Do genetic polymorphisms in a beta-carotene metabolising key enzyme influence dietary Vitamin A requirements?

Lead Research Organisation: Newcastle University
Department Name: Agriculture Food and Rural Development


Vitamin A (retinol) is an essential nutrient for vision, embryonic development, maturity of organs, cellular proliferation and the immune response. It is obtained from the diet per se or through provitamin A sources that are cleaved enzymatically in the body to produce retinol. Vitamin A deficiency occurs largely due to increases in physiological requirements (growth, pregnancy, lactation, infection) together with a low dietary intake. Currently, the mean daily intake of vitamin A is below the recommended intake levels for men and women aged 19 to 24 years. This means that young individuals in the UK rely to over 50% on provitamin A sources, beta-carotene being the main one, to cover their vitamin A needs. Since a significant proportion of young British individuals have a low vitamin A intake and since the Expert Group on Vitamins and Minerals has recommended to limit the use of beta-carotene in food supplements, there is growing concern that young men and women in the UK might develop subclinical deficiencies, therefore increasing their susceptibility to infectious diseases. This concern is especially valid for young pregnant women who will experience a higher physiological need for vitamin A. Given the fact that a high proportion of the British population relies on beta carotene to cover their vitamin A needs, it is important to note that the amount of retinol produced after ingestion and conversion of beta-carotene is highly variable between healthy individuals. Several studies have shown a large disparity between individuals who are efficient or inefficient converters of beta-carotene, with variations of up to 8 fold. It is possible that the observed differences in obtaining retinol from beta-carotene may be due to genetic polymorphisms in genes involved in aspects of beta-carotene conversion. Indeed, results from our laboratory have shown that two genetic variants exists in the key enzyme responsible for beta-carotene conversion with high frequencies, and that individuals inheriting this genetic trait (1 in 4 of the UK population) might need to increase their intake of preformed Vitamin A sources. The proposed study therefore plans to carry out a human intervention trial using state-of-the art whole body metabolic measurements to identify if individuals carrying this genetic variation might be at a higher risk of developing subclinical vitamin A deficiency. The study is designed to answer the following questions: 1) Have individuals with a genetic variation a reduced capacity of producing retinol from beta carotene when it is consumed at concentrations found in the diet? 2) Are there differences between men and women in their ability to produce retinol from provitamin A sources? 3) Is the intestine the main organ responsible for producing retinol from provitamin A sources, or is the conversion also occuring in other organs at comparable rates?

Technical Summary

Chronic intake of foods low in vitamin A (retinol) and provitamin A forming an unbalanced diet with little variety is common in young individuals in the UK population and can lead to subclinical micronutrient deficiency. Provitamin A sources such as beta-carotene are cleaved centrally by the beta-carotene 15,15' monooxygenase (BCMO1) into retinal, the precursor of retinol. However, the amount of beta-carotene and retinol produced after ingestion of beta-carotene is highly variable between healthy individuals, with approximately 40% of the subjects being classified as low responders. Several stable isotope studies have shown a large disparity between the most efficient converters and the most inefficient converters of beta-carotene with variations of up to 8 fold. It is possible that differences in beta-carotene response may be due to genetic polymorphisms in genes involved in aspects of beta-carotene conversion. Indeed, our latest results have shown that the recombinant R267S/A379V double variant of BCMO1 has only half the activity of the wild type. More importantly, 34% of the western population have the R267S/A379V haplotype (HapMap). A high percentage of the Western population may therefore not be able to achieve adequate vitamin A intake if dietary beta-carotene is a major source of their vitamin A. This is of particular relevance to vegetarians, to young individuals aged 19-24 years who have lower intakes of preformed retinol than any other age group, and to pregnant women. To verify if the current in vitro findings can be extended to the physiological situation, we propose to carry out a human intervention trial using a double tracer study design. The aim of this study is to identify if the R267S/A379V double variant in BCMO1 is indicative of the low responder trait observed in other studies.


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Description The main outcome of the funded BBSRC study so far is the development of a novel, rapid and sensitive LCMS method for high-throughput analysis of human plasma samples for _-carotene and vitamin A stable isotope studies. The method offers the advantage of a single extraction step from plasma and single 7 minute analytical LCMS run to quantify administered 13C10 _-carotene and derived metabolites. Another important advantage of this new method is that intestinal-derived retinol can be distinguished from hepatic-derived retinol due to the different way retinol is exported from these organs to the blood: retinyl esters in chylomicrons from the intestine, and retinol bound to retinol binding protein (RBP) from the liver. Since retinol and retinyl palmitate can both be quantified by the method, we can accurately determine the flux of retinol from these tissue compartments. We are in the process of submitting the method for publication and expect it to become the new standard analytical method in this area.

The study further indicated that a single physiological dose of both 13C10 labelled _-carotene as well as 13C10 retinyl acetate can be detected for up to 14 days. The method therefore shows great sensitivity and specificity, allowing the study of pro-vitamin A conversion and its importance in health and disease in free living volunteers. We also observed gender differences in circulating retinoids. Since this is not caused by different cleavage activities, it highlights the importance of vitamin A homeostasis and intensive metabolic flux between different tissues, which clearly would need further study. More importantly, the project was able to show that the intestine was the main site for provitamin A conversion. 33% of the total 13C5 retinoids produced from 13C10 _-carotene were secreted from the liver as 13C5 retinol within the first 24h, with a range of 16% to 46%. The large inter-individual differences in metabolite flux could potentially be caused through differing tissue uptake and storage, but unlikely via extra-intestinal conversion of _-carotene within the first 24 hours. Future modelling approaches will enable us to investigate this further.

Our group has shown that genetic variability in _-carotene metabolism may provide an explanation for the molecular basis of the poor converter phenotype within the population. Assessment of the responsiveness to a pharmacological dose of _-carotene in female volunteers confirmed that SNPs 5' upstream from the BCMO1 gene reduced the catalytic activity of BCMO1 in female volunteers by 59, 51, and 48%, respectively. Preliminary results indicate that metabolic effects of polymorphisms upstream of BCMO1 can be detected under physiological conditions. We are currently exploring this further by testing if a daily dose of 7mg _-carotene could overcome the low responder phenotype through a follow on project funded by DSM Nutritional Products. Once these data become available, we are in a position to publish these novel findings.
Exploitation Route The study results are important in relation to supplement producers as it will be important to investigate if a higher intake of provitamin A carotenoids could overcome the low responder phenotype. The new method allows a sensitive and accurate analysis of conversion at physiological concentrations and above.

Results from this study could also be of importance to international agencies such as FAO, WHO as well as Governmental Health Organisations. Detection of low responders in low income countries will allow to re-address public health policies on how to treat vitamin A deficiency through supplementation, fortification or food diversification.
The new LCMS method will allow a fast, efficient, sensitive and affordable measurement of total vitamin A status. This will be important for National Diet Surveys who currently rely on plasma retinol concentrations, which is not a reliable indicator of vitamin A status. The method will also be important for future studies on vitamin A status, and could be applied in studies investigating the importance of vitamin A in relation to immunology, pregnancy, lactation and bone health.
Sectors Agriculture, Food and Drink,Healthcare

Description The findings of the research award have allowed our research team to develop a novel LC/MS/MS method that has now been applied in two studies and is currently being employed in a large international investigation on vitamin A nutrition funded by the Bill and Melinda Gates Foundation. We have further been contacted by researchers from Indonesia, The Netherlands, Mexico as well as the Unites States to conduct stable isotope labelled vitamin A analysis of their serum samples using the newly established LC/MS/MS method. Thus, the research finding of the original award has now generated further income from 4 research grants and is employed in international research collaborations.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Healthcare
Description Assessing the risk of vitamin A toxicity due to large scale food fortification and other interventions
Amount $2,072,059 (USD)
Funding ID OPP1115464 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 01/2015 
End 01/2018
Description Effect of SNPs in the beta-carotene 15,15'-monooxygenase (BCMO1) enzyme on retinol formation and beta-carotene plasma responses
Amount £248,000 (GBP)
Organisation DSM 
Department DSM Nutritional Products
Sector Private
Country Switzerland
Start 01/2011 
End 12/2013
Description Effects of environmental enteropathy on micronutrient absorption and retention in young children, with emphasis on zinc homeostasis
Amount $59,494 (USD)
Funding ID OPP1113134 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 11/2015 
End 06/2017
Description LC/MS/MS analysis of beta-carotene and retinol and SNP analysis in the BCMO1 gene
Amount $108,380 (USD)
Organisation University of California, Davis 
Department School of Medicine
Sector Academic/University
Country United States
Start 11/2015 
End 06/2017
Title LC/MS/MS method for detecting stable isotopes of vitamin A in blood and tissue samples 
Description We have developed a new sensitive analytical method that allows the simultaneous study of beta-carotene bioefficacy and vitamin A status in human subjects at physiological doses. The simple extraction procedure and single 7 min LC/MS run-time for all analytes makes the method applicable to the high throughput of samples generated in large human intervention studies. Furthermore, the new method is more sensitive than comparable LC/MS methods currently employed in other laboratories. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact The method allowed us to be involved in a range of follow on funds received from DSM Nutritional products, The Gates Foundation as well as direct funds obtained from UC Davis in the United States. The method was also recognised by the IAEA in Vienna and lead to our involvement in the latest research efforts by the IAEA to harmonise the approaches of using stable isotopes in the analysis of vitamin A status.