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Compositional Analysis of Genetically Engineered GR2E “Golden Rice” in Comparison to That of Conventional Rice

  • B. P. Mallikarjuna Swamy*
    B. P. Mallikarjuna Swamy
    International Rice Research Institute, Los Banos, Laguna 4031, Philippines
    *E-mail for B.P.M.S.: [email protected]
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    Orcidhttp://orcid.org/0000-0003-3125-4225
  • , 
  • Mercy Samia
    Mercy Samia
    International Rice Research Institute, Los Banos, Laguna 4031, Philippines
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  • , 
  • Raul Boncodin
    Raul Boncodin
    International Rice Research Institute, Los Banos, Laguna 4031, Philippines
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  • , 
  • Severino Marundan
    Severino Marundan
    International Rice Research Institute, Los Banos, Laguna 4031, Philippines
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  • , 
  • Democrito B. Rebong
    Democrito B. Rebong
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
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  • , 
  • Reynante L. Ordonio
    Reynante L. Ordonio
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
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  • , 
  • Ronalyn T. Miranda
    Ronalyn T. Miranda
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
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  • , 
  • Anna T. O. Rebong
    Anna T. O. Rebong
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
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  • , 
  • Anielyn Y. Alibuyog
    Anielyn Y. Alibuyog
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
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  • , 
  • Cheryl C. Adeva
    Cheryl C. Adeva
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    More by Cheryl C. Adeva
  • , 
  • Russell Reinke
    Russell Reinke
    International Rice Research Institute, Los Banos, Laguna 4031, Philippines
    The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    Donald Danforth Plant Science Center, Saint Louis, Missouri 63132, United States
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  • , and 
  • Donald J. MacKenzie
    Donald J. MacKenzie
    Donald Danforth Plant Science Center, Saint Louis, Missouri 63132, United States
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Cite this: J. Agric. Food Chem. 2019, 67, 28, 7986–7994
Publication Date (Web):June 17, 2019
https://doi.org/10.1021/acs.jafc.9b01524

Copyright © 2019 American Chemical Society. This publication is licensed under CC-BY.

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Abstract

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Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of β-carotene and other provitamin A carotenoids in the grain. Except for β-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89–113% and 57–99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

KEYWORDS:

1. Introduction

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Vitamin A refers to a group of fat-soluble retinoids, (1) including retinol, retinal, and retinyl esters, that help regulate immune function, reduce morbidity of infectious diseases, and are required for normal functioning of the visual system, maintenance of cell function for growth, epithelial integrity, production of red blood cells, immunity, and reproduction. (2) Humans must obtain vitamin A in the diet, either as preformed vitamin A in animal products, such as meat, eggs, and dairy, or as provitamin A carotenoids, mainly β-carotene, in plant products, such as green leafy and many types of yellow-colored vegetables and orange-colored fruit. In Southeast Asian countries, the average per capita rice consumption is 130 kg/year, providing nearly two-thirds of caloric intake. (3) The high consumption of rice implies a lack of dietary diversity that in combination with the poor micronutrient content of polished rice in general, and lack of any provitamin A specifically, is a risk factor for vitamin A deficiency (VAD). Prolonged or severe deficiency can lead to disorders such as childhood blindness, anemia, and weakened host resistance to infection, which can increase the severity of infectious diseases and risk of death. (4,5)
Rice leaves, and indeed the photosynthetic tissues of all plants, produce and accumulate β-carotene. However, rice in its milled form, as it is usually consumed, is characterized by the complete absence of provitamin A carotenoids. The carotenoid biosynthetic pathway in immature rice endosperm functions up to the synthesis of geranylgeranyl diphosphate. While this compound is not solely devoted to carotenogenesis, it can be used as a substrate to produce the uncolored carotenoid phytoene by expressing the heterologous enzyme phytoene synthase (psy) in rice endosperm. (6) Completion of the pathway leading to the synthesis of all-trans-lycopene via expression of the phytoene desaturase (crti) gene from Pantoea ananatis was described by Ye et al. (7) Initial proof of concept work utilized the psy gene from Narcissus pseudonarcissus (daffodil), which was later found to be rate-limiting for the accumulation of β-carotene. Paine et al. (8) described improved constructs incorporating the Zea mays phytoene synthase gene (Zmpsy1) that yielded transformants, including event GR2E, subsequently designated as IR-ØØGR2E-5 based on the unique identification scheme developed by the OECD. (9) The GR2E transformation event in the Japonica cultivar Kaybonnet expressed 11.4 μg/g total carotenoids in T2 generation seed, of which ca. 81% were mixed isomers of β-carotene. (8)
For new varieties without purposefully altered nutritional properties, which include the vast majority of currently authorized genetically engineered crops, the compositional assessment is part of the weight of evidence approach for evaluating whether there were any unanticipated consequences of the genetic modification. (10,11) The experience over the past two decades with genetically engineered crop plants with introduced traits conferring insect resistance and/or herbicide tolerance has indicated that the incorporation of these traits has little biologically meaningful effect on the composition of key nutrients and antinutrients. (12) As with products of conventional plant breeding, most compositional variation is due to environmental and agronomic factors and the base genetics of the plant variety. (13,14)
The compositional evaluation of genetically modified crops is to determine any significant changes in nutrient composition in comparison to its traditional counterpart and to assess the safety of the intended or unintended changes. (11,15) In order to determine whether the genetic modification resulting in provitamin A biofortified GR2E rice resulted in any additional biologically meaningful changes in key nutrients or antinutrients, comprehensive compositional analyses were performed on samples of paddy rice, straw, and bran derived from GR2E and nontransgenic, near-isogenic, control rice cultivated over two growing seasons in the Philippines during 2015 and 2016 at four locations representing typical rice-growing conditions.

2. Materials and Methods

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2.1. Experimental Design

The field phase was conducted over two growing seasons (2015 wet season and 2016 dry season) at four sites in the Philippines: PhilRice-Batac Station, Batac City, Ilocos Norte; Robert S. Zeigler Experimental Station, Los Banos, Laguna; PhilRice Central Experimental Station, Munoz, Nueva Ecija; PhilRice Isabela Station, San Mateo, Isabela. Planting and plot maintenance practices, including pest and disease control measures, were done according to local agronomic practices for rice production. Three blocks (replicates) of each entry (event GR2E in PSBRc82 background and near-isogenic, nontransgenic control PSBRc82) were established at each test site in a randomized complete block design. Each entry was planted in 10-row plots of 5 m in length (20 cm × 20 cm spacing) for a total plot area of 10 m2. There were 25 plants per row, totalling 250 plants per entry per plot.

2.2. Rice (Oryza sativa) Samples

Grain and straw samples were collected from matured rice plants, the stage when typical grain harvest would occur. Grain was collected from at least 150 plants per plot, excluding the outer rows, and pooled into a single sample per plot, totalling three samples per treatment at each location. Straw was collected from at least eight plants per plot, randomly selected and excluding the outer rows, and pooled into a single sample per replicated block. Following collection, samples were shipped frozen to EPL Bio-Analytical Services (Niantic, IL) for processing and analysis.

2.3. Compositional Parameters

Consistent with guidance contained within the OECD consensus document on compositional considerations for new rice varieties, (16) the nutrients and antinutrients were analyzed in replicate samples of paddy rice, straw, and bran (Table 1).
Table 1. Compositional Parameters Analyzed in the Paddy Rice, Rice Straw, and Bran of GR2E and Control Rice
compoundpaddy rice
proximates and fibermoisture, crude protein, crude fat, ash, acid detergent fiber (ADF), neutral detergent fiber fire (NDF), crude fiber fiber, total dietary fiber (TDF), and carbohydrate.
mineralscalcium, phosphorus, magnesium, potassium, zinc, manganese, copper, iron, and sodium
vitaminsthiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9), and α-tocopherol (E)
polysaccharidestotal starch and amylose
fatty acidscaprylic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), pentadecanoic (C15:0), palmitic (C16:0), palmitoleic (C16:1 Δ9), heptadecanoic (C17:0), stearic (C18:0), oleic (C18:1 Δ9), linoleic (C18:2 Δ9,12), α-linolenic (C18:3 Δ9,12,15), arachidic (C20:0), eicosenoic (C20:1), eicosadienoic (C20:2Δ11,14), eicosatrienoic (C20:3 Δ11,14,17), arachidonic (C20:4 Δ5,8,11,14), behenic (C22:0), erucic (C22:1 Δ13), lignoceric (C24:0), and nervonic (C24:1 Δ15)
amino acidslysine, arginine, glycine, histidine, isoleucine, leucine, phenylalanine, threonine, valine, alanine, aspartic acid, glutamic acid, proline, serine, tyrosine, cysteine, methionine, and tryptophan
carotenoidsβ-cryptoxanthin, all-trans-α-carotene, all-trans-β-carotene, 9′-cis-β-carotene, and total carotenoids
bioactive compoundsphytic acid and trypsin inhibitor
compoundrice straw and bran
proximates and fibermoisture, crude protein, crude fat, ash, ADF, NDF, crude fiber, and carbohydrate
mineralscalcium and phosphorus

2.4. Compositional Analyses

With the exception of carotenoids, all compositional analyses were performed by EPL Bio-Analytical Services (Niantic, IL) using methods published by the Association of Official Analytical Chemists (AOAC), the American Oil Chemists’ Society (AOCS), or the American Association of Cereal Chemists (AACC).

2.4.1. Proximate and Mineral Analyses

Samples were analyzed to determine the percentage of moisture by gravimetric measurement of weight loss after drying in a forced air oven. The ash content was determined by gravimetric measurement of the weight loss after ignition in a muffle furnace. (17) The analytical procedure for crude protein determination in rice straw, bran, and paddy rice grain utilized an automated Kjeldahl technique based on a method provided by the manufacturer. (18) For the determination of crude fat, samples were hydrolyzed with 3 N HCl at 90 °C for 60 or 80 min, respectively, for rice grain or straw and bran. The hydrolysates were extracted with a petroleum ether/ethyl ether/ethyl alcohol solution at 90 °C for 60 min. After extraction, the samples were oven-dried and the crude fat content was determined gravimetrically. (19,20) Crude fiber determination in rice grain, straw, and bran was based on methods provided by the manufacturer of the extraction apparatus. (21) Samples were analyzed to determine the percentage of neutral detergent fiber (NDF) by digesting with a neutral detergent solution, sodium sulfite, and α-amylase. After the sample was rinsed with water, the remaining residue was dried and weighed to determine the NDF content. (22) Acid detergent fiber (ADF) was determined following digestion with an acid detergent solution. (23) For the determination of total dietary fiber (TDF) in rice grain, duplicate samples were gelatinized with heat-stable α-amylase and then enzymatically digested with protease and with amyloglucosidase to remove protein and starch, respectively. Soluble dietary fiber was precipitated with ethanol, and the dried residue was quantified gravimetrically. Protein analysis was performed on one of the duplicate samples, while the other duplicate sample was analyzed for ash. The weight of the protein and ash was subtracted from the weight of the residue to yield the weight of TDF. (18,24) The carbohydrate (CHO) content of rice straw, bran, and paddy rice grain was calculated using the formula (25)
Minerals were analyzed by inductively coupled plasma optical emission spectroscopy. (26,27)

2.4.2. Total Starch and Amylose

The analytical procedure for total starch determination in paddy rice grain was based on a method provided by the AOAC (2000i). (28) Nonresistant starch was extracted and converted to d-glucose by using pancreatic α-amylase and amyloglucosidase. Resistant starch was dissolved in 2 M KOH with vigorous stirring, neutralized, and hydrolyzed to d-glucose with amyloglucosidase. The d-glucose in nonresistant and resistant starch was measured with glucose oxidase/peroxidase reagent (GOPOD), and the absorbance was measured on a spectrophotometer at 510 nm. Amylose determination was based on a Megazyme test kit. (29) The samples were defatted using ethanol and were treated with Concanavalin-A to precipitate and remove the amylopectin. The amylose was enzymatically hydrolyzed to d-glucose by using α-amylase and amyloglucosidase. The total starch, in a separate aliquot, was similarly hydrolyzed to d-glucose. Both aliquots were measured calorimetrically using GOPOD. The concentration of amylose was estimated as the ratio of GOPOD absorbance at 510 nm for the amylose versus that of starch.

2.4.3. Amino Acids

Along with tryptophan, cysteine, and methionine, 15 additional amino acids were determined. The analytical procedure for amino acid analysis of paddy rice grain samples was based on internationally adopted methods. (30,31) Following acid hydrolysis, free acids were converted to their 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatives, which were analyzed by reverse-phase ultrahigh-performance liquid chromatography (UPLC) with UV detection. (32)m-Tryptophan was determined on the basis of an established LiOH hydrolysis procedure with reverse-phase UPLC with UV detection. Following acid oxidation and hydrolysis, cysteine and methionine were converted to cysteic acid and methionine sulfone, respectively, as above and analyzed by reverse-phase UPLC with UV detection.

2.4.4. Fatty Acids

Following ether extraction and saponification with 0.5 N sodium hydroxide in methanol, free fatty acids were converted to their fatty acid methyl ester (FAME) derivatives and analyzed by GC-FID. (33) Fatty acids are reported as percent total fatty acids.

2.4.5. B Vitamins and α-Tocopherol

Thiamine (B1) and riboflavin (B2) were analyzed by reverse-phase HPLC tandem mass spectrometry (MS/MS) following extraction with 10% HOAc/4.3% TCA solution. (34) Niacin (B3), pantothenic acid (B5), pyridoxine (B6), and folic acid (B9) were determined using microbiological assays as previously described. (35−38) α-Tocopherol was extracted with hot hexane, and the extracts were analyzed by normal-phase UPLC with fluorescence detection. (39)

2.4.6. Carotenoids

Carotenoids were determined according to Gemmecker et al. (40) All work was performed under low-light conditions. Dehulled and polished rice grains were ground to a fine powder, and weighed amounts (target weight 500 mg) were suspended by sonication in 2 mL of distilled water in 15 mL Falcon tubes and incubated for 10 min at 60 °C. Cooled samples were centrifuged (3000g, 5 min) and the pellets extracted with 2 mL of acetone containing 100 μL of a 20 μg/mL stock solution of the lipophilic metalloorganic dye VIS682A (QCR Solutions Corporation) as an internal standard. Following centrifugation, pellets were re-extracted twice with 2 mL of acetone and the resulting supernatant fractions were combined. A 2 mL portion of petroleum ether (PE)/diethyl ether (DE) (2/1 v/v) was added to each combined supernatant fraction (ca. 8 mL), and volumes were adjusted to 14 mL with distilled water. After vortexing, phase separation was achieved by centrifugation (3000g, 5 min). The organic phase was recovered and the aqueous phase re-extracted with another 2 mL of PE/DE (2/1 v/v), followed by centrifugation (3000g, 5 min). The combined organic phases were dried using a vacuum concentrator (Eppendorf Concentrator 5301) and redissolved in 100 μL of chloroform, 5 μL aliquots of which were analyzed by HPLC using a 5 μm (particle size) C30-RP YMC reversed-phase column (150 × 3 mm, i.d.). The separation of carotenoids was achieved with a linear gradient from 57% A to 95% A using the solvent system A, MeOH/methyl tert-butyl ether (MTBE) (1/1), and B, MeOH/MTBE/H2O (5/1/1). Carotenoids were detected at 450 nm and the VIS682A internal standard at 680 nm using a photodiode array detector. Peak areas were converted into amounts using a response curve with β-carotene (Sigma) and normalized to the VIS682A internal standard recovery to account for unspecific losses during sample processing.

2.4.7. Antinutrients

Concentrations of phytic acid and trypsin inhibitor were determined in samples of paddy rice grain using methods published by the AOAC (2000g) (41) and AOCS (1997), (42) respectively.

2.5. Statistical Analysis

All statistical analyses were conducted (43) using the “lmerTest” package. For a given compositional analyte, data were analyzed using the linear mixed model (44)
where μi denotes the mean of the ith entry (fixed effect), lj denotes the effect of the jth site (random effect), sk denotes the effect of the sth season (random effect), (μL)ij denotes the interaction between the entries and sites (random effect), (μs)ik denotes the interaction between the entries and growing seasons (random effect), (ls)jk denotes the interaction between sites and growing seasons (random effect), (μLs)i denotes the interaction between entries, locations, and growing seasons (random effect), and εijk denotes the residual error (random effect).
The linear mixed effect models “lmer” procedure from the “lmerTest” package was used to fit the linear mixed model and to generate estimates of variance components and p values. For each compositional analyte, the least-squares (LS)-mean value across years and sites was estimated from the corresponding statistical model for GR2E rice and the control PSBRc82 rice using the “lsmeans” package.

2.5.1. Statistical Comparisons and Interpretations

The first step in the evaluation was to test for differences in LS-mean values between GR2E rice and the control PSBRc82 rice. Where a statistically significant difference (p value <0.05) was identified in the multiyear combined-site analysis, further context for interpreting the possible biological significance of the difference was gathered through comparisons with the range of values for each analyte reported in the published literature or available from the ILSI Crop Composition Database. (45) Analyte values for GR2E rice that fell within the combined literature range for that analyte were considered to be within the range of normal variability of conventional rice.

3. Results and Discussion

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3.1. Proximates, Fiber, and Minerals

Fiber is the predominant form of carbohydrate present in rice straw, with measured fractions comprising neutral detergent fiber (NDF; insoluble lignin, cellulose, and hemicellulose), acid detergent fiber (ADF; cellulose and lignin), and total dietary fiber (TDF) consisting of insoluble and soluble fiber (pectin). The macro minerals include calcium, phosphorus, and potassium. The micronutrient minerals, iron, copper, sodium, and zinc are incorporated in plant tissues in only trace amounts.
In the multiyear combined-site analysis, comparisons of proximates, fiber, and minerals in grain (paddy), straw, and bran samples derived from GR2E and control PSBRc82 rice resulted in no statistically significant differences in any of the measured parameters (Table 2).
Table 2. Proximate, Fiber, and Mineral Composition of Grain, Straw, and Bran Samples Derived from GR2E and Control Rice
 GR2Econtrol PSBRc82  
componentmeanarangemeanrangep valueblit. rangec
Grain Samples
ash (%DB)5.89(4.95–7.17)6.02(5.00–7.06)0.3743.61–8.6
crude fat (%DB)1.42(0.84–2.16)1.34(0.56–1.98)0.7110.56–3.47
crude protein (%DB)8.1(6.07–11.2)8.26(6.03–11.40)0.5455.9–11.8
CHOd(%DB)84.6(81.0–86.9)84.4(81.1–86.40)0.56080.0–86.4
moisture (%FW)12.3(11.1–13.8)12.3(10.9–13.60)0.8027.6–28.4
ADF (%DB)18.5(15.7–21.7)17.7(15.6–18.80)0.35210.8–18.8
NDF (%DB)22.1(17.5–35.5)20.6(16.2–32.80)0.47715–32.8
crude fiber (%DB)12.0(10.1–14.6)11.1(10.1–12.30)0.2138.6–18.13
TDF (%DB)17.0(12.8–20.3)16.9(11.4–21.40)0.95511.4–23.0
amylose (%DB)12.9(7.31–18.6)12.8(6.76–18.60)0.9556.76–18.6
starch (%DB)59.5(32.8–71.5)61.1(28.1–73.90)0.68928.1–73.9
Ca (mg/100 g DB)22.5(14.2–35.0)21.4(15.3–29.8)0.55410–150
Cu (mg/100 g DB)0.39(0.18–0.68)0.37(0.22–0.51)0.5440.2–1.3
Fe (mg/100 g DB)3.96(2.37–10.6)4.57(2.58–9.08)0.5141.6–9.08
Mg (mg/100 g DB)131(87.5–185)133(102–157)0.63130–170
Mn (mg/100 g DB)6.61(4.33–8.39)6.47(4.85–7.78)0.6742–11.7
P (mg/100 g DB)327(211–461)329(241–383)0.870190–470
K (mg/100 g DB)346(236–597)339(222–472)0.666170–472
Na (mg/100 g DB)1.5(0.56–3.81)1.3(0.54–3.07)0.4670–100
Zn (mg/100 g DB)2.31(1.63–3.21)2.19(1.73–2.78)0.5690.2–3.6
Straw Samples
ash (%DB)25.7(21.1–30.4)25.3(20.6–30.2)0.46310.8–24
crude fat (%DB)2.58(1.31–4.8)2.72(1.0–6.15)0.7670.9–6.15
crude protein (%DB)6.13(3.16–11.3)6.12(2.85–10.2)0.9742.4–10.2
CHO (%DB)66.1(60.5–69.2)66.4(62.1–70.5)0.73562.1–81.6
Moisture (%FW)12(9.24–14)12.6(8.12–22.1)0.5933.5–73.7
ADF (%DB)52.6(46.1–58.0)51.6(44.7–59.3)0.52236.1–59.3
NDF (%DB)62.5(56.3–68.9)62.2(50.2–69.7)0.81850.2–78.6
crude fiber (%DB)30.1(26.9–35.8)29.7(26.0–34.9)0.65926.0–41.5
Ca (g/kg DB)3.77(1.75–7.06)3.64(1.79–5.43)0.7031.7–5.43
P (g/kg DB)1.52(0.82–3.61)1.47(0.81–3.47)0.6170.5–3.47
Bran Samples
ash (%DB)10.3(10.0–10.6)11.4(10.7–12.0)0.2816.5–14
crude fat (%DB)24.1(23.8–24.4)25.3(24.6–26.0)0.27211.3–26.0
crude protein (%DB)15.1(14.7–15.6)14.6(14.1–15.2)0.54311.5–18.3
CHO (%DB)50.4(49.4–51.4)48.6(47.7–49.5)0.31533–63.8
moisture (%FW)11.5(10.8–12.2)11.9(10.9–12.9)0.7826.9–12.9
ADF (%DB)16.0(11.6–20.4)18.0(13.8–22.1)0.7746.6–22.1
NDF (%DB)23.7(23.7–23.8)25.8(24.0–27.6)0.36616.1–29.6
crude fiber (%DB)8.97(8.31–9.63)9.05(9.01–9.08)0.9235.6–12.2
Ca (g/kg DB)0.62(0.46–0.77)0.59(0.55–0.63)0.8730.2–2.9
P (g/kg DB)25.9(25.8–26.0)26.7(25.5–27.8)0.5648.6–27.8
a

Except for bran, values represent the least-squares mean of three replicate samples collected over two growing seasons from each of four locations in the Philippines (n = 24 for each entry). For each analyte, the lowest and highest individual values across years and locations are shown in parentheses. All values are expressed on a dry basis (DB) except for moisture, which is expressed on a fresh weight (FW) basis. Bran samples were produced from composite grain samples pooled across all trial site locations, one each for GR2E and PSBRc82 per year, and means are arithmatic means.

b

Statistical significance was assigned at p < 0.05. Data from bran samples were analyzed using Student’s t test.

c

The combined literature range and ILSI crop composition database. (45)

d

Abbreviations: CHO = carbohydrate by calculation; ADF = acid detergent fiber; NDF = neutral detergent fiber; TDF = total dietary fiber; DW = dry weight; FW = fresh weight.

3.2. Amino Acids

A comparison of the amino acid composition of event GR2E and control PSBRc82 rice grain is shown in Table 3. Across locations and growing seasons, there were no statistically significant differences in the concentrations of any amino acids between samples of GR2E and control PSBRc82 rice.
Table 3. Amino Acid Composition of Grain Samples Derived from GR2E and Control Rice
 GR2Econtrol PSBRc82  
componentmeanarangemeanrangep valueblit. rangec
Essential Amino Acids (mg/100 g DB)
histidine212(162–276)215(157–281)0.659140–281
isoleucine329(241–446)332(237–464)0.787240–460
leucine644(464–886)651(462–921)0.743460–920
lysine299(216–443)294(211–434)0.711210–430
methionine169(124–228)166(127–215)0.602130–310
phenylalanine440(324–593)444(317–622)0.799280–620
threonine309(235–400)308(217–409)0.942220–410
tryptophan73.4(44.2–107)74.9(50.9–101)0.79550–180
valine468(341–627)474(338–653)0.699340–650
Nonessential Amino Acids (mg/100 g DB)
alanine455(329–625)460(331–628)0.74330–630
arginine564(409–737)564(408–782)0.988410–850
aspartic acid708(493–1010)710(497–994)0.960500–990
cystine156(117–214)155(113–198)0.767100–260
glutamic acid1354(942–1980)1360(890–1990)0.898890–1990
glycine389(293–495)393(292–511)0.716290–510
proline376(276–510)381(278–521)0.678280–540
serine401(296–540)401(231–556)0.998230–560
tyrosine214(158–282)207(133–291)0.531130–480
a

Values represent the least-squares mean of three replicate samples collected over two growing seasons from each of four locations in the Philippines (n = 24 for each entry). For each analyte, the lowest and highest individual values across years and locations are shown in parentheses. All values are expressed on a dry basis (DB).

b

Statistical significance was assigned at p < 0.05.

c

The combined literature range was derived from the literature and ILSI crop composition database. (45)

3.3. Fatty Acids

The complete fatty acid profile of grain from GR2E and control rice was determined, and the results are summarized in Table 4. The concentrations of a number of fatty acids occurring in trace amounts in both GR2E and PSBRc82 control rice grain samples were below the limit of quantification and are not reported (these are itemized in the footnotes to Table 4). In the combined-site analysis over both growing seasons, the only statistically significant difference observed between GR2E and control PSBRc82 rice samples was in the concentration of stearic (C18:0) acid, which was approximately 6.5% higher for GR2E rice but was still within the combined literature range for this parameter.
Table 4. Fatty Acid Composition of Grain Samples Derived from GR2E and Control Rice
 GR2Econtrol PSBRc82  
componentmeanarangemeanrangep valueblit. rangec
Saturated Fatty Acids (% Total Fatty Acids)
myristic (C14:0)0.44(0.35–0.52)0.39(0.25–0.49)0.1980.25–1.10
palmitic (C16:0)19.50(18.80–20.40)18.5(15.8–19.1)0.22214.90–31.20
stearic (C18:0)2.25(1.95–2.78)2.11(1.71–2.68)0.0491.50–2.80
arachidic (C20:0)0.86(0.74–0.99)0.89(0.74–1.02)0.1970.40–1.02
behenic (C22:0)0.51(0.41–0.59)0.54(0.48–0.61)0.1420.20–0.82
lignoceric (C24:0)0.93(0.69–1.25)1.00(0.81–1.19)0.1160.40–1.34
Unsaturated Fatty Acids (% Total Fatty Acids)
palmitoleic (C16:1)0.19(0.16–0.22)0.19(0.15–0.22)0.3610.10–0.93
oleic (C18:1)39.70(38.40–41.30)40.20(38.4–45.9)0.48730.90–45.90
linoleic (C18:2)33.5(32.40–34.40)34.00(30.1–35.4)0.39426.10–39.00
α-linolenic (C18:3)1.63(1.34–2.31)1.64(1.27–2.7)0.8870.90–2.70
eicosenoic (C20:1)0.48(0.41–0.54)0.52(0.44–0.77)0.2860.40–0.77
a

Values represent the least-squares mean of three replicate samples collected over two growing seasons from each of four locations in the Philippines (n = 24 for each entry). For each analyte, the lowest and highest individual values across years and locations are shown in parentheses. The concentrations of the following fatty acids were below the lower limit of quantification and are not reported: caprylic (C8:0); capric (C10:0); lauric (C12:0); pentadecanoic (C15:0); heptadecanoic (C17:0); eicosadienoic (C20:2); eicosatrienoic (C20:3); arachidonic (C20:4); erucic (C22:1); nervonic (C24:1).

b

Statistical significance was assigned at p < 0.05.

c

The combined literature range ILSI crop composition database. (45)

3.4. Vitamins, Carotenoids, and Antinutrients

Samples of event GR2E and control PSBRc82 rice grain were analyzed for concentrations of the water-soluble B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, and folic acid) and α-tocopherol (vitamin E). With the exception of riboflavin, where concentrations were below the limit of quantification (LOQ) for all samples, there were no statistically significant differences noted in concentrations of B vitamins and vitamin E between GR2E and control PSBRc82 rice (Table 5). Samples of milled rice prepared from grain (paddy) samples were also analyzed at IRRI for β-carotene and other provitamin A carotenoids. In these analyses, the concentrations of all-trans-β-carotene ranged between 1.96 and 7.31 μg/g across locations and years (Table 5) and on average comprised ca. 59% of the total carotenoids as determined by HPLC. Concentrations of provitamin A carotenoids in samples derived from control PSBRc82 rice were below the LOQ in each case.
Table 5. Concentrations of Vitamins, Carotenoids, and Antinutrients in Grain Samples Derived from GR2E and Control Rice
 GR2Econtrol  
componentmeanarangemeanrangep valueblit. rangec
Vitamins (mg/kg DB)
thiamine (B1)3.13(2.33–3.77)3.08(2.35–3.81)0.6342.35–6.25
niacin (B3)36.9(23.4–58.3)32.6(20.2–48.8)0.41720.2–65
pantothenic acid (B5)9.15(7.31–11.9)9.13(7.22–11.4)0.9297.22–14.0
pyridoxine (B6)2.72(2.22–3.3)2.75(2.1–5.42)0.9192.1–8.0
folic acid (B9)0.91(0.56–2.56)0.88(0.39–1.54)0.8810.39–1.54
α-tocopherol2.98(2.47–3.87)2.75(2.1–3.5)0.3362.1–23
Carotenoids (mg/kg DB)
β-cryptoxanthin0.31(0.23–0.46)<LOQd   
all-trans-α-carotene0.71(0.35–1.32)<LOQ   
all-trans-β-carotene3.57(1.96–7.31)<LOQ   
9′-cis-β-carotene0.76(0.5–1.32)<LOQ   
total carotenoids5.88(3.5–10.9)<LOQ   
Antinutrients
phytic acid (%DB)0.86(0.58–1.1)0.88(0.61–1.23)0.6220.6–1.23
trypsin inhibitor (TIU/mg)0.92(0.28–1.71)1.0(0.03–4.17)0.8280.03–4.17
a

Values represent the least-squares mean of three replicate samples collected over two growing seasons from each of four locations in the Philippines (n = 24 for each entry). For each analyte, the lowest and highest individual values across years and locations are shown in parentheses. Values for riboflavin (vitamin B2) were below the LOQ of 0.9 mg/kg dry weight for all samples tested and are not included in this table.

b

Statistical significance was assigned at p < 0.05.

c

The combined literature range and ILSI crop composition database. (45)

d

LOQ = limit of quantification.

Rice contains few compounds that are not favorable for human or animal nutrition, and these are generally not present at concentrations that would raise any safety concerns. (16) Both phytic acid and trypsin inhibitors are considered antinutritional factors present in a range of food crops, including rice.
Phytic acid (inositol hexakisphosphate; phytate when in salt form) is the main storage form of phosphorus in plant tissues, which is not in a bioavailable form for monogastric animals that lack the digestive enzyme phytase. Phytic acid also has strong binding affinity to important minerals such as calcium, magnesium, iron, and zinc, thus reducing the absorption of these minerals.
Trypsin inhibitors are proteins known to interfere with digestion and ultimately absorption of food material and are typical antinutritional components in soybeans, cereals, and potatoes. (16) In rice, both phytic acid and trypsin inhibitors are mainly concentrated in the bran, which is a component of rice grain (paddy).
Grain samples from GR2E and control PSBRc82 rice were analyzed for phytic acid and trypsin inhibitor activity. There were no statistically significant differences in the concentrations of phytic acid or in the levels of trypsin inhibitor between samples of GR2E and PSBRc82 control rice (Table 5).

3.5. Nutritional Effect of Elevated β-Carotene in GR2E Rice

β-Carotene is the major carotenoid present in the human diet and is an effective source of vitamin A in both conventional foods and vitamin supplements. It is generally considered virtually nontoxic because humans tolerate high dietary dosages without apparent harm (46) and there are no reports of adverse effects arising from the consumption of naturally occurring β-carotene in food. There is similarly no evidence that conversion of β-carotene to vitamin A contributes to vitamin A toxicity, even when β-carotene is ingested in large amounts. Standard toxicological tests, including teratogenic, mutagenic, and carcinogenic assays, have been performed on β-carotene without any evidence of harmful effects, as reviewed in ref (47).
On the basis of a pooled analysis of 10 prospective cohort studies in North America and Europe that included more than 500000 women, the mean daily intake of β-carotene is in the range of 2.7–6.4 mg. (48) Other data from the National Health and Nutrition Examination Survey (NHANES) published by the US Department of Agriculture indicate that average daily intake of β-carotene from food sources by adult men and women living in the United States is ca. 2.5 mg.
The conversion efficiency of dietary β-carotene to retinol is in the range of (3.6–28):1 by weight (49) and is greatly influenced by the type of study, the vitamin A status, disease and genetic background of the individuals under study, and the food matrix. Rice has a simple and easily digestible food matrix, which allows for a high bioavailability and bioconversion of β-carotene to vitamin A. The bioconversion efficiency of β-carotene in golden rice was found to be 3.8:1 for healthy adults (50) and 2.3:1 for children in China. (51) A bioconversion efficiency of 4.4:1 was reported for β-carotene biofortified cassava (52) and 3.2:1 for yellow maize with high β-carotene. (53) A vitamin A equivalency for β-carotene of approximately 4:1 is applicable for biofortified cassava, yellow maize, and golden rice. (54)
Using mean concentrations of all-trans-β-carotene (3.57 mg/kg) measured in samples of milled rice, it was estimated that substitution of total daily rice consumption with rice containing event GR2E could potentially contribute 89–113% and 57–99% of the estimated average requirement (EAR) for vitamin A for children in Bangladesh and the Philippines, respectively (Table 6). Expressed another way, a 100 g (uncooked weight) portion of GR2E rice could supply 30–40% of the recommended daily intake (RDI) of vitamin A for children and 11–13% of the RDI for nonpregnant or pregnant adult women.
Table 6. Potential Contribution of Rice Containing Event GR2E to Meeting Vitamin A Needs in Bangladesh and the Philippines
 mean daily rice consumption (g, raw)aEARb (μg/day RAEc)RDId (μg/day RAE)β-carotene equive from GR2E Rice (μg/day)% of EARf% of RDI
Bangladesh
preschool-aged children1572103005618962
school-aged children (6–14 years)26127540093211378
nonpregnant, nonlactating women (15–49 years)36050070012856446
Philippines
preschool-aged children (6 months–5 years)1002103003575740
school-aged children (6–12 years)2292754008189968
adult (19–59 years)33250070011865942
pregnant women28755080010254732
lactating women342800110012213828
a

Mean daily rice consumption data for Bangladesh and the Philippines were from Leyvraz et al. (55) and the seventh National Nutrition Survey published by the Food and Nutrition Research Institute of the Philippines (http://www.fnri.dost.gov.ph/index.php/19-nutrition-statistic/108-seventh-national-nutrition-survey), respectively .

b

The EAR (estimated average requirement) is the median daily intake value that is estimated to meet the requirement of half the healthy individuals in a life-stage and gender group. At this level of intake, the other half of the individuals in the specified group would not have their needs met. The EAR is used to calculate the RDI. It is also used to assess the adequacy of nutrient intakes and can be used to plan the intake of groups. Values for EAR and RDI were taken from the nutrient reference values for Australia and New Zealand (https://www.nrv.gov.au/nutrients/vitamin-a).

c

RAE = retinol activity equivalent.

d

The RDI (recommended daily intake) is the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97–98%) healthy individuals in a particular life-stage and gender group. The RDI is the goal for usual intake by an individual.

e

β-Carotene equivalent units were calculated by summing the mean concentration of all-trans-β-carotene and half the mean concentrations of other provitamin A carotenoids (e.g., all-trans-α-carotene, β-cryptoxanthin, and other isomers of β-carotene) measured in samples of milled GR2E rice (Table 5). This was to take into account that the conversion efficiency of these latter carotenoids is approximately 50% of all-trans-β-carotene (i.e.: 3.57 + (0.5 × (0.31 + 0.71 + 0.76)) = 4.46 μg/g). The values for β-carotene equivalents were further adjusted to reflect ca. 20% loss during cooking (unpublished data, IRRI).

f

For the calculation of percent EAR and percent RDI, bioconversion ratios of 3:1 for children and 4:1 for adults were assumed. These are conservative estimates based on mean values reported for adults of 3.6:1 (49) and 2.3:1 for children. (51)

Practically, nutritional effects will be significantly influenced by substitution rates of conventional rice with rice containing event GR2E, varietal (background germplasm) effects on provitamin A expression, actual bioconversion efficiency in target populations, and the known loss of β-carotene over time due to storage, processing, and cooking methods. Assessment of the realistically achievable contribution of rice varieties containing event GR2E to improving vitamin A status in at-risk populations will require additional bioefficacy testing that can only be accomplished following the regulatory authorization of event GR2E for food use in the target geographies.
Over all, among the 62 compositional parameters quantified in samples derived from GR2E rice grain, and components that were measured in samples of straw and derived bran, there were no meaningful differences between GR2E and control rice, except for the intended production of provitamin A carotenoids measured in milled GR2E rice. With the exception of provitamin A carotenoids, the compositional parameters measured in samples of GR2E rice were within or similar to ranges reported in the literature for conventional rice varieties with a history of safe consumption. In those instances where the analyte concentrations measured for GR2E rice were slightly outside the combined literature range for that parameter, there were no significant differences between GR2E rice and the control rice. Overall, no consistent patterns emerged to suggest that biologically meaningful changes in composition or nutritive value of the grain, straw, or bran had occurred as an unexpected, unintended, consequence of the genetic modification. Other than the intended production of β-carotene and related provitamin A carotenoids in the endosperm, GR2E rice was found to be compositionally equivalent to conventional rice.

Author Information

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  • Corresponding Author
  • Authors
    • Mercy Samia - International Rice Research Institute, Los Banos, Laguna 4031, Philippines
    • Raul Boncodin - International Rice Research Institute, Los Banos, Laguna 4031, Philippines
    • Severino Marundan - International Rice Research Institute, Los Banos, Laguna 4031, Philippines
    • Democrito B. Rebong - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Reynante L. Ordonio - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Ronalyn T. Miranda - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Anna T. O. Rebong - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Anielyn Y. Alibuyog - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Cheryl C. Adeva - The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines
    • Russell Reinke - International Rice Research Institute, Los Banos, Laguna 4031, Philippines;  The Philippines Rice Research Institute, Science City of Munoz, 3119 Nueva Ecija, Philippines;  Donald Danforth Plant Science Center, Saint Louis, Missouri 63132, United States
    • Donald J. MacKenzie - Donald Danforth Plant Science Center, Saint Louis, Missouri 63132, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was supported by the Bill & Melinda Gates Foundation, Seattle, WA (Global Development Grant Number OPPGD1383) and by the United States Agency for International Development.

References

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    Herman, R. A.; Price, W. D. Unintended compositional changes in genetically modified (GM) crops: 20 years of research. J. Agric. Food Chem. 2013, 61, 11695– 701,  DOI: 10.1021/jf400135r
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    Unintended Compositional Changes in Genetically Modified (GM) Crops: 20 Years of Research
    Herman, Rod A.; Price, William D.
    Journal of Agricultural and Food Chemistry (2013), 61 (48), 11695-11701CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    A review. The compositional equivalency between genetically modified (GM) crops and nontransgenic comparators has been a fundamental component of human health safety assessment for 20 years. During this time, a large amt. of information has been amassed on the compositional changes that accompany both the transgenosis process and traditional breeding methods; addnl., the genetic mechanisms behind these changes have been elucidated. After two decades, scientists are encouraged to objectively assess this body of literature and det. if sufficient scientific uncertainty still exists to continue the general requirement for these studies to support the safety assessment of transgenic crops. It is concluded that suspect unintended compositional effects that could be caused by genetic modification have not materialized on the basis of this substantial literature. Hence, compositional equivalence studies uniquely required for GM crops may no longer be justified on the basis of scientific uncertainty.
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    Harrigan, G. G.; Stork, L. G.; Riordan, S. G.; Reynolds, T. L.; Ridley, W. P.; Masucci, J. D.; MacIsaac, S.; Halls, S. C.; Orth, R.; Smith, R. G.; Wen, L.; Brown, W. E.; Welsch, M.; Riley, R.; McFarland, D.; Pandravada, A.; Glenn, K. C. Impact of genetics and environment on nutritional and metabolite components of maize grain. J. Agric. Food Chem. 2007, 55, 6177– 6185,  DOI: 10.1021/jf070494k
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    Impact of Genetics and Environment on Nutritional and Metabolite Components of Maize Grain
    Harrigan, George G.; Stork, LeAnna G.; Riordan, Susan G.; Reynolds, Tracey L.; Ridley, William P.; Masucci, James D.; MacIsaac, Susan; Halls, Steven C.; Orth, Robert; Smith, Ronald G.; Wen, Li; Brown, Wayne E.; Welsch, Michael; Riley, Rochelle; McFarland, David; Pandravada, Anand; Glenn, Kevin C.
    Journal of Agricultural and Food Chemistry (2007), 55 (15), 6177-6185CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnol.-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnol.-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly assocd. with a newly introduced trait. Therefore, steps were initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against 2 different testers. These were grown at 3 different, but geog. similar, locations in the United States. In addn. to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, org. acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant org. acid was citric acid, with only minor amts. of other org. acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and org. acids appeared to be markedly higher than that obsd. for the OECD analytes.
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    Assessment of GE food safety using '-omics' techniques and long-term animal feeding studies
    Ricroch, Agnes E.
    New Biotechnology (2013), 30 (4), 349-354CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)
    A review. Despite the fact that a thorough, lengthy and costly evaluation of genetically engineered (GE) crop plants (including compositional anal. and toxicol. tests) is imposed before marketing, some European citizens remain sceptical of the safety of GE food and feed. In this context, are addnl. tests necessary, and if so, what can we learn from them. To address these questions, we examd. data from 60 recent high-throughput '-omics' comparisons between GE and non-GE crop lines and 17 recent long-term animal feeding studies (longer than the classical 90-day subchronic toxicol. tests), as well as 16 multigenerational studies on animals. The '-omics' comparisons revealed that the genetic modification has less impact on plant gene expression and compn. than that of conventional plant breeding. Moreover, environmental factors (such as field location, sampling time, or agricultural practices) have a greater impact than transgenesis. None of these '-omics' profiling studies has raised new safety concerns about GE varieties; neither did the long-term and multigenerational studies on animals. Therefore, there is no need to perform such long-term studies in a case-by-case approach, unless reasonable doubt still exists after conducting a 90-day feeding test. In addn., plant compositional anal. and '-omics' profiling do not indicate that toxicol. tests should be mandatory. We discuss what complementary fundamental studies should be performed and how to choose the most efficient exptl. design to assess risks assocd. with new GE traits. The possible need to update the current regulatory framework is discussed.
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    Jiao, Z.; Si, X.X.; Li, G. K.; Zhang, Z. M.; Xu, X. P. Unintended Compositional Changes in Transgenic Rice Seeds (Oryza sativa L.) Studied by Spectral and Chromatographic Analysis Coupled with Chemometrics Methods. J. Agric. Food Chem. 2010, 58, 1746– 1754,  DOI: 10.1021/jf902676y
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    Unintended Compositional Changes in Transgenic Rice Seeds (Oryza sativa L.) Studied by Spectral and Chromatographic Analysis Coupled with Chemometrics Methods
    Jiao, Zhe; Si, Xiao-xi; Li, Gong-ke; Zhang, Zhuo-min; Xu, Xin-ping
    Journal of Agricultural and Food Chemistry (2010), 58 (3), 1746-1754CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    Unintended compositional changes in transgenic rice seeds were studied by near-IR reflectance, GC-MS, HPLC, and ICP-AES coupled with chemometrics strategies. Three kinds of transgenic rice with resistance to fungal diseases or insect pests were comparatively studied with the non-transgenic counterparts in terms of key nutrients such as protein, amino acids, fatty acids, vitamins, elements, and antinutrient phytic acid recommended by the Organization for Economic Co-operation and Development (OECD). The compositional profiles were discriminated by chemometrics methods, and the discriminatory compds. were protein, three amino acids, two fatty acids, two vitamins, and several elements. Significance of differences for these compds. was proved by anal. of variance, and the variation extent ranged from 20 to 74% for amino acids, from 19 to 38% for fatty acids, from 25 to 57% for vitamins, from 20 to 50% for elements, and 25% for protein, whereas phytic acid content did not change significantly. The unintended compositional alterations as well as unintended change of phys. characteristic in transgenic rice compared with non-transgenic rice might be related to the genetic transformation, the effect of which needs to be elucidated by addnl. studies.
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    A new isocratic HPLC method was developed and tested for detg. tryptophan in feeds. Samples were hydrolyzed for 20 h at 120° under exclusion of O2 in 4N LiOH, microfiltered, and then analyzed by HPLC. Sepn. of amino acids was achieved using a C18 reversed-phase column eluted with 0.02M KH2PO4/methanol/water (20:3:2 by vol.). Three wavelengths (217, 254, and 280 nm) were compared for suitable absorbance by tryptophan. Aspects of the hydrolysis phase of anal. were also tested. This method permits rapid sample throughput (6 samples per h) and yields reproducible, high recovery values for all feed tested (98.9 ± 1.2%). All three wavelengths tested gave suitable detection of tryptophan from the feed hydrolyzates. Defatting was necessary to maintain max. recovery of tryptophan from the samples. Adjustment of hydrolyzate pH prior to anal. was not necessary and may even interfere with detection of tryptophan. Hydrolyzates stored at room temp. for 24 h had no detectable loss of tryptophan.
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    Simultaneous determination of tocopherols and tocotrienols in hazelnuts by a normal phase liquid chromatographic method
    Amaral, Joana S.; Casal, Susana; Torres, Duarte; Seabra, Rosa M.; Oliveira, Beatriz P. P.
    Analytical Sciences (2005), 21 (12), 1545-1548CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
    A normal-phase high-performance liq. chromatog. (NP-HPLC) method for the detn. of tocopherols and tocotrienols in hazelnuts is reported. Three extn. procedures (with and without sapon.) were assayed; the best results were obtained with a simple solid-liq. extn. procedure. Chromatog. sepn. was achieved using an Inertsil 5 SI column using isocratic elution with hexane/1,4-dioxane (95.5:4.5, vol./vol.) at a flow rate of 0.7 mL/min. The effluent was monitored by a series arrangement of a diode-array followed by a fluorescence detector. All compds. were sepd. in a short period of time (17 min). The method proved to be rapid, sensitive, reproducible, and accurate, allowing the simultaneous detn. of all vitamin E homologues.
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    Koushik, A.; Hunter, D. J.; Spiegelman, D.; Anderson, K. E.; Buring, J. E.; Freudenheim, J. L.; Goldbohm, R. A.; Hankinson, S. E.; Larsson, S. C.; Leitzmann, M.; Marshall, J. R.; McCullough, M.; Miller, A. B.; Rodriguez, C.; Rohan, T. E.; Ross, J. A.; Schatzkin, A.; Schouten, L. J.; Willett, W. C.; Wolk, A.; Zhang, S. M.; Smith-Warner, S. A. Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies. Int. J. Cancer 2006, 119, 2148– 2154,  DOI: 10.1002/ijc.22076
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    Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies
    Koushik, Anita; Hunter, David J.; Spiegelman, Donna; Anderson, Kristin E.; Buring, Julie E.; Freudenheim, Jo L.; Goldbohm, R. Alexandra; Hankinson, Susan E.; Larsson, Susanna C.; Leitzmann, Michael; Marshall, James R.; McCullough, Marjorie L.; Miller, Anthony B.; Rodriguez, Carmen; Rohan, Thomas E.; Ross, Julie A.; Schatzkin, Arthur; Schouten, Leo J.; Willett, Walter C.; Wolk, Alicja; Zhang, Shumin M.; Smith-Warner, Stephanie A.
    International Journal of Cancer (2006), 119 (9), 2148-2154CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
    Carotenoids in fruits and vegetables may protect against cancer because of their properties, including their functions as precursors of vitamin A and antioxidants. We examd. the assocns. between dietary intakes of α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin, and lycopene and the risk of invasive epithelial ovarian cancer using pooled primary data from 10 prospective cohort studies in North America and Europe. The carotenoid intakes were estd. from validated food frequency questionnaires used at baseline in each study. Study-specific relative risks (RR) were estd. using the Cox proportional hazards model and then combined using a random-effects model. Among 521,911 women, 2012 cases of ovarian cancer occurred during a follow-up of 7-22 years across studies. The major carotenoids were not much assocd. with the risk of ovarian cancer. The pooled multivariate RR values (95% confidence intervals) were 1.00 (0.95-1.05) for 600 μg/day increase in α-carotene intake, 0.96 (0.93-1.03) for 2500 μg/day increase in β-carotene intake, 0.99 (0.97-1.02) for 100 μg/day increase in β-cryptoxanthin intake, 0.98 (0.94-1.03) for 2500 μg/day increase in lutein/zeaxanthin intake, and 1.01 (0.97-1.05) for 4000 μg/day increase in lycopene intake. These assocns. did not appreciably differ among the studies. The obsd. assocns. did not much vary by population subgroups or histol. type of ovarian cancer. Thus, consumption of the major carotenoids during adulthood may not play a major role in the incidence of ovarian cancer.
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    Tang, G. Bioconversion of dietary provitamin A carotenoids to vitamin A in humans. Am. J. Clin. Nutr. 2010, 91, 1468S– 1473S,  DOI: 10.3945/ajcn.2010.28674G
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    Bioconversion of dietary provitamin A carotenoids to vitamin A in humans
    Tang, Guangwen
    American Journal of Clinical Nutrition (2010), 91 (5S), 1468S-1473SCODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
    A review. Recent progress in the measurement of the bioconversion of dietary provitamin A carotenoids to vitamin A is reviewed in this article. Methods to assess the bioavailability and bioconversion of provitamin A carotenoids have advanced significantly in the past 10 y, specifically through the use of stable isotope methodol., which includes the use of labeled plant foods. The effects of the food matrix on the bioconversion of provitamin A carotenoids to vitamin A, dietary fat effects, and the effect of genotype on the absorption and metab. of β-carotene have been reported recently. A summary of the major human studies that detd. conversion factors for dietary β-carotene to retinol is presented here, and these data show that the conversion efficiency of dietary β-carotene to retinol is in the range of 3.6-28:1 by wt. There is a wide variation in conversion factors reported not only between different studies but also between individuals in a particular study. These findings show that the vitamin A value of individual plant foods rich in provitamin A carotenoids may vary significantly and need further investigation.
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    Tang, G.; Tang, G.; Qin, J.; Dolnikowski, G. G.; Russell, R. M.; Grusak, M. A. Golden rice is an effective source of vitamin A. Am. J. Clin. Nutr. 2009, 89 (6), 1776– 1783,  DOI: 10.3945/ajcn.2008.27119
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    Golden Rice is an effective source of vitamin A
    Tang, Guangwen; Qin, Jian; Dolnikowski, Gregory G.; Russell, Robert M.; Grusak, Michael A.
    American Journal of Clinical Nutrition (2009), 89 (6), 1776-1783CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
    Genetically engineered "Golden Rice" contains up to 35 μg β-carotene per g of rice. It is important to det. the vitamin A equivalency of Golden Rice β-carotene to project the potential effect of this biofortified grain in rice-consuming populations that commonly exhibit low vitamin A status. The objective was to det. the vitamin A value of intrinsically labeled dietary Golden Rice in humans. Golden Rice plants were grown hydroponically with heavy water (deuterium oxide) to generate deuterium-labeled [2H]β-carotene in the rice grains. Golden Rice servings of 65-98 g (130-200 g cooked rice) contg. 0.99-1.53 mg β-carotene were fed to 5 healthy adult volunteers (3 women and 2 men) with 10 g butter. A ref. dose of [13C10]retinyl acetate (0.4-1.0 mg) in oil was given to each volunteer 1 wk before ingestion of the Golden Rice dose. Blood samples were collected over 36 d. The authors' results showed that the mean (± SD) area under the curve for the total serum response to [2H]retinol was 39.9 ± 20.7 μg/d after the Golden Rice dose. Compared with that of the [13C10]retinyl acetate ref. dose (84.7 ± 34.6 μg/d), Golden Rice β-carotene provided 0.24-0.94 mg retinol. Thus, the conversion factor of Golden Rice β-carotene to retinol is 3.8 ± 1.7 to 1 with a range of 1.9-6.4 to 1 by wt., or 2.0 ± 0.9 to 1 with a range of 1.0-3.4 to 1 by moles. β-Carotene derived from Golden Rice is effectively converted to vitamin A in humans.
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  51. 51
    Tang, G.; Hu, Y.; Yin, S.; Wang, Y.; Dallal, G. E.; Grusak, M. A.; Russell, R. M. β-carotene in Golden Rice is as good as β-carotene in oil at providing vitamin A to children. Am. J. Clin. Nutr. 2012, 96, 658– 664,  DOI: 10.3945/ajcn.111.030775
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    51
    β-Carotene in golden rice is as good as β-carotene in oil at providing vitamin A to children
    Tang, Guangwen; Hu, Yuming; Yin, Shi-an; Wang, Yin; Dallal, Gerard E.; Grusak, Michael A.; Russell, Robert M.
    American Journal of Clinical Nutrition (2012), 96 (3), 658-664CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
    Background: Golden Rice (GR) has been genetically engineered to be rich in β-carotene for use as a source of vitamin A. Objective: The objective was to compare the vitamin A value of β-carotene in GR and in spinach with that of pure β-carotene in oil when consumed by children. Design: Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient soln. contg. 23 atom% 2H2O) or [2H8]β-carotene in an oil capsule. The GR and spinach β-carotene were enriched with deuterium (2H) with the highest abundance mol. mass (M) at Mβ-C+2H10. [13C10]Retinyl acetate in an oil capsule was administered as a ref. dose. Serum samples collected from subjects were analyzed by using gas chromatog. electron-capture neg. chem. ionization mass spectrometry for the enrichments of labeled retinol: Mretinol+4 (from [2H8]β-carotene in oil), Mretinol+5 (from GR or spinach [2H10]β-carotene), and Mretinol+10 (from [13C10]retinyl acetate). Results: Using the response to the dose of [13C10]retinyl acetate (0.5 mg) as a ref., our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure β-carotene (0.5 mg), GR β-carotene (0.6 mg), and spinach β-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by wt., resp. Conclusions: The β-carotene in GR is as effective as pure β-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ∼100 to 150 g cooked GR (50 g dry wt.) can provide ∼60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.
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  52. 52
    La Frano, M. R.; Woodhouse, L. R.; Burnett, D. J.; Burri, B. J. Biofortified cassava increases β-carotene and vitamin A concentrations in the TAG-rich plasma layer of American women. Br. J. Nutr. 2013, 110 (2), 310– 20,  DOI: 10.1017/S0007114512005004
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    52
    Biofortified cassava increases β-carotene and vitamin A concentrations in the TAG-rich plasma layer of American women
    La Frano, Michael R.; Woodhouse, Leslie R.; Burnett, Dustin J.; Burri, Betty J.
    British Journal of Nutrition (2013), 110 (2), 310-320CODEN: BJNUAV; ISSN:0007-1145. (Cambridge University Press)
    Biofortification of cassava with the provitamin A carotenoid β-carotene is a potential mechanism for alleviating vitamin A deficiency. Cassava is a staple food in the African diet, but data regarding the human bioavailability of β-carotene from this food are scarce. The objective of the present study was to evaluate provitamin A-enhanced cassava as a source of β-carotene and vitamin A for healthy adult women. The study was a randomized, cross-over trial of ten American women. The subjects consumed three different porridges sepd. by 2 wk washout periods. Treatment meals (contg. 100 g cassava) included: biofortified cassava (2 mg β-carotene) porridge with added oil (15 mL peanut or rapeseed oil, 20 g total fat); biofortified cassava porridge without added oil (6 g total fat); unfortified white cassava porridge with a 0·3 mg retinyl palmitate ref. dose and added oil (20 g total fat). Blood was collected six times from - 0·5 to 9·5 h post-feeding. TAG-rich lipoprotein (TRL) plasma was sepd. by ultracentrifugation and analyzed using HPLC with coulometric array electrochem. detection. The AUC for retinyl palmitate increased after the biofortified cassava meals were fed (P< 0·05). Vitamin A conversion was 4·2 (sd 3·1) and 4·5 (sd 3·1) μg β-carotene:1 μg retinol, with and without added oil, resp. These results show that biofortified cassava increases β-carotene and retinyl palmitate TRL plasma concns. in healthy well-nourished adult women, suggesting that it is a viable intervention food for preventing vitamin A deficiency.
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  53. 53
    Muzhingi, T.; Gadaga, T. H.; Siwela, A. H.; Grusak, M. A.; Russell, R. M.; Tang, G. Yellow maize with high beta-carotene is an effective source of vitamin A in healthy Zimbabwean men. Am. J. Clin. Nutr. 2011, 94 (2), 510– 519,  DOI: 10.3945/ajcn.110.006486
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    53
    Yellow maize with high β-carotene is an effective source of vitamin A in healthy Zimbabwean men
    Muzhingi, Tawanda; Gadaga, Tendekayi H.; Siwela, Andrew H.; Grusak, Michael A.; Russell, Robert M.; Tang, Guangwen
    American Journal of Clinical Nutrition (2011), 94 (2), 510-519CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
    The bioconversion efficiency of yellow corn β-carotene to retinol in humans is unknown. The objective of this study was to det. the vitamin A value of yellow corn β-carotene in humans. High β-carotene-contg. yellow corn was grown in a hydroponic medium with 23 at,% 2H2O during grain development. Yellow corn β-carotene showed the highest abundance of enrichment as [2H9]β-carotene. 8 Healthy Zimbabwean men volunteered for the study. On day 1 after a fasting blood draw, subjects consumed 300 g yellow corn porridge contg. 1.2 mg β-carotene, 20 g butter, and a 0.5-g corn oil capsule. On day 8, fasting blood was drawn, and subjects consumed 1 mg [13C10]retinyl acetate in a 0.5-g corn oil capsule and 300 g white corn porridge with 20 g butter. 36 Blood samples were collected from each subject over 36 d. Concns. and enrichments of retinol and β-carotene in labeled doses and serum were detd. with the use of HPLC, gas chromatog.-mass spectrometry, and liq. chromatog.-tandem mass spectrometry. The area under the curve (AUC) of retinol from 1.2 mg yellow corn β-carotene was 72.9 nmol/d, and the AUC of retinol from 1 mg retinyl acetate 13C10 was 161.1 nmol/d. The conversion factor of yellow corn β-carotene to retinol by wt. was 3.2 ± 1.5 to 1. In 8 healthy Zimbabwean men, 300 g cooked yellow corn contg. 1.2 mg β-carotene that was consumed with 20.5 g fat showed the same vitamin A activity as 0.38 mg retinol and provided 40-50% of the adult vitamin A Recommended Dietary Allowance.
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    Van Loo-Bouwman, C. A.; Naber, T. H. J.; Schaafsma, G. A review of vitamin A equivalency of β-carotene in various food matrices for human consumption. Br. J. Nutr. 2014, 111 (12), 2153– 2166,  DOI: 10.1017/S0007114514000166
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    A review of vitamin A equivalency of β-carotene in various food matrices for human consumption
    Van Loo-Bouwman, Carolien A.; Naber, Ton H. J.; Schaafsma, Gertjan
    British Journal of Nutrition (2014), 111 (12), 2153-2166CODEN: BJNUAV; ISSN:0007-1145. (Cambridge University Press)
    A review. Vitamin A equivalency of β-carotene (VEB) is defined as the amt. of ingested β-carotene in μg that is absorbed and converted into 1 μg retinol (vitamin A) in the human body. The objective of the present review was to discuss the different ests. for VEB in various types of dietary food matrixes. Different methods are discussed such as mass balance, dose-response and isotopic labeling. The VEB is currently estd. by the US Institute of Medicine (IOM) as 12:1 in a mixed diet and 2:1 in oil. For humans consuming β-carotene dissolved in oil, a VEB between 2:1 and 4:1 is feasible. A VEB of approx. 4:1 is applicable for biofortified cassava, yellow maize and Golden Rice, which are specially bred for human consumption in developing countries. We propose a range of 9:1-16:1 for VEB in a mixed diet that encompasses the IOM VEB of 12:1 and is realistic for a Western diet under Western conditions. For a 'prudent' (i.e. non-Western) diet including a variety of commonly consumed vegetables, a VEB could range from 9:1 to 28:1 in a mixed diet.
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    Leyvraz, M.; Laillou, A.; Rahman, S.; Ahmed, T.; Rahman, A. S.; Alam, N.; Ireen, S.; Panagides, D. An assessment of the potential impact of fortification of staples and condiments on micronutrient intake of young children and women of reproductive age in Bangladesh. Nutrients 2016, 8, 541,  DOI: 10.3390/nu8090541
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    55
    An assessment of the potential impact of fortification of staples and condiments on micronutrient intake of young children and women of reproductive age in Bangladesh
    Leyvraz, Magali; Laillou, Arnaud; Rahman, Sabuktagin; Ahmed, Tahmeed; Rahman, Ahmed Shafiqur; Alam, Nurul; Ireen, Santhia; Panagides, Dora
    Nutrients (2016), 8 (9), 541/1-541/12CODEN: NUTRHU; ISSN:2072-6643. (MDPI AG)
    Bangladesh has experienced rapid economic growth and achieved major health improvements in the past decade, but malnutrition rates remain high. A nationally representative study conducted in 2011 assessed the dietary habits of 841 children 24-59 mo old, 1428 children 6-14 years old, and 1412 nonpregnant, nonlactating women. The study's objective was to assess dietary intakes of key micronutrients and the consumption pattern of potentially fortifiable foods, and then to model the potential impact of the fortification of key staple foods. The current intakes of several micronutrients-namely, iron, zinc, folate, vitamin A, and vitamin B12-were found to be insufficient to meet the needs of Bangladesh's children and women. The fortification of rice with iron and zinc and edible oil with vitamin A has the potential to fill a significant part of the nutrient gap, as these are consumed widely and in significant amts. Wheat flour and sugar are not as promising food vehicles in the Bangladeshi context, as they were consumed by a smaller portion of the population and in smaller amts. In conclusion, fortification of rice and oil is recommended to address the large gap in micronutrient intakes.
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      Annals of the New York Academy of Sciences (2014), 1324 (Technical Considerations for Rice Fortification in Public Health), 55-66CODEN: ANYAA9; ISSN:0077-8923. (John Wiley & Sons, Inc.)
      Micronutrient deficiencies are often linked to low dietary diversity. Rice fortification could substantially increase micronutrient intake in countries where rice is a staple food. The World Health Organization (WHO) interim consensus statement on maize and wheat flour fortification is based on the premise of the public health importance of specific micronutrient deficiencies and evidence of the benefits of increased micronutrient intake. Since this rationale for maize and wheat flour also applies to rice, it is recommended to fortify rice with iron, folic acid, vitamin B12, vitamin A, and zinc, as well as with thiamin, vitamin B6, and niacin, as polished rice has low levels of these micronutrients. To achieve intake that meets the estd. av. requirement of adults, the following levels are recommended where rice consumption is 150-300 g/cap/day (mg/100 g): iron, 7; folic acid, 0.13; vitamin B12, 0.001; vitamin A, 0.15; zinc, 6; thiamin, 0.5; niacin, 7; and vitamin B6, 0.6. These concns. can be achieved at a 1:100 blending ratio of fortified:unfortified kernels. The costs of rice fortification are largely detd. by the manufg. of the fortified kernels rather than by the no. of micronutrients that are added. These are general recommendations that can be adjusted locally, and monitoring and impact evaluation should accompany the introduction of rice fortification.
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      Scrimshaw, N. S.; SanGiovanni, J. P. Synergism of nutrition, infection, and immunity: an overview. Am. J. Clin. Nutr. 1997, 66, 464S– 477S,  DOI: 10.1093/ajcn/66.2.464S
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      Synergism of nutrition, infection, and immunity: an overview
      Scrimshaw, Nevin S.; SanGiovanni, John Paul
      American Journal of Clinical Nutrition (1997), 66 (2), 464S-477SCODEN: AJCNAC; ISSN:0002-9165. (American Society for Clinical Nutrition)
      Infections, no matter how mild, have adverse effects on nutritional status. The significance of these effects depends on the previous nutritional status of the individual, the nature and duration of the infection, and the diet during the recovery period. Conversely, almost any nutrient deficiency, if sufficiently severe, will impair resistance to infection. Iron deficiency and protein-energy malnutrition, both highly prevalent, have the greatest public health importance in this regard. Remarkable advances in immunol. of recent decades have increased insights into the mechanisms responsible for the effects of infection. These include impaired antibody formation; loss of delayed cutaneous hypersensitivity; reduced Ig concns.; decreased thymic and splenic lymphocytes; reduced complement formation, secretory IgA, and interferon; and lower T cells and T cell subsets (helper, suppressor-cytotoxic, and natural killer cells) and interleukin 2 receptors. The effects obsd. with single or multiple nutrient deficiencies are due to some combination of these responses. In general, cell-mediated and nonspecific immunity are more sensitive than humoral immunity.
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      Christian, P.; Schulze, K.; Stoltzfus, R. J.; West, K. P. Hyporetinolemia, illness symptoms, and acute phase protein response in pregnant women with and without night blindness. Am. J. Clin. Nutr. 1998, 67, 1237– 43,  DOI: 10.1093/ajcn/67.6.1237
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      Hyporetinolemia, illness symptoms, and acute phase protein response in pregnant women with and without night blindness
      Christian, Parul; Schulze, Kerry; Stoltzfus, Rebecca J.; West, Keith P. Jr.
      American Journal of Clinical Nutrition (1998), 67 (6), 1237-1243CODEN: AJCNAC; ISSN:0002-9165. (American Society for Clinical Nutrition)
      We examd. the assocn. among elevations in acute phase proteins, reported illness, and hyporetinolemia in 234 pregnant Nepali women with (cases) and without (controls) night blindness. Serum α1-acid glycoprotein (AGP) and C-reactive protein (CRP) were inversely assocd. with serum retinol concns. Elevations in the concn. of CRP in both cases and controls and of AGP in cases were assocd. with significant redns. (≈0.2-0.3 μmol/L) in serum retinol. The risk of a low serum retinol concn. (< 0.7 μmol/L) with elevated AGP (≥ 1 g/L) and CRP (≥ 5 mg/L) concns. was significantly higher in cases (odds ratios = 8.6 and 4.3, resp.) than in controls (odd ratios = 1.9 and 2.4, resp.). A 7-d morbidity history indicated that cases were significantly more likely than controls to report symptoms of infections of the urinary, reproductive, and gastrointestinal tracts. Only a few of these symptoms (diarrhea, nausea, and vomiting) were significantly assocd. with low serum retinol concns. Illness in the previous week and elevated CRP or AGP concns. were synergistically assocd. with lower serum retinol. For example, the redn. in serum retinol in women with diarrhea and elevated AGP was 0.54 μmol/L, compared with a redn. of 0.03 μmol/L in those with diarrhea only. AGP and CRP may provide useful information about the effect of reported illness on hyporetinolemia in pregnancy. Infection-related hyporetinolemia may predispose women to night blindness during pregnancy in Nepal.
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      Burkhardt, P. K.; Beyer, P.; Wünn, J.; Kloti, A.; Armstrong, G. A.; Schledz, M.; Von Lintig, J. Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis. Plant Journal 1997, 11, 1071– 1078,  DOI: 10.1046/j.1365-313X.1997.11051071.x
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      Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudo-narcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis
      Burkhardt, Peter K.; Beyer, Peter; Wunn, Joachim; Kloti, Andreas; Armstrong, Gregory A.; Schledz, Michael; Von Lintig, Johannes; Potrykus, Ingo
      Plant Journal (1997), 11 (5), 1071-1078CODEN: PLJUED; ISSN:0960-7412. (Blackwell)
      Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither β-carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make β-carotene into rice endosperm tissue. Investigation of the biochem. properties of immature rice endosperm using 14C-labeled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two mols. of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for β-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudo-narcissus) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a crit. step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for long-term prospects of overcoming worldwide vitamin A deficiency.
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      Ye, X.; Al-Babili, S.; Kloti, A.; Zhang, J.; Lucca, P.; Beyer, P.; Potrykus, I. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science (New York, N.Y.) 2000, 287, 303– 305,  DOI: 10.1126/science.287.5451.303
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      Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm
      Ye X; Al-Babili S; Kloti A; Zhang J; Lucca P; Beyer P; Potrykus I
      Science (New York, N.Y.) (2000), 287 (5451), 303-5 ISSN:0036-8075.
      Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
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      Paine, J.; Shipton, C.; Chaggar, S.; Howells, R. M.; Kennedy, M. J.; Vernon, G.; Wright, S. Y.; Hinchliffe, E.; Adams, J. L.; Silverstone, A. L.; Drake, R. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat. Biotechnol. 2005, 23, 482– 487,  DOI: 10.1038/nbt1082
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      Improving the nutritional value of Golden Rice through increased pro-vitamin A content
      Paine, Jacqueline A.; Shipton, Catherine A.; Chaggar, Sunandha; Howells, Rhian M.; Kennedy, Mike J.; Vernon, Gareth; Wright, Susan Y.; Hinchliffe, Edward; Adams, Jessica L.; Silverstone, Aron L.; Drake, Rachel
      Nature Biotechnology (2005), 23 (4), 482-487CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      'Golden Rice' is a variety of rice engineered to produce β-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher β-carotene content. The authors hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in β-carotene accumulation. Through systematic testing of other plant psys, the authors identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. The authors went on to develop 'Golden Rice 2' introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. The authors obsd. an increase in total carotenoids of up to 23-fold (max. 37 μg/g) compared to the original Golden Rice and a preferential accumulation of β-carotene.
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      Codex. Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants CAC/GL45-2003; 2003.
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      Herman, R. A.; Price, W. D. Unintended compositional changes in genetically modified (GM) crops: 20 years of research. J. Agric. Food Chem. 2013, 61, 11695– 701,  DOI: 10.1021/jf400135r
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      Unintended Compositional Changes in Genetically Modified (GM) Crops: 20 Years of Research
      Herman, Rod A.; Price, William D.
      Journal of Agricultural and Food Chemistry (2013), 61 (48), 11695-11701CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A review. The compositional equivalency between genetically modified (GM) crops and nontransgenic comparators has been a fundamental component of human health safety assessment for 20 years. During this time, a large amt. of information has been amassed on the compositional changes that accompany both the transgenosis process and traditional breeding methods; addnl., the genetic mechanisms behind these changes have been elucidated. After two decades, scientists are encouraged to objectively assess this body of literature and det. if sufficient scientific uncertainty still exists to continue the general requirement for these studies to support the safety assessment of transgenic crops. It is concluded that suspect unintended compositional effects that could be caused by genetic modification have not materialized on the basis of this substantial literature. Hence, compositional equivalence studies uniquely required for GM crops may no longer be justified on the basis of scientific uncertainty.
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      Harrigan, G. G.; Stork, L. G.; Riordan, S. G.; Reynolds, T. L.; Ridley, W. P.; Masucci, J. D.; MacIsaac, S.; Halls, S. C.; Orth, R.; Smith, R. G.; Wen, L.; Brown, W. E.; Welsch, M.; Riley, R.; McFarland, D.; Pandravada, A.; Glenn, K. C. Impact of genetics and environment on nutritional and metabolite components of maize grain. J. Agric. Food Chem. 2007, 55, 6177– 6185,  DOI: 10.1021/jf070494k
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      Impact of Genetics and Environment on Nutritional and Metabolite Components of Maize Grain
      Harrigan, George G.; Stork, LeAnna G.; Riordan, Susan G.; Reynolds, Tracey L.; Ridley, William P.; Masucci, James D.; MacIsaac, Susan; Halls, Steven C.; Orth, Robert; Smith, Ronald G.; Wen, Li; Brown, Wayne E.; Welsch, Michael; Riley, Rochelle; McFarland, David; Pandravada, Anand; Glenn, Kevin C.
      Journal of Agricultural and Food Chemistry (2007), 55 (15), 6177-6185CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnol.-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnol.-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly assocd. with a newly introduced trait. Therefore, steps were initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against 2 different testers. These were grown at 3 different, but geog. similar, locations in the United States. In addn. to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, org. acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant org. acid was citric acid, with only minor amts. of other org. acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and org. acids appeared to be markedly higher than that obsd. for the OECD analytes.
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      Ricroch, A. E. Assessment of GE food safety using ’-omics’ techniques and long-term animal feeding studies. New Biotechnol. 2013, 30, 349– 354,  DOI: 10.1016/j.nbt.2012.12.001
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      Assessment of GE food safety using '-omics' techniques and long-term animal feeding studies
      Ricroch, Agnes E.
      New Biotechnology (2013), 30 (4), 349-354CODEN: NBEIBR; ISSN:1871-6784. (Elsevier B.V.)
      A review. Despite the fact that a thorough, lengthy and costly evaluation of genetically engineered (GE) crop plants (including compositional anal. and toxicol. tests) is imposed before marketing, some European citizens remain sceptical of the safety of GE food and feed. In this context, are addnl. tests necessary, and if so, what can we learn from them. To address these questions, we examd. data from 60 recent high-throughput '-omics' comparisons between GE and non-GE crop lines and 17 recent long-term animal feeding studies (longer than the classical 90-day subchronic toxicol. tests), as well as 16 multigenerational studies on animals. The '-omics' comparisons revealed that the genetic modification has less impact on plant gene expression and compn. than that of conventional plant breeding. Moreover, environmental factors (such as field location, sampling time, or agricultural practices) have a greater impact than transgenesis. None of these '-omics' profiling studies has raised new safety concerns about GE varieties; neither did the long-term and multigenerational studies on animals. Therefore, there is no need to perform such long-term studies in a case-by-case approach, unless reasonable doubt still exists after conducting a 90-day feeding test. In addn., plant compositional anal. and '-omics' profiling do not indicate that toxicol. tests should be mandatory. We discuss what complementary fundamental studies should be performed and how to choose the most efficient exptl. design to assess risks assocd. with new GE traits. The possible need to update the current regulatory framework is discussed.
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      Jiao, Z.; Si, X.X.; Li, G. K.; Zhang, Z. M.; Xu, X. P. Unintended Compositional Changes in Transgenic Rice Seeds (Oryza sativa L.) Studied by Spectral and Chromatographic Analysis Coupled with Chemometrics Methods. J. Agric. Food Chem. 2010, 58, 1746– 1754,  DOI: 10.1021/jf902676y
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      Unintended Compositional Changes in Transgenic Rice Seeds (Oryza sativa L.) Studied by Spectral and Chromatographic Analysis Coupled with Chemometrics Methods
      Jiao, Zhe; Si, Xiao-xi; Li, Gong-ke; Zhang, Zhuo-min; Xu, Xin-ping
      Journal of Agricultural and Food Chemistry (2010), 58 (3), 1746-1754CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Unintended compositional changes in transgenic rice seeds were studied by near-IR reflectance, GC-MS, HPLC, and ICP-AES coupled with chemometrics strategies. Three kinds of transgenic rice with resistance to fungal diseases or insect pests were comparatively studied with the non-transgenic counterparts in terms of key nutrients such as protein, amino acids, fatty acids, vitamins, elements, and antinutrient phytic acid recommended by the Organization for Economic Co-operation and Development (OECD). The compositional profiles were discriminated by chemometrics methods, and the discriminatory compds. were protein, three amino acids, two fatty acids, two vitamins, and several elements. Significance of differences for these compds. was proved by anal. of variance, and the variation extent ranged from 20 to 74% for amino acids, from 19 to 38% for fatty acids, from 25 to 57% for vitamins, from 20 to 50% for elements, and 25% for protein, whereas phytic acid content did not change significantly. The unintended compositional alterations as well as unintended change of phys. characteristic in transgenic rice compared with non-transgenic rice might be related to the genetic transformation, the effect of which needs to be elucidated by addnl. studies.
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      Determination of tryptophan from feedstuffs using reverse phase high-performance liquid chromatography
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      A new isocratic HPLC method was developed and tested for detg. tryptophan in feeds. Samples were hydrolyzed for 20 h at 120° under exclusion of O2 in 4N LiOH, microfiltered, and then analyzed by HPLC. Sepn. of amino acids was achieved using a C18 reversed-phase column eluted with 0.02M KH2PO4/methanol/water (20:3:2 by vol.). Three wavelengths (217, 254, and 280 nm) were compared for suitable absorbance by tryptophan. Aspects of the hydrolysis phase of anal. were also tested. This method permits rapid sample throughput (6 samples per h) and yields reproducible, high recovery values for all feed tested (98.9 ± 1.2%). All three wavelengths tested gave suitable detection of tryptophan from the feed hydrolyzates. Defatting was necessary to maintain max. recovery of tryptophan from the samples. Adjustment of hydrolyzate pH prior to anal. was not necessary and may even interfere with detection of tryptophan. Hydrolyzates stored at room temp. for 24 h had no detectable loss of tryptophan.
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      Simultaneous determination of tocopherols and tocotrienols in hazelnuts by a normal phase liquid chromatographic method
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      A normal-phase high-performance liq. chromatog. (NP-HPLC) method for the detn. of tocopherols and tocotrienols in hazelnuts is reported. Three extn. procedures (with and without sapon.) were assayed; the best results were obtained with a simple solid-liq. extn. procedure. Chromatog. sepn. was achieved using an Inertsil 5 SI column using isocratic elution with hexane/1,4-dioxane (95.5:4.5, vol./vol.) at a flow rate of 0.7 mL/min. The effluent was monitored by a series arrangement of a diode-array followed by a fluorescence detector. All compds. were sepd. in a short period of time (17 min). The method proved to be rapid, sensitive, reproducible, and accurate, allowing the simultaneous detn. of all vitamin E homologues.
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      American Journal of Clinical Nutrition (1990), 52 (2), 183-202CODEN: AJCNAC; ISSN:0002-9165.
      A review, with 224 refs., on the toxicity of excessive levels of vitamin A and carotenes in humans and animals.
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      EFSA Scientific Opinion on the re-evaluation of mixed carotenes (E 160a (i)) and beta-carotene (E 160a (ii)) as a foodadditive. EFSA Journal 2012, 10, 2593,  DOI: 10.2903/j.efsa.2012.2593
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      Koushik, A.; Hunter, D. J.; Spiegelman, D.; Anderson, K. E.; Buring, J. E.; Freudenheim, J. L.; Goldbohm, R. A.; Hankinson, S. E.; Larsson, S. C.; Leitzmann, M.; Marshall, J. R.; McCullough, M.; Miller, A. B.; Rodriguez, C.; Rohan, T. E.; Ross, J. A.; Schatzkin, A.; Schouten, L. J.; Willett, W. C.; Wolk, A.; Zhang, S. M.; Smith-Warner, S. A. Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies. Int. J. Cancer 2006, 119, 2148– 2154,  DOI: 10.1002/ijc.22076
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      Intake of the major carotenoids and the risk of epithelial ovarian cancer in a pooled analysis of 10 cohort studies
      Koushik, Anita; Hunter, David J.; Spiegelman, Donna; Anderson, Kristin E.; Buring, Julie E.; Freudenheim, Jo L.; Goldbohm, R. Alexandra; Hankinson, Susan E.; Larsson, Susanna C.; Leitzmann, Michael; Marshall, James R.; McCullough, Marjorie L.; Miller, Anthony B.; Rodriguez, Carmen; Rohan, Thomas E.; Ross, Julie A.; Schatzkin, Arthur; Schouten, Leo J.; Willett, Walter C.; Wolk, Alicja; Zhang, Shumin M.; Smith-Warner, Stephanie A.
      International Journal of Cancer (2006), 119 (9), 2148-2154CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
      Carotenoids in fruits and vegetables may protect against cancer because of their properties, including their functions as precursors of vitamin A and antioxidants. We examd. the assocns. between dietary intakes of α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin, and lycopene and the risk of invasive epithelial ovarian cancer using pooled primary data from 10 prospective cohort studies in North America and Europe. The carotenoid intakes were estd. from validated food frequency questionnaires used at baseline in each study. Study-specific relative risks (RR) were estd. using the Cox proportional hazards model and then combined using a random-effects model. Among 521,911 women, 2012 cases of ovarian cancer occurred during a follow-up of 7-22 years across studies. The major carotenoids were not much assocd. with the risk of ovarian cancer. The pooled multivariate RR values (95% confidence intervals) were 1.00 (0.95-1.05) for 600 μg/day increase in α-carotene intake, 0.96 (0.93-1.03) for 2500 μg/day increase in β-carotene intake, 0.99 (0.97-1.02) for 100 μg/day increase in β-cryptoxanthin intake, 0.98 (0.94-1.03) for 2500 μg/day increase in lutein/zeaxanthin intake, and 1.01 (0.97-1.05) for 4000 μg/day increase in lycopene intake. These assocns. did not appreciably differ among the studies. The obsd. assocns. did not much vary by population subgroups or histol. type of ovarian cancer. Thus, consumption of the major carotenoids during adulthood may not play a major role in the incidence of ovarian cancer.
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    49. 49
      Tang, G. Bioconversion of dietary provitamin A carotenoids to vitamin A in humans. Am. J. Clin. Nutr. 2010, 91, 1468S– 1473S,  DOI: 10.3945/ajcn.2010.28674G
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      Bioconversion of dietary provitamin A carotenoids to vitamin A in humans
      Tang, Guangwen
      American Journal of Clinical Nutrition (2010), 91 (5S), 1468S-1473SCODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
      A review. Recent progress in the measurement of the bioconversion of dietary provitamin A carotenoids to vitamin A is reviewed in this article. Methods to assess the bioavailability and bioconversion of provitamin A carotenoids have advanced significantly in the past 10 y, specifically through the use of stable isotope methodol., which includes the use of labeled plant foods. The effects of the food matrix on the bioconversion of provitamin A carotenoids to vitamin A, dietary fat effects, and the effect of genotype on the absorption and metab. of β-carotene have been reported recently. A summary of the major human studies that detd. conversion factors for dietary β-carotene to retinol is presented here, and these data show that the conversion efficiency of dietary β-carotene to retinol is in the range of 3.6-28:1 by wt. There is a wide variation in conversion factors reported not only between different studies but also between individuals in a particular study. These findings show that the vitamin A value of individual plant foods rich in provitamin A carotenoids may vary significantly and need further investigation.
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    50. 50
      Tang, G.; Tang, G.; Qin, J.; Dolnikowski, G. G.; Russell, R. M.; Grusak, M. A. Golden rice is an effective source of vitamin A. Am. J. Clin. Nutr. 2009, 89 (6), 1776– 1783,  DOI: 10.3945/ajcn.2008.27119
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      Golden Rice is an effective source of vitamin A
      Tang, Guangwen; Qin, Jian; Dolnikowski, Gregory G.; Russell, Robert M.; Grusak, Michael A.
      American Journal of Clinical Nutrition (2009), 89 (6), 1776-1783CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
      Genetically engineered "Golden Rice" contains up to 35 μg β-carotene per g of rice. It is important to det. the vitamin A equivalency of Golden Rice β-carotene to project the potential effect of this biofortified grain in rice-consuming populations that commonly exhibit low vitamin A status. The objective was to det. the vitamin A value of intrinsically labeled dietary Golden Rice in humans. Golden Rice plants were grown hydroponically with heavy water (deuterium oxide) to generate deuterium-labeled [2H]β-carotene in the rice grains. Golden Rice servings of 65-98 g (130-200 g cooked rice) contg. 0.99-1.53 mg β-carotene were fed to 5 healthy adult volunteers (3 women and 2 men) with 10 g butter. A ref. dose of [13C10]retinyl acetate (0.4-1.0 mg) in oil was given to each volunteer 1 wk before ingestion of the Golden Rice dose. Blood samples were collected over 36 d. The authors' results showed that the mean (± SD) area under the curve for the total serum response to [2H]retinol was 39.9 ± 20.7 μg/d after the Golden Rice dose. Compared with that of the [13C10]retinyl acetate ref. dose (84.7 ± 34.6 μg/d), Golden Rice β-carotene provided 0.24-0.94 mg retinol. Thus, the conversion factor of Golden Rice β-carotene to retinol is 3.8 ± 1.7 to 1 with a range of 1.9-6.4 to 1 by wt., or 2.0 ± 0.9 to 1 with a range of 1.0-3.4 to 1 by moles. β-Carotene derived from Golden Rice is effectively converted to vitamin A in humans.
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    51. 51
      Tang, G.; Hu, Y.; Yin, S.; Wang, Y.; Dallal, G. E.; Grusak, M. A.; Russell, R. M. β-carotene in Golden Rice is as good as β-carotene in oil at providing vitamin A to children. Am. J. Clin. Nutr. 2012, 96, 658– 664,  DOI: 10.3945/ajcn.111.030775
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      51
      β-Carotene in golden rice is as good as β-carotene in oil at providing vitamin A to children
      Tang, Guangwen; Hu, Yuming; Yin, Shi-an; Wang, Yin; Dallal, Gerard E.; Grusak, Michael A.; Russell, Robert M.
      American Journal of Clinical Nutrition (2012), 96 (3), 658-664CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
      Background: Golden Rice (GR) has been genetically engineered to be rich in β-carotene for use as a source of vitamin A. Objective: The objective was to compare the vitamin A value of β-carotene in GR and in spinach with that of pure β-carotene in oil when consumed by children. Design: Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient soln. contg. 23 atom% 2H2O) or [2H8]β-carotene in an oil capsule. The GR and spinach β-carotene were enriched with deuterium (2H) with the highest abundance mol. mass (M) at Mβ-C+2H10. [13C10]Retinyl acetate in an oil capsule was administered as a ref. dose. Serum samples collected from subjects were analyzed by using gas chromatog. electron-capture neg. chem. ionization mass spectrometry for the enrichments of labeled retinol: Mretinol+4 (from [2H8]β-carotene in oil), Mretinol+5 (from GR or spinach [2H10]β-carotene), and Mretinol+10 (from [13C10]retinyl acetate). Results: Using the response to the dose of [13C10]retinyl acetate (0.5 mg) as a ref., our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure β-carotene (0.5 mg), GR β-carotene (0.6 mg), and spinach β-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by wt., resp. Conclusions: The β-carotene in GR is as effective as pure β-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ∼100 to 150 g cooked GR (50 g dry wt.) can provide ∼60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.
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    52. 52
      La Frano, M. R.; Woodhouse, L. R.; Burnett, D. J.; Burri, B. J. Biofortified cassava increases β-carotene and vitamin A concentrations in the TAG-rich plasma layer of American women. Br. J. Nutr. 2013, 110 (2), 310– 20,  DOI: 10.1017/S0007114512005004
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      52
      Biofortified cassava increases β-carotene and vitamin A concentrations in the TAG-rich plasma layer of American women
      La Frano, Michael R.; Woodhouse, Leslie R.; Burnett, Dustin J.; Burri, Betty J.
      British Journal of Nutrition (2013), 110 (2), 310-320CODEN: BJNUAV; ISSN:0007-1145. (Cambridge University Press)
      Biofortification of cassava with the provitamin A carotenoid β-carotene is a potential mechanism for alleviating vitamin A deficiency. Cassava is a staple food in the African diet, but data regarding the human bioavailability of β-carotene from this food are scarce. The objective of the present study was to evaluate provitamin A-enhanced cassava as a source of β-carotene and vitamin A for healthy adult women. The study was a randomized, cross-over trial of ten American women. The subjects consumed three different porridges sepd. by 2 wk washout periods. Treatment meals (contg. 100 g cassava) included: biofortified cassava (2 mg β-carotene) porridge with added oil (15 mL peanut or rapeseed oil, 20 g total fat); biofortified cassava porridge without added oil (6 g total fat); unfortified white cassava porridge with a 0·3 mg retinyl palmitate ref. dose and added oil (20 g total fat). Blood was collected six times from - 0·5 to 9·5 h post-feeding. TAG-rich lipoprotein (TRL) plasma was sepd. by ultracentrifugation and analyzed using HPLC with coulometric array electrochem. detection. The AUC for retinyl palmitate increased after the biofortified cassava meals were fed (P< 0·05). Vitamin A conversion was 4·2 (sd 3·1) and 4·5 (sd 3·1) μg β-carotene:1 μg retinol, with and without added oil, resp. These results show that biofortified cassava increases β-carotene and retinyl palmitate TRL plasma concns. in healthy well-nourished adult women, suggesting that it is a viable intervention food for preventing vitamin A deficiency.
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    53. 53
      Muzhingi, T.; Gadaga, T. H.; Siwela, A. H.; Grusak, M. A.; Russell, R. M.; Tang, G. Yellow maize with high beta-carotene is an effective source of vitamin A in healthy Zimbabwean men. Am. J. Clin. Nutr. 2011, 94 (2), 510– 519,  DOI: 10.3945/ajcn.110.006486
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      53
      Yellow maize with high β-carotene is an effective source of vitamin A in healthy Zimbabwean men
      Muzhingi, Tawanda; Gadaga, Tendekayi H.; Siwela, Andrew H.; Grusak, Michael A.; Russell, Robert M.; Tang, Guangwen
      American Journal of Clinical Nutrition (2011), 94 (2), 510-519CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
      The bioconversion efficiency of yellow corn β-carotene to retinol in humans is unknown. The objective of this study was to det. the vitamin A value of yellow corn β-carotene in humans. High β-carotene-contg. yellow corn was grown in a hydroponic medium with 23 at,% 2H2O during grain development. Yellow corn β-carotene showed the highest abundance of enrichment as [2H9]β-carotene. 8 Healthy Zimbabwean men volunteered for the study. On day 1 after a fasting blood draw, subjects consumed 300 g yellow corn porridge contg. 1.2 mg β-carotene, 20 g butter, and a 0.5-g corn oil capsule. On day 8, fasting blood was drawn, and subjects consumed 1 mg [13C10]retinyl acetate in a 0.5-g corn oil capsule and 300 g white corn porridge with 20 g butter. 36 Blood samples were collected from each subject over 36 d. Concns. and enrichments of retinol and β-carotene in labeled doses and serum were detd. with the use of HPLC, gas chromatog.-mass spectrometry, and liq. chromatog.-tandem mass spectrometry. The area under the curve (AUC) of retinol from 1.2 mg yellow corn β-carotene was 72.9 nmol/d, and the AUC of retinol from 1 mg retinyl acetate 13C10 was 161.1 nmol/d. The conversion factor of yellow corn β-carotene to retinol by wt. was 3.2 ± 1.5 to 1. In 8 healthy Zimbabwean men, 300 g cooked yellow corn contg. 1.2 mg β-carotene that was consumed with 20.5 g fat showed the same vitamin A activity as 0.38 mg retinol and provided 40-50% of the adult vitamin A Recommended Dietary Allowance.
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    54. 54
      Van Loo-Bouwman, C. A.; Naber, T. H. J.; Schaafsma, G. A review of vitamin A equivalency of β-carotene in various food matrices for human consumption. Br. J. Nutr. 2014, 111 (12), 2153– 2166,  DOI: 10.1017/S0007114514000166
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      54
      A review of vitamin A equivalency of β-carotene in various food matrices for human consumption
      Van Loo-Bouwman, Carolien A.; Naber, Ton H. J.; Schaafsma, Gertjan
      British Journal of Nutrition (2014), 111 (12), 2153-2166CODEN: BJNUAV; ISSN:0007-1145. (Cambridge University Press)
      A review. Vitamin A equivalency of β-carotene (VEB) is defined as the amt. of ingested β-carotene in μg that is absorbed and converted into 1 μg retinol (vitamin A) in the human body. The objective of the present review was to discuss the different ests. for VEB in various types of dietary food matrixes. Different methods are discussed such as mass balance, dose-response and isotopic labeling. The VEB is currently estd. by the US Institute of Medicine (IOM) as 12:1 in a mixed diet and 2:1 in oil. For humans consuming β-carotene dissolved in oil, a VEB between 2:1 and 4:1 is feasible. A VEB of approx. 4:1 is applicable for biofortified cassava, yellow maize and Golden Rice, which are specially bred for human consumption in developing countries. We propose a range of 9:1-16:1 for VEB in a mixed diet that encompasses the IOM VEB of 12:1 and is realistic for a Western diet under Western conditions. For a 'prudent' (i.e. non-Western) diet including a variety of commonly consumed vegetables, a VEB could range from 9:1 to 28:1 in a mixed diet.
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    55. 55
      Leyvraz, M.; Laillou, A.; Rahman, S.; Ahmed, T.; Rahman, A. S.; Alam, N.; Ireen, S.; Panagides, D. An assessment of the potential impact of fortification of staples and condiments on micronutrient intake of young children and women of reproductive age in Bangladesh. Nutrients 2016, 8, 541,  DOI: 10.3390/nu8090541
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      55
      An assessment of the potential impact of fortification of staples and condiments on micronutrient intake of young children and women of reproductive age in Bangladesh
      Leyvraz, Magali; Laillou, Arnaud; Rahman, Sabuktagin; Ahmed, Tahmeed; Rahman, Ahmed Shafiqur; Alam, Nurul; Ireen, Santhia; Panagides, Dora
      Nutrients (2016), 8 (9), 541/1-541/12CODEN: NUTRHU; ISSN:2072-6643. (MDPI AG)
      Bangladesh has experienced rapid economic growth and achieved major health improvements in the past decade, but malnutrition rates remain high. A nationally representative study conducted in 2011 assessed the dietary habits of 841 children 24-59 mo old, 1428 children 6-14 years old, and 1412 nonpregnant, nonlactating women. The study's objective was to assess dietary intakes of key micronutrients and the consumption pattern of potentially fortifiable foods, and then to model the potential impact of the fortification of key staple foods. The current intakes of several micronutrients-namely, iron, zinc, folate, vitamin A, and vitamin B12-were found to be insufficient to meet the needs of Bangladesh's children and women. The fortification of rice with iron and zinc and edible oil with vitamin A has the potential to fill a significant part of the nutrient gap, as these are consumed widely and in significant amts. Wheat flour and sugar are not as promising food vehicles in the Bangladeshi context, as they were consumed by a smaller portion of the population and in smaller amts. In conclusion, fortification of rice and oil is recommended to address the large gap in micronutrient intakes.
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