooxygenase (BCMO1), and retinoic acid arising from oxidation of retinal. However, the possibility exists that β-carotene in its intact form can also affect adipocyte function. Development of a knock out model and identification of a loss-of-function mutation have pointed out BCMO1 as being probably the sole enzyme responsible for provitamin A conversion into retinal in mammals. The utilisation of BCMO1−/−mice should provide insights on β-carotene effect on its own in the future. In humans, intervention studies have highlighted the huge interindividual variation of β-carotene conversion efficiency, possibly due to genetic polymorphisms, which might impact on response to β-carotene. This brief review discusses the processes involved in β-carotene conversion and the effect of cleavage products on body fat and adipose tissue function.
doi:10.1007/s12263-009-0128-3
PMCID: PMC2745744  PMID: 19557453
β-Carotene; Vitamin A; Adipose tissue; Metabolic diseases; Genetic variants
8.  Which Circulating Antioxidant Vitamins Are Confounded by Socioeconomic Deprivation? The MIDSPAN Family Study 
PLoS ONE  2010;5(6):e11312.
Background
Antioxidant vitamins are often described as having “independent” associations with risk of cancer, cardiovascular disease (CVD) and mortality. We aimed to compare to what extent a range of antioxidant vitamins and carotenoids are associated with adulthood and childhood markers of socioeconomic deprivation and to adverse lifestyle factors.
Methods and Findings
Socioeconomic and lifestyle measures were available in 1040 men and 1298 women from the MIDSPAN Family Study (30–59 years at baseline) together with circulating levels of vitamins A, C, E, and carotenoids (α-carotene, β-carotene, lutein and lycopene). Markers of socioeconomic deprivation in adulthood were consistently as strongly associated with lower vitamin C and carotenoid levels as markers of adverse lifestyle; the inverse association with overcrowding was particularly consistent (vitamin C and carotenoids range from 19.1% [95% CI 30.3–6.0] to 38.8% [49.9–25.3] lower among those in overcrowded residencies). These associations were consistent after adjusting for month, classical CVD risk factors, body mass index, physical activity, vitamin supplements, dietary fat and fibre intake. Similar, but weaker, associations were seen for childhood markers of deprivation. The association of vitamin A or E were strikingly different; several adult adverse lifestyle factors associated with higher levels of vitamin A and E, including high alcohol intake for vitamin A (9.5% [5.7–13.5]) and waist hip ratio for vitamin E (9.5% [4.8–14.4]), with the latter associations partially explained by classical risk factors, particularly cholesterol levels.
Conclusions
Plasma vitamin C and carotenoids have strong inverse associations with adulthood markers of social deprivation, whereas vitamin A and E appear positively related to specific adverse lifestyle factors. These findings should help researchers better contextualize blood antioxidant vitamin levels by illustrating the potential limitations associated with making causal inferences without consideration of social deprivation.
doi:10.1371/journal.pone.0011312
PMCID: PMC2892487  PMID: 20593021
9.  High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type 2 diabetes 
Background
Patients with type 2 diabetes have increased levels of oxidative stress and inflammation. A high fruit and vegetable intake may be beneficial.
Objective
To study whether fruit and vegetable intake and levels of plasma antioxidants relate to markers of oxidative stress and inflammation in a group of patients with type 2 diabetes. Further, to investigate whether plasma antioxidants are good biomarkers for intake of fruit and vegetables.
Design
Patients with type 2 diabetes were studied. Their dietary intake and levels of plasma antioxidants, and markers of oxidative stress and inflammation were analysed.
Results
Fruit and vegetable intake was inversely related to oxidative stress. Plasma carotenoids were negatively correlated with inflammation. The plasma levels of α-carotene and β-carotene showed strongly positive associations with fruit and vegetable intake.
Conclusions
The results suggest that fruit and vegetable intake may decrease oxidative stress and inflammation in this group of patients. An increased intake of fruit and vegetables can therefore be beneficial for patients with type 2 diabetes, since these patients are documented to have raised oxidative stress and inflammation. The study support the usefulness of plasma α-carotene and β-carotene as biomarkers for fruit and vegetable intake.
doi:10.1080/17482970701737285
PMCID: PMC2606994
antioxidants; fruit; inflammatory cytokine; oxidative stress; type 2 diabetes; vegetables
10.  Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase 
BMC Biochemistry  2009;10:31.
Background
β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system.
Results
Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.
Conclusions
Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.
doi:10.1186/1471-2091-10-31
PMCID: PMC2801523  PMID: 20003456
11.  A nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene is tightly associated with accumulation of carotenoids in adipose tissue in sheep (Ovis aries) 
BMC Genetics  2010;11:10.
Background
Sheep carcasses with yellow fat are sporadically observed at Norwegian slaughter houses. This phenomenon is known to be inherited as a recessive trait, and is caused by accumulation of carotenoids in adipose tissue. Two enzymes are known to be important in carotenoid degradation in mammals, and are therefore potential candidate genes for this trait. These are beta-carotene 15,15'-monooxygenase 1 (BCMO1) and the beta-carotene oxygenase 2 (BCO2).
Results
In the present study the coding region of the BCMO1 and the BCO2 gene were sequenced in yellow fat individuals and compared to the corresponding sequences from control animals with white fat. In the yellow fat individuals a nonsense mutation was found in BCO2 nucleotide position 196 (c.196C>T), introducing a stop codon in amino acid position 66. The full length protein consists of 575 amino acids. In spite of a very low frequency of this mutation in the Norwegian AI-ram population, 16 out of 18 yellow fat lambs were found to be homozygous for this mutation.
Conclusion
In the present study a nonsense mutation (c.196C>T) in the beta-carotene oxygenase 2 (BCO2) gene is found to strongly associate with the yellow fat phenotype in sheep. The existence of individuals lacking this mutation, but still demonstrating yellow fat, suggests that additional mutations may cause a similar phenotype in this population. The results demonstrate a quantitatively important role for BCO2 in carotenoid degradation, which might indicate a broad enzyme specificity for carotenoids. Animals homozygous for the mutation are not reported to suffer from any negative health or development traits, pointing towards a minor role of BCO2 in vitamin A formation. Genotyping AI rams for c.196C>T can now be actively used in selection against the yellow fat trait.
doi:10.1186/1471-2156-11-10
PMCID: PMC2828402  PMID: 20122251
12.  The Biochemical Characterization of Ferret Carotene-9′, 10′-Monooxygenase Catalyzing Cleavage of Carotenoids in Vitro and in Vivo* 
The Journal of biological chemistry  2006;281(28):19327-19338.
Previous studies have shown that β-carotene 15,15′-monooxygenase catalyzes the cleavage of β-carotene at the central carbon 15,15′-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9′,10′-monooxygenase (CMO2) in β-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10′-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9′,10′-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of β-carotene/lycopene cleavage into β-apo-10′-carotenal/apo-10′-lycopenal in vitro and the formation of apo-10′-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-β-carotene and the 5-cis- and 13-cis-isomers of lycopene at the 9′,10′-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with β-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10′-lycopenol, as well as up-regulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10′-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10′-lycopenoic acid and apo-10′-lycopenol, respectively, depending upon the presence of NAD+ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10′-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.
doi:10.1074/jbc.M512095200
PMCID: PMC1819471  PMID: 16672231
13.  Carotenoids and alcoholic liver disease 
Chronic and excessive consumption of alcohol leads to the development of alcoholic liver disease. The depletion of vitamin A is a well-known consequence of alcohol consumption, and may be associated with the observed alcohol-induced hepatic injury. The provitamin A carotenoid β-carotene has been demonstrated to increase alcohol-induced hepatic injury when given in high doses, while low dose supplementation provides protection against hepatic injury. However, it is unknown if the hepatoprotective effects of low dose β-carotene are due to the protective actions of β-carotene itself or if the alterations are due to restored vitamin A levels. Future studies are needed to provide further insight into the specific mechanisms by which β-carotene exerts its protective effect. Further, supplementation studies utilizing high doses of β-carotene in the presence of alcohol must be done with caution.
doi:10.3978/j.issn.2304-3881.2013.10.01
PMCID: PMC3924697  PMID: 24570953
Carotenoids; β-carotene; vitamin A; alcoholic liver disease; hepatic injury
14.  Beta-Carotene Reduces Body Adiposity of Mice via BCMO1 
PLoS ONE  2011;6(6):e20644.
Evidence from cell culture studies indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15′-oxygenase (Bcmo1) and the BC-9′,10′-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into β-10′-apocarotenal and β-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1-/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and β-10′-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite β-10′-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARγ activity in adipocytes
doi:10.1371/journal.pone.0020644
PMCID: PMC3106009  PMID: 21673813
15.  The Interplay Between Uric Acid and Antioxidants in Relation to Physical Function in Older Persons 
OBJECTIVES
To investigate the relationship between circulating uric acid (UA) levels and plasma antioxidants and whether antioxidant levels modulate the association between UA and physical function.
DESIGN
Cross-sectional.
SETTING
Community-based.
PARTICIPANTS
Nine hundred sixty-six elderly persons participating in the baseline assessment of the Invecchiare in Chianti Study.
MEASUREMENTS
UA, carotenoid, tocopherol, and selenium concentrations were assayed. Physical function was measured using the Short Physical Performance Battery (SPPB) and difficulties in instrumental activities of daily living (IADLs). Potential confounders were assessed using standardized methods.
RESULTS
Total carotenoids (P =.008), in particular α-carotene (P =.02), lutein (P<.001), zeaxanthin (P<.001), lycopene (P =.07), cryptoxanthin (P =.29), and selenium (P =.04) were inversely associated with UA levels. Total tocopherols (P =.06) and α-tocopherol (P =.10) had a positive trend across UA levels. SPPB (P =.01) and IADL disability (P =.002) were nonlinearly distributed across the UA quintiles. Participants within the middle UA quintile (4.8–5.3 mg/dL) were less disabled in IADLs and had better SPPB scores than those in the extreme UA quintiles. There was a significant interaction between UA and selenium in the model predicting SPPB score (P =.02).
CONCLUSION
UA levels are inversely associated with circulating carotenoids and selenium. Participants with intermediate UA levels had a higher prevalence of good physical functions, higher SPPB scores, and lower IADL disability. This study suggests that older subjects with intermediate UA levels may have an optimum balance between proinflammatory and antioxidant compounds that may contribute to better physical performance.
doi:10.1111/j.1532-5415.2007.01260.x
PMCID: PMC2669302  PMID: 17661959
uric acid; antioxidants; metabolism; inflammation; physical function; disability
16.  Circulating carotenoids, mammographic density, and subsequent risk of breast cancer 
Cancer research  2009;69(24):9323.
Mammographic density is one of the strongest predictors of breast cancer risk. Recently, it has been suggested that reactive oxygen species may influence breast cancer risk through its influence on mammographic density. In the current study, we addressed this hypothesis and also assessed if the association between carotenoids and breast cancer risk varies by mammographic density. We conducted a nested case-control study consisting of 604 breast cancer cases and 626 controls with prospectively measured circulating carotenoid levels and mammographic density in the Nurses’ Health Study. Circulating levels of α-carotene, β-carotene, β-cryptoxanthin, lycopene, and lutein/zeaxanthin were measured. We utilized a computer-assisted thresholding method to measure percent mammographic density. We found no evidence that circulating carotenoids are inversely associated with mammographic density. However, mammographic density significantly modified the association between total circulating carotenoids and breast cancer (P-heterogeneity=0.008). Overall, circulating total carotenoids was inversely associated with breast cancer risk (p-trend=0.01). Among women in the highest tertile of mammographic density, total carotenoids was associated with a 50% reduction in breast cancer risk (OR=0.5; 95% CI 0.3-0.8). In contrast, there was no inverse association between carotenoids and breast cancer risk among women with low mammographic density. Similarly, among women in the highest tertile of mammographic density, high levels of circulating α-carotene, β-cryptoxanthin, lycopene and lutein/zeaxanthin were associated with a significant 40-50% reduction in breast cancer risk (P-trend<0.05). Our results suggest that plasma levels of carotenoids may play a role in reducing breast cancer risk, particularly among women with high mammographic density.
doi:10.1158/0008-5472.CAN-09-1018
PMCID: PMC2820729  PMID: 19934322
Circulating carotenoids; mammographic density; antioxidants; breast cancer; oxidative stress
17.  Associations Between α-Tocopherol, β-Carotene, and Retinol and Prostate Cancer Survival 
Cancer research  2009;69(9):3833-3841.
Previous studies suggest that carotenoids and tocopherols (vitamin E compounds) may be inversely associated with prostate cancer risk, yet little is known about how they affect prostate cancer progression and survival. We investigated whether serum α-tocopherol, β-carotene, and retinol concentrations, or the α-tocopherol and β-carotene trial supplementation, affected survival of men diagnosed with prostate cancer during the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized, double-blind, placebo-controlled primary prevention trial testing the effects of β-carotene and α-tocopherol supplements on cancer incidence in adult male smokers in southwestern Finland (n=29,133). Prostate cancer survival was examined using the Kaplan-Meier method with deaths from other causes treated as censoring, and using Cox proportional hazards regression models with hazard ratios (HR) and 95% confidence intervals (95% CI) adjusted for family history of prostate cancer, age at randomization, benign prostatic hyperplasia, age and stage at diagnosis, height, BMI, and serum cholesterol. As of April 2005, 1,891 men were diagnosed with prostate cancer and 395 died of their disease. Higher serum α-tocopherol at baseline was associated with improved prostate cancer survival (HR=0.67, 0.45–1.00), especially among cases who had received the trial’s α-tocopherol intervention and who were in the highest quintile of α-tocopherol at baseline (HR=0.51, 0.20–0.90) or at the 3-year follow-up measurement (HR=0.26, 0.09–0.71). Serum β-carotene, serum retinol, and supplemental β-carotene had no apparent effects on survival. These findings suggest that higher α-tocopherol (and not β-carotene or retinol) status increases overall prostate cancer survival. Further investigations, possibly including randomized studies, are needed to confirm this observation.
doi:10.1158/0008-5472.CAN-08-4640
PMCID: PMC2787239  PMID: 19383902
18.  Differential Expression of the Demosponge (Suberites domuncula) Carotenoid Oxygenases in Response to Light: Protection Mechanism Against the Self-Produced Toxic Protein (Suberitine) 
Marine Drugs  2012;10(1):177-199.
The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the β,β-carotene-15,15′-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only β-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade β-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with β-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from β-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin.
doi:10.3390/md10010177
PMCID: PMC3280542  PMID: 22363229
Suberitine; β-carotene; retinal; β-carotene dioxygenase; sponges; Suberites domuncula
19.  Impact of Circulating Vitamin D Binding Protein Levels on the Association Between 25-Hydroxyvitamin D and Pancreatic Cancer Risk: A Nested Case-Control Study 
Cancer Research  2012;72(5):1190-1198.
High concentrations of circulating 25-hydroxyvitamin D [25(OH)D] have been associated with elevated pancreatic cancer risk. As this is contrary to an expected inverse association between vitamin D status and cancer, we examined whether vitamin D binding protein (DBP), the primary carrier of vitamin D compounds in circulation, plays a role in this relationship. Prediagnostic serum DBP and 25(OH)D were studied in relation to risk of pancreatic cancer in a nested case-control study of 234 pancreatic cancer cases and 234 controls in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish men. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression, and statistical tests were two-sided. We found that DBP and 25(OH)D were correlated (r=0.27; p<0.0001), and DBP was inversely associated with pancreatic cancer risk (OR=0.66, 95% CI=0.39–1.12, for the highest vs. lowest quartile; p-trend=0.02). Importantly, this association appeared to have a threshold between quartiles 2–4 and quartile 1, and was primarily evident among men with concurrent high 25(OH)D concentrations (OR=0.33, 95% CI=0.16–0.70 for highest vs. lowest quartile; p-trend=0.002), with no association in men with lower serum 25(OH)D (OR=1.28, 95% CI=0.62–2.61 for highest vs. lowest quartile, p-trend 0.63, p-interaction= 0.01). Men with higher 25(OH)D concentrations and serum DBP below the median showed greatly elevated risk of pancreatic cancer (OR=5.01, 95% CI 2.33–10.78, for highest vs. lowest quartile; p-trend < 0.0001), while risk was weakly inversely associated with serum 25(OH)D when DBP concentrations were higher (p-interaction = 0.001). Taken together, our findings indicate that higher DBP concentrations may sequester more 25(OH)D and reduce free 25(OH)D bioavailability. Simultaneous examination of DBP and 25(OH)D may be important in determining the association of vitamin D with cancer risk.
doi:10.1158/0008-5472.CAN-11-2950
PMCID: PMC3294078  PMID: 22232734
Vitamin D Binding Protein; 25-Hydroxyvitamin D; Pancreatic Cancer; serum biomarkers; prospective study
20.  Detection of a Cis eQTL Controlling BMCO1 Gene Expression Leads to the Identification of a QTG for Chicken Breast Meat Color 
PLoS ONE  2011;6(7):e14825.
Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: AJ271386), encoding the β-carotene 15, 15′-monooxygenase, a key enzyme in the conversion of β-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of β-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A.
doi:10.1371/journal.pone.0014825
PMCID: PMC3130028  PMID: 21750696
21.  Enzymatic Formation of Apo-Carotenoids from the Xanthophyll Carotenoids Lutein, Zeaxanthin and β-Cryptoxanthin by Ferret Carotene-9’,10’-Monooxygenase 
Xanthophyll carotenoids, such as lutein, zeaxanthin and β-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15’-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9’,10’-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirus-generated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and β-cryptoxanthin. Utilizing HPLC, LC-MS and GC-MS, we identified both volatile and non-volatile apocarotenoid products including 3-OH-β-ionone, 3-OH-α-ionone, β-ionone, 3-OH-α-apo-10’-carotenal, 3-OH-β-apo-10’-carotenal, and β-apo-10’-carotenal, indicating cleavage at both the 9,10 and 9’,10’ carbon-carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over β-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-β-apo-10’-carotenal with CMO2 lysate resulted in the formation of 3-OH-β-ionone. In the presence of NAD+, in vitro incubation of 3-OH-β-apo-10’-carotenal with ferret hepatic homogenates formed 3-OH-β-apo-10’-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.
doi:10.1016/j.abb.2010.11.005
PMCID: PMC3026080  PMID: 21081106
Xanthophyll; metabolism; CMO1; CMO2; Apo-carotenoid
22.  Genetic evidence that raised sex hormone binding globulin (SHBG) levels reduce the risk of type 2 diabetes 
Human Molecular Genetics  2009;19(3):535-544.
Epidemiological studies consistently show that circulating sex hormone binding globulin (SHBG) levels are lower in type 2 diabetes patients than non-diabetic individuals, but the causal nature of this association is controversial. Genetic studies can help dissect causal directions of epidemiological associations because genotypes are much less likely to be confounded, biased or influenced by disease processes. Using this Mendelian randomization principle, we selected a common single nucleotide polymorphism (SNP) near the SHBG gene, rs1799941, that is strongly associated with SHBG levels. We used data from this SNP, or closely correlated SNPs, in 27 657 type 2 diabetes patients and 58 481 controls from 15 studies. We then used data from additional studies to estimate the difference in SHBG levels between type 2 diabetes patients and controls. The SHBG SNP rs1799941 was associated with type 2 diabetes [odds ratio (OR) 0.94, 95% CI: 0.91, 0.97; P = 2 × 10−5], with the SHBG raising allele associated with reduced risk of type 2 diabetes. This effect was very similar to that expected (OR 0.92, 95% CI: 0.88, 0.96), given the SHBG-SNP versus SHBG levels association (SHBG levels are 0.2 standard deviations higher per copy of the A allele) and the SHBG levels versus type 2 diabetes association (SHBG levels are 0.23 standard deviations lower in type 2 diabetic patients compared to controls). Results were very similar in men and women. There was no evidence that this variant is associated with diabetes-related intermediate traits, including several measures of insulin secretion and resistance. Our results, together with those from another recent genetic study, strengthen evidence that SHBG and sex hormones are involved in the aetiology of type 2 diabetes.
doi:10.1093/hmg/ddp522
PMCID: PMC2798726  PMID: 19933169
23.  Plasma retinol, carotene and vitamin E concentrations and lung function in a crocidolite-exposed cohort from Wittenoom, Western Australia: a cohort study 
Nutrition Journal  2005;4:16.
Background
Increased rates of death from asbestos related diseases have been reported for people previously employed in the mining and milling operations at Wittenoom (Western Australia), and people who lived in the nearby town, where they were environmentally exposed to crocidolite.
Methods
Annual measurements of forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) and plasma concentrations of retinol, carotene and vitamin E have been made since 1992. Mixed effects models were used to examine the associations between lung function and the plasma vitamin levels of retinol, carotene and vitamin E.
Results
After adjusting for potential confounders, higher plasma retinol and carotene concentrations were significantly associated with higher levels of lung function at entry into the study, while vitamin E concentrations were associated with lower entry lung function. Retinol was associated with a less steep decline of lung function over time, while carotene concentrations were associated with an increased decline of lung function over time and vitamin E levels were not associated with changes of lung function over time.
Conclusion
These results support a beneficial relationship between plasma concentrations of retinol on the levels and rates of change of lung function, while showing no such consistent beneficial effect for plasma levels of beta-carotene or vitamin E.
doi:10.1186/1475-2891-4-16
PMCID: PMC1156941  PMID: 15888203
24.  Influence of vitamin D binding protein on the association between circulating vitamin D and risk of bladder cancer 
British Journal of Cancer  2012;107(9):1589-1594.
Background:
There is little research investigating the role of vitamin D binding protein (DBP) in the association between 25-hydroxyvitamin D (25(OH)D) and disease risk.
Methods:
Within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, 250 bladder cancer cases were randomly sampled and matched 1:1 to controls on age and date of blood collection. Odds ratios (OR) and 95% confidence intervals (CI) of bladder cancer were estimated by quartiles of DBP (measured by ELISA), 25(OH)D and the molar ratio of 25(OH)D:DBP, a proxy for free circulating 25(OH)D. Analyses were also conducted stratifying 25(OH)D by DBP (median split) and vice versa.
Results:
We found no direct association between circulating DBP levels and bladder cancer risk (P-trend=0.83). The inverse association between 25(OH)D and bladder cancer risk was unchanged after adjustment for DBP (Q4 vs Q1 OR=0.61, 95% CI=0.36–1.05; P-trend=0.04), and was stronger among men with lower DBP (low DBP: 25(OH)D Q4 vs Q1 OR=0.47, 95% CI=0.23–1.00; high DBP: 25(OH)D Q4 vs Q1 OR=0.83, 95% CI=0.40–1.75; P for interaction=0.11).
Conclusion:
Our findings provide additional support for an aetiologic role for vitamin D in bladder cancer and suggest that free, rather than total, circulating vitamin D may be a more relevant exposure when examining bladder and, perhaps, other cancers.
doi:10.1038/bjc.2012.417
PMCID: PMC3493763  PMID: 22990651
urinary bladder neoplasms; 25-hydroxyvitamin D; vitamin D binding protein; case–control study
25.  Genome-wide association study of circulating retinol levels 
Human Molecular Genetics  2011;20(23):4724-4731.
Retinol is one of the most biologically active forms of vitamin A and is hypothesized to influence a wide range of human diseases including asthma, cardiovascular disease, infectious diseases and cancer. We conducted a genome-wide association study of 5006 Caucasian individuals drawn from two cohorts of men: the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study and the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. We identified two independent single-nucleotide polymorphisms associated with circulating retinol levels, which are located near the transthyretin (TTR) and retinol binding protein 4 (RBP4) genes which encode major carrier proteins of retinol: rs1667255 (P =2.30× 10−17) and rs10882272 (P =6.04× 10−12). We replicated the association with rs10882272 in RBP4 in independent samples from the Nurses’ Health Study and the Invecchiare in Chianti Study (InCHIANTI) that included 3792 women and 504 men (P =9.49× 10−5), but found no association for retinol with rs1667255 in TTR among women, thus suggesting evidence for gender dimorphism (P-interaction=1.31× 10−5). Discovery of common genetic variants associated with serum retinol levels may provide further insight into the contribution of retinol and other vitamin A compounds to the development of cancer and other complex diseases.
doi:10.1093/hmg/ddr387
PMCID: PMC3209826  PMID: 21878437

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