For my Agricultural Biochemistry class we had to write a research paper that focused on Golden Rice ( A genetically modified organism (GMO)) as a good source of Vitamin A. A long story short this rice was developed to provide Vitamin A to poor countries that have citizens with vitamin A deficiencies. The rice is created by taking genes from other plants and bacteria that synthesize beta carotene. These genes are then added to the rice's genome to create this rice that is Golden in color hence the name. Beta carotene is a precursor of Vitamin A. Your body must convert the Beta Carotene to Vitamin A by biochemical processes that require the utilization of fat. So my paper discusses some of the issues of Beta Carotene as a source of Vitamin A. Enjoy!
The Bioavailability and Bioefficacy of
Beta-carotene as a Source of Vitamin A
by Nadia Ruffin
Biochemistry 818 Fall Semester
Golden rice is a genetically modified organism that has incorporated beta-carotene genes from bacteria and dafodils into rice. The combination of beta-carotene into a staple food like rice could be the cure to ending Vitamin A deficiency thus combating blindness and reproductive issues in third world countries . While the methodology has shown promising results there is still much research that needs to be done on a biochemical level in regarding to the efficiency of beta-carotene being used as a main source of vitamin A. In my paper I decided to look at the bioavailability of beta-carotene in food. Then I looked into the bioefficacy of converting beta-carotene into retinol and how different factors can affect the outcome. Finally I looked at how absorption of retinol can be effected by varying levels of oil in the diet.
Vitamin A is a fat soluble vitamin. The name vitamin A is given to a group of organic compounds known as retinoids: these includes retinol, retinal, retinoic acid, and provitamin A carotenoids. The main dietary forms of preformed vitamin A are carotenoids (most common Beta-carotene) in fruits and vegetables and long-chain Fatty Acid esters of retinol in foods of animal origin (Plack, 1965). It is interesting how beta-carotene rich foods are always recommended as a good source of vitamin A. In actuality carotenoids are a precursor for vitamin A and must be converted into retinol before the body can utilize it. Animal products like liver, dairy or eggs would be better sources of retinol because they are already bioavailable and do not require them to them be converted. It is at this point that utilizing carotenoids as a source of vitamin A may become a problem.
Bioavailability is defined as the fraction of an ingested nutrient from food that is available for absorption in the intestine and metabolic process and storage (Jackson, 1997). There are many factors that affect bioavailability of beta-carotene like source of food, how it is prepared etc. In a study conducted by Dr. Hedren and colleagues they performed an in-vitro digestion simulation to see how much beta-carotene is actually extracted from carrots. Their setup consisted of batches of raw carrots just cut, pulped carrots with cooking oil, pulped carrots without oil and pulped cooked carrots with cooking oil added. They added pepsin-HCl dropping the pH to 2.0 and then raised the pH to 7.5 and a pancreatin-bile extract mixture was added at the end. It has been shown that bile salts play a role in extracting carotenoids from food. What they found is that three percent of the total beta-carotene content was released from raw carrots in pieces. When homogenized (pulped) 21% of beta-carotene was released. Cooking the pulp increased the accessibility to 27%. Addition of cooking oil to the cooked pulp further increased the released amount to 39% (Hedren et al, 2002). It was interesting that the amount of beta-carotene released after cooking and adding oil increased. While cooking beta-carotene rich vegetables does release more beta carotene there can also be some loss. According to the USDA vegetables can lose roughly 5 percent of their total beta-carotene through baking or roasting. When compared these cooking methods to boiling, the total loss of beta-carotene can be between 10 to 15 percent. The longer you boil food the more you give it the opportunity for beta-carotene to leach into the water. Also simply mashing the vegetables can release more beta-carotene than in it raw unadulterated form.
So how is this relevant to golden rice? Well to prepare rice it must be cooked for extensive period of time to get it to the desired tenderness. There was a study by Guangwen Tang et al. on Golden Rice that made a claim that it did not lose it's beta-carotene content even after cooking, based off their analysis, but they never explained how they analyzed it. Their study also yielded an estimated 80% conversion rate of beta-carotene to retinol. This feeding trial was performed on only five healthy US volunteers, showed the effective conversion of Golden Rice β-carotene, even though all individuals were of normal vitamin A status (Tang et.al, 2009). This study and other golden rice studies do not test the conversion of beta-carotene to retinol in vitamin A deficient populations which brings up the next point converting beta-carotene to retinol.
The mechanism to cleave beta-carotene in half uses an enzyme called dioxygenase, the product is two molecules of retinal that is further reduced using reductase to retinol. While this sounds simple it's not that cut and dry. Bioefficacy is the capability of an individual —governed by individual internal factors— to convert β-carotene (provitamin A) into retinal, retinol (vitamin A) and retinyl esters. It is expressed as the amount of β-carotene required to yield one retinol activity equivalent (1 RAE) (Yeun, 2002). To explain this further 1 mcg of available retinol is equivalent to the following amounts from animal or plant dietary sources: 1 mcg of retinol, 12 mcg of beta-carotene, and 24 mcg of alpha-carotene or beta-cryptoxanthin. There are many factors that affect the conversion of beta-carotene to retinol. Some examples of these are gender, availability of carotenoids vs retinoids and most recently scientists have found that genetics plays a role as well.
In two separate studies by different researchers each looked at how gender played a role in beta-carotene conversion. Hinkenbottom et al. studied the conversion of beta-carotene to retinol in men and found that conversion rates were variable and surprisingly low. Using a double tracer study 11 healthy men were given orally 30 micromol hexadeuterated (D6) retinyl acetate (all-trans-19,19,19,20,20,20-[2H6]retinyl acetate) and then were given 37 micromol D6 beta-carotene (19,19,19,19',19',19'-[2H6]beta-carotene) one week later. All doses were given during breakfast. Plasma samples from each of the participants were analyzed for levels of D6 retinol, D6 beta-carotene, and trideuterated (D3) retinol (derived from D6 beta-carotene) (Hinkenbottom et al, 2002). Using HPLC they were able to see the conversion of D6 beta-carotene to D3 retinol.. Only 6 of the 11 men had measurable levels. The mean absorption of D6 beta-carotene in these 6 subjects was 4.097 +/- 1.208%, and the mean conversion ratio was 0.0540 +/- 0.0128 mol retinol to 1 mol beta-carotene (Hinkenbottom et al, 2002). This same study was conducted two years earlier on women by Lin, a colleague of Hinkenbottom. The results for this study were very similar to the results of the men's study. Women had low conversion and it was variable. Eleven women were selected and of them only 6 were able to convert D6 beta-carotene to D3 retinol. The mean absorption of D(6) beta-carotene in these 6 subjects was 6.1 +/- 0.02% and their conversion ratio was 1.47 +/- 0.49 mol D(3) retinol to 1 mol D(6) beta-carotene. The remaining 5 subjects were low responders with </=0.01% absorption and a mean conversion ratio of 0.014 +/- 0.004 mol D(3) retinol to 1 mol D(6) beta-carotene (Lin, 2000). In both studies there were measurable levels of D6 retinol. This retinol was not derived from beta-carotene and did not require the conversion to be absorbed. Looking at these results it shows that getting the recommended amount of vitamin A from beta-carotene may not be a good source. Why is the conversion so varied and low for so many people?
Genetics plays a role in determine who can efficiently convert beta-carotene and who cannot. A study conducted in the UK by Wing Leung and colleagues investigated the effects of the four identified SNP 5’ (rs6420424, rs8044334, rs11645428, and rs6564851) upstream from the BCMO1 gene on beta-carotene bioconversion or conversion efficiency. Recent studies have shown that BCMO1 gene can affect circulating carotenoid concentrations. Twenty-eight women volunteered to be part of this study. The women fasted for 12 hours and were given a 120 mg β-carotene (10% SWS from DSM Nutritional Products) together with a fat-rich meal the consisted of a muffin, fruit yoghurt and water (Leung et al., 2009). Blood samples were taken before the supplement and meal was given and again three hours after the meal. A total of 36% of women had retinol equivalent intakes above the recommended nutrient intake of 600 μg/d and 14% had intakes below the lower recommended nutrient intake of 250 μg/d. Fasting mean plasma concentrations for β-carotene and retinol were 0.36 μmol/L (95% CI = 0.29–0.45 μmol/L) and 1.52 μmol/L (95% CI = 1.36–1.69 μmol/L), respectively (Leung et al., 2009). SNP analysis was performed using PCR on plasma samples collected from the blood. The women were categorized by either being responders or poor responders on their ability to convert beta-carotene. Women who carried these three alleles: A allele (rs6420424), G allele (rs11645428), and G allele (rs6564851) of these intronic SNPs showed a reduction in conversion efficiency by up to 59% (Leung, 2009). They found that there is variation among races and very well could be variations amongst genders. How is this relevant to golden rice? Providing beta-carotene as the only source of vitamin A, to people from different backgrounds, may be beneficial for some and not to others on a nutritional basis. Other issues like an individual’s overall health and stored retinol levels in liver will affect how beta-carotene is utilized in the body. Once again this study used healthy individuals. It would be interesting to see how conversion is affected in the population of people the rice is intended for. It has been suggested that a person with a vitamin A deficiency will convert beta-carotene to retinol better than someone who is not deficient. This brings up the final issue of retinol absorption.
Once the beta-carotene has been released from the food, converted to retinol it must be absorbed. Remember vitamin A or retinol is fat soluble. There needs to be some type of unsaturated fat in the diet for the retinol to be absorbed into the intestines so it can be distributed throughout the body via the lymphatic system or stored in the liver. Fat-soluble micronutrients including vitamin A and carotenoids are assumed to follow the fate of lipids in the upper gastrointestinal tract (Borel, 2003). Before retinol can be absorbed it must be esterified. These retinyl esters are then incorporated into chylomicrons. Chylomicrons are created from lipids obtained from the diet and intestinal lipoproteins. These chylomicrons are then secreted into the lymph via enterocytes.
A study conducted by Melody Brown et al. examined how fat-free, low-fat and full-fat salad dressings affected the absorption of carotenoids by extracting chylomicrons. Seven individuals were used in the study (a mixture of both males and females). Each person was fed 3 salads with equal amounts of spinach, romaine lettuce, cherry tomatoes, and carrots with salad dressings containing 0, 6, or 28 g canola oil. The salads were not all fed at once but randomly over a 2 week period. After eat feeding blood samples were taken hourly from time 0-12hrs. Chylomicrons were isolated from the samples and using HPLC the carotenoid absorption was determined. What they found is that when no fat was added plasma carotenoid levels found in chylomicrons were low but as fat was increased the carotenoid levels increase significantly with full-fat salad having the most. This in significant because if golden rice is going to be the staple food then for the people to get the full benefit of the beta carotene they are going to have fat incorporated into the diet. On the golden rice website they claim the addition or subtraction of fat in the diet did not matter and that the conversion of beta-carotene and the absorption of retinol would still be significant. I would disagree due the findings from other studies showing that the presence of oil is necessary. Again all their trials were conducted on health individuals who did not have a vitamin A deficiency. The absorption of retinol derived from golden rice would need to be studied on the group of people the rice is intended for. Also there would need to be some way to incorporate oil into the diet whether it be animal or plant based.
After looking at the bioavailability of beta-carotene in plant based foods, the bioefficacy of converting beta-carotene into retinol and the absorption of retinol the findings suggests that beta-carotene alone may not be a valuable source for populations with a vitamin A deficiency problem. All the tests conducted were on healthy individuals or in-vitro and gave variable low results. While golden rice seems to have it's benefits to me it would have been simpler for them to teach people how grow beta-carotene rich foods like sweet potatoes and spinach. It would be even more beneficial if there could be a way to raise livestock like cattle or egg laying chickens so that people have a bio-available source of retinol that does not require conversion.
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