Overlapping Vitamin A Interventions with Provitamin A Carotenoids and Preformed Vitamin A Cause Excessive Liver Retinol Stores in Male Mongolian Gerbils.

BACKGROUND: Vitamin A (VA) deficiency is a public health problem in some countries. Fortification, supplementation, and increased provitamin A consumption through biofortification are efficacious, but monitoring is needed due to risk of excessive VA intake when interventions overlap. OBJECTIVES: Two studies in 28-36-d-old male Mongolian gerbils simulated exposure to multiple VA interventions to determine the effects of provitamin A carotenoid consumption from biofortified maize and carrots and preformed VA fortificant on status. METHODS: Study 1 was a 2 × 2 × 2 factorial design (n = 85) with high-ß-carotene maize, orange carrots, and VA fortification at 50% estimated gerbil needs, compared with white maize and white carrot controls. Study 2 was a 2 × 3 factorial design (n = 66) evaluating orange carrot and VA consumption through fortification at 100% and 200% estimated needs. Both studies utilized 2-wk VA depletion, baseline evaluation, 9-wk treatments, and liver VA stores by HPLC. Intestinal scavenger receptor class B member 1 (Scarb1), ß-carotene 15,15'-dioxygenase (Bco1), ß-carotene 9',10'-oxygenase (Bco2), intestine-specific homeobox (Isx), and cytochrome P450 26A1 isoform α1 (Cyp26a1) expression was analyzed by qRT-PCR in study 2. RESULTS: In study 1, liver VA concentrations were significantly higher in orange carrot (0.69 ± 0.12 µmol/g) and orange maize groups (0.52 ± 0.21 µmol/g) compared with baseline (0.23 ± 0.069 µmol/g) and controls. Liver VA concentrations from VA fortificant alone (0.11 ± 0.053 µmol/g) did not differ from negative control. In study 2, orange carrot significantly enhanced liver VA concentrations (0.85 ± 0.24 µmol/g) relative to baseline (0.43 ± 0.14 µmol/g), but VA fortificant alone (0.42 ± 0.21 µmol/g) did not. Intestinal Scarb1 and Bco1 were negatively correlated with increasing liver VA concentrations (P < 0.01, r2 = 0.25-0.27). Serum retinol concentrations did not differ. CONCLUSIONS: Biofortified carrots and maize without fortification prevented VA deficiency in gerbils. During adequate provitamin A dietary intake, preformed VA intake resulted in excessive liver stores in gerbils, despite downregulation of carotenoid absorption and cleavage gene expression.

Maize Milling Method Affects Growth and Zinc Status but Not Provitamin A Carotenoid Bioefficacy in Male Mongolian Gerbils.

Background: Vitamin A (VA) and zinc deficiencies are prevalent. Maize is a common staple, and milling affects nutrient and nutrient-modifier profiles.Objective: We investigated the interaction of maize milling methods (i.e., whole grain compared with refined) in male Mongolian gerbils aged 29-35 d with conventionally bred provitamin A-biofortified (orange) or white maize on VA and zinc status.Methods: Study 1 (n = 67) was a 2 × 3 milling (whole compared with refined) by VA [no-vitamin A placebo group (VA-), orange, and VA-supplemented group (VA+)] design, with 4 wk of VA depletion followed by six 4-wk treatments (n = 10/treatment). Study 2 (n = 33) was a 2 × 2 milling-by-zinc [no-zinc placebo group (Zn-) compared with zinc-supplemented group (Zn+)] design, including 2 wk of VA depletion followed by four 3-wk treatments (n = 8-9/treatment). For study 1, positive and negative control groups were given supplemental VA at equimolar amounts to ß-carotene equivalents consumed by the orange groups (74 ± 5 nmol/d) or placebo, respectively. For study 2, positive and negative control groups were given 152 µg Zn/d or placebo, respectively.Results: Milling significantly affected zinc concentration, providing 44-45% (whole grain) or 9-14% (refined) NRC requirements. In study 1, orange maize improved liver VA concentrations (mean ± SD: 0.28 ± 0.08 µmol/g) compared with the white maize groups (0.072 ± 0.054 µmol/g). Provitamin A bioefficacy was similar. In study 2, neither zinc nor milling influenced liver retinol. Refined Zn- gerbils weighed less than others by day 14 (46.6 ± 7.1 compared with 56.5 ± 3.5 g, respectively; P < 0.0001). Milling affected pancreas zinc concentrations (refined Zn-: 21.1 ± 1.8 µg Zn/g; whole Zn-: 32.5 ± 5.8 µg Zn/g).Conclusions: Whole-grain intake improved zinc and did not affect provitamin A bioefficacy. Other factors affected by milling (e.g., shelf life, preference, aflatoxin fractioning) need to be considered to maximize health.

13C Natural Abundance of Serum Retinol Is a Novel Biomarker for Evaluating Provitamin A Carotenoid-Biofortified Maize Consumption in Male Mongolian Gerbils.

BACKGROUND: Crops such as maize, sorghum, and millet are being biofortified with provitamin A carotenoids to ensure adequate vitamin A (VA) intakes. VA assessment can be challenging because serum retinol concentrations are homeostatically controlled and more sensitive techniques are resource-intensive. OBJECTIVES: We investigated changes in serum retinol relative differences of isotope amount ratios of (13)C/(12)C (δ(13)C) caused by natural (13)C fractionation in C3 compared with C4 plants as a biomarker to detect provitamin A efficacy from biofortified (orange) maize and high-carotene carrots. METHODS: The design was a 2 × 2 × 2 maize (orange compared with white) by carrot (orange compared with white) by a VA fortificant (VA+ compared with VA-) in weanling male Mongolian gerbils (n = 55), which included a 14-d VA depletion period and a 62-d treatment period (1 baseline and 8 treatment groups; n = 5-7/group). Liver VA and serum retinol were quantified, purified by HPLC, and analyzed by GC combustion isotope ratio mass spectrometry for (13)C. RESULTS: Treatments affected liver VA concentrations (0.048 ± 0.039 to 0.79 ± 0.24 µmol/g; P < 0.0001) but not overall serum retinol concentrations (1.38 ± 0.22 µmol/L). Serum retinol and liver VA δ(13)C were significantly correlated (R(2) = 0.92; P < 0.0001). Serum retinol δ(13)C differentiated control groups that consumed white maize and white carrots (-27.1 ± 1.2 δ(13)C‰) from treated groups that consumed orange maize and white carrots (-21.6 ± 1.4 δ(13)C‰ P < 0.0001) and white maize and orange carrots (-30.6 ± 0.7 δ(13)C‰ P < 0.0001). A prediction model demonstrated the relative contribution of orange maize to total dietary VA for groups that consumed VA from mixed sources. CONCLUSIONS: Provitamin A efficacy and quantitative estimation of the relative contribution to dietary VA were demonstrated with the use of serum retinol δ(13)C. This method could be used for maize efficacy or effectiveness studies and with other C4 crops biofortified with provitamin A carotenoids (e.g., millet, sorghum). Advantages include no extrinsic tracer dose, 1 blood sample, and higher sensitivity than serum retinol concentrations alone.

Genome-wide association analysis reveals new targets for carotenoid biofortification in maize.

KEY MESSAGE: Genome-wide association analysis in CIMMYT's association panel revealed new favorable native genomic variations in/nearby important genes such as hydroxylases and CCD1 that have potential for carotenoid biofortification in maize. Genome-wide association studies (GWAS) have been used extensively to identify allelic variation for genes controlling important agronomic and nutritional traits in plants. Provitamin A (proVA) enhancing alleles of lycopene epsilon cyclase (LCYE) and ß-carotene hydroxylase 1 (CRTRB1), previously identified through candidate-gene based GWAS, are currently used in CIMMYT's maize breeding program. The objective of this study was to identify genes or genomic regions controlling variation for carotenoid concentrations in grain for CIMMYT's carotenoid association mapping panel of 380 inbred maize lines, using high-density genome-wide platforms with ~476,000 SNP markers. Population structure effects were minimized by adjustments using principal components and kinship matrix with mixed models. Genome-wide linkage disequilibrium (LD) analysis indicated faster LD decay (3.9 kb; r (2) = 0.1) than commonly reported for temperate germplasm, and therefore the possibility of achieving higher mapping resolution with our mostly tropical diversity panel. GWAS for various carotenoids identified CRTRB1, LCYE and other key genes or genomic regions that govern rate-critical steps in the upstream pathway, such as DXS1, GGPS1, and GGPS2 that are known to play important roles in the accumulation of precursor isoprenoids as well as downstream genes HYD5, CCD1, and ZEP1, which are involved in hydroxylation and carotenoid degradation. SNPs at or near all of these regions were identified and may be useful target regions for carotenoid biofortification breeding efforts in maize; for example a genomic region on chromosome 2 explained ~16% of the phenotypic variance for ß-carotene independently of CRTRB1, and a variant of CCD1 that resulted in reduced ß-cryptoxanthin degradation was found in lines that have previously been observed to have low proVA degradation rates.

High-provitamin A carotenoid (Orange) maize increases hepatic vitamin A reserves of offspring in a vitamin A-depleted sow-piglet model during lactation.

The relationship of dietary vitamin A transfer from mother to fetus is not well understood. The difference in swine offspring liver reserves was investigated between single-dose vitamin A provided to the mother post-conception compared with continuous provitamin A carotenoid dietary intake from biofortified (enhanced provitamin A) orange maize (OM) fed during gestation and lactation. Vitamin A-depleted sows were fed OM (n = 5) or white maize (WM) + 1.05 mmol retinyl palmitate administered at the beginning of gestation (n = 6). Piglets (n = 102) were killed at 0, 10, 20, and 28 d after birth. Piglets from sows fed OM had higher liver retinol reserves (P < 0.0001) and a combined mean concentration from d 10 to 28 of 0.11 ± 0.030 µmol/g. Piglets from sows fed WM had higher serum retinol concentrations (0.56 ± 0.25 µmol/L; P = 0.0098) despite lower liver retinol concentrations of 0.068 ± 0.026 µmol/g from d 10 to 28. Milk was collected at 0, 5, 10, 20, and 28 d. Sows fed OM had a higher milk retinol concentration (1.36 ± 1.30 µmol/L; P = 0.038), than those fed WM (0.93 ±1.03 µmol/L). Sow livers were collected at the end of the study (n = 3/group) and had identical retinol concentrations (0.22 ± 0.05 µmol/g). Consumption of daily provitamin A carotenoids by sows during gestation and lactation increased liver retinol status in weanling piglets, illustrating the potential for provitamin A carotenoid consumption from biofortified staple foods to improve vitamin A reserves. Biofortified OM could have a measurable impact on vitamin A status in deficient populations if widely adopted.

Genetic variation among elite inbred lines suggests potential to breed for BNI-capacity in maize.

Biological nitrification inhibition (BNI) is a plant function where root systems release antibiotic compounds (BNIs) specifically aimed at suppressing nitrifiers to limit soil-nitrate formation in the root zone. Little is known about BNI-activity in maize (Zea mays L.), the most important food, feed, and energy crop. Two categories of BNIs are released from maize roots; hydrophobic and hydrophilic BNIs, that determine BNI-capacity in root systems. Zeanone is a recently discovered hydrophobic compound with BNI-activity, released from maize roots. The objectives of this study were to understand/quantify the relationship between zeanone activity and hydrophobic BNI-capacity. We assessed genetic variability among 250 CIMMYT maize lines (CMLs) characterized for hydrophobic BNI-capacity and zeanone activity, towards developing genetic markers linked to this trait in maize. CMLs with high BNI-capacity and ability to release zeanone from roots were identified. GWAS was performed using 27,085 SNPs (with unique positions on the B73v.4 reference genome, and false discovery rate = 10), and phenotypic information for BNI-capacity and zeanone production from root systems. Eighteen significant markers were identified; three associated with specific BNI-activity (SBNI), four with BNI-activity per plant (BNIPP), another ten were common between SBNI and BNIPP, and one with zeanone release. Further, 30 annotated genes were associated with the significant SNPs; most of these genes are involved in pathways of "biological process", and one (AMT5) in ammonium regulation in maize roots. Although the inbred lines in this study were not developed for BNI-traits, the identification of markers associated with BNI-capacity suggests the possibility of using these genomic tools in marker-assisted selection to improve hydrophobic BNI-capacity in maize.

Redesigning crop varieties to win the race between climate change and food security.

Climate change poses daunting challenges to agricultural production and food security. Rising temperatures, shifting weather patterns, and more frequent extreme events have already demonstrated their effects on local, regional, and global agricultural systems. Crop varieties that withstand climate-related stresses and are suitable for cultivation in innovative cropping systems will be crucial to maximize risk avoidance, productivity, and profitability under climate-changed environments. We surveyed 588 expert stakeholders to predict current and novel traits that may be essential for future pearl millet, sorghum, maize, groundnut, cowpea, and common bean varieties, particularly in sub-Saharan Africa. We then review the current progress and prospects for breeding three prioritized future-essential traits for each of these crops. Experts predict that most current breeding priorities will remain important, but that rates of genetic gain must increase to keep pace with climate challenges and consumer demands. Importantly, the predicted future-essential traits include innovative breeding targets that must also be prioritized; for example, (1) optimized rhizosphere microbiome, with benefits for P, N, and water use efficiency, (2) optimized performance across or in specific cropping systems, (3) lower nighttime respiration, (4) improved stover quality, and (5) increased early vigor. We further discuss cutting-edge tools and approaches to discover, validate, and incorporate novel genetic diversity from exotic germplasm into breeding populations with unprecedented precision, accuracy, and speed. We conclude that the greatest challenge to developing crop varieties to win the race between climate change and food security might be our innovativeness in defining and boldness to breed for the traits of tomorrow.

Comparative intake of white- versus orange-colored maize by Zambian children in the context of promotion of biofortified maize.

BACKGROUND: Vitamin A deficiency is associated with poor health outcomes related to reproduction, growth, vision, and immunity. Biofortification of staple crops is a novel strategy for combating vitamin A deficiency in high-risk populations where staple food intakes are high. African populations are proposed beneficiaries of maize (Zea mays) biofortified with provitamin A carotenoids, often called "orange maize" because of its distinctive deep yellow-orange kernels. The color facilitates ready recognition but presents a cultural challenge to maize-consuming populations, including those in much of Africa, who traditionally eat white varieties. OBJECTIVE: This study explores the intake patterns of, as well as adaptation to, traditional foods made with provitamin A-biofortified maize compared with white maize in rural Zambian children 3 to 5 years of age (n = 189) during a 3-month feeding trial. METHODS: The subjects were fed a breakfast of maize porridge (sweet mush), a lunch of maize nshima (stiff mush) with various side dishes, and an afternoon snack based on a 6-day rotating menu. The trial was conducted in 2010. The orange maize used in the trial came from three different sources. O1 maize was from the 2009 harvest and was stored in a freezer until use in 2010. O2 maize was also from the 2009 harvest and was stored in a cold room until 2010. O3 ("fresh") maize was from the 2010 harvest and was fed immediately after harvest in week 9 of the study and then stored in a freezer until milling for the final four weeks. RESULTS: Consumption of menu items, except snacks, was influenced by week (p < .0084). The intakes of porridge and nshima made with orange maize equaled those of porridge and nshima made with white maize from week 2 onward. The intakes of porridge and nshima prepared from O1 and O2 did not differ, but intakes became significantly higher when meals made from O3 were introduced (p < .014 for porridge and p < or = .013 for nshima). CONCLUSIONS: These results demonstrate quick adaptation to orange maize, a preference for recently harvested maize, and an optimistic outlook for similar adaptation patterns in other biofortified-maize target countries.

Toward product-based regulation of crops.

Current process-based approaches to regulation are no longer fit for purpose.

Provitamin a carotenoid bioavailability:what really matters?

Micronutrient malnutrition, or “hidden hunger,” afflicts a large part of the world’s population, with vitamin A deficiency among the most prevalent public health problems. Provitamin A carotenoids in plant foods are a source of vitamin A for humans; however, several factors, including species of carotenoids, host status, and effectors of absorption can negatively, positively, or in yet undetermined ways affect the bioavailability of these compounds. Staple foods biofortified with provitamin A carotenoids have shown more efficient bioconversion to retinol than generally observed for vegetables (e. g., 3 - 6 versus 10 - 80 beta-carotene to 1 µg retinol). Staple foods such as maize, rice, and cassava, are generally more accessible than meat or vegetable sources of retinol or provitamin A carotenoids to poor consumers, who are most likely to suffer micronutrient malnutrition. Interdisciplinary teamwork, including plant breeders, nutritionists, government and local agencies, seed companies, and communities, is needed to avail biofortified crops to needy populations. Key steps include developing, validating the nutritional effects of, providing nutrition education concerning, and promoting the use of biofortified crops. Provitamin A carotenoid biofortification of sweet potato, maize, cassava, and rice are at different stages along this continuum. Close linkages between agriculture, nutrition, and health, are essential in the quest to eradicate hunger among the poor.

Provitamin A Carotenoids in Grain Reduce Aflatoxin Contamination of Maize While Combating Vitamin A Deficiency.

Aflatoxin contamination of maize grain and products causes serious health problems for consumers worldwide, and especially in low- and middle-income countries where monitoring and safety standards are inconsistently implemented. Vitamin A deficiency (VAD) also compromises the health of millions of maize consumers in several regions of the world including large parts of sub-Saharan Africa. We investigated whether provitamin A (proVA) enriched maize can simultaneously contribute to alleviate both of these health concerns. We studied aflatoxin accumulation in grain of 120 maize hybrids formed by crossing 3 Aspergillus flavus resistant and three susceptible lines with 20 orange maize lines with low to high carotenoids concentrations. The hybrids were grown in replicated, artificially-inoculated field trials at five environments. Grain of hybrids with larger concentrations of beta-carotene (BC), beta-cryptoxanthin (BCX) and total proVA had significantly less aflatoxin contamination than hybrids with lower carotenoids concentrations. Aflatoxin contamination had negative genetic correlation with BCX (-0.28, p < 0.01), BC (-0.18, p < 0.05), and proVA (-0.23, p < 0.05). The relative ease of breeding for increased proVA carotenoid concentrations as compared to breeding for aflatoxin resistance in maize suggests using the former as a component of strategies to combat aflatoxin contamination problems for maize. Our findings indicate that proVA enriched maize can be particularly beneficial where the health burdens of exposure to aflatoxin and prevalence of VAD converge with high rates of maize consumption.

Genome Editing, Gene Drives, and Synthetic Biology: Will They Contribute to Disease-Resistant Crops, and Who Will Benefit?

Genetically engineered crops have been grown for more than 20 years, resulting in widespread albeit variable benefits for farmers and consumers. We review current, likely, and potential genetic engineering (GE) applications for the development of disease-resistant crop cultivars. Gene editing, gene drives, and synthetic biology offer novel opportunities to control viral, bacterial, and fungal pathogens, parasitic weeds, and insect vectors of plant pathogens. We conclude that there will be no shortage of GE applications totackle disease resistance and other farmer and consumer priorities for agricultural crops. Beyond reviewing scientific prospects for genetically engineered crops, we address the social institutional forces that are commonly overlooked by biological scientists. Intellectual property regimes, technology regulatory frameworks, the balance of funding between public- and private-sector research, and advocacy by concerned civil society groups interact to define who uses which GE technologies, on which crops, and for the benefit of whom. Ensuring equitable access to the benefits of genetically engineered crops requires affirmative policies, targeted investments, and excellent science.

Carotenoid and Tocochromanol Profiles during Kernel Development Make Consumption of Biofortified "Fresh" Maize an Option to Improve Micronutrient Nutrition.

Biofortification is a strategy to reduce micronutrient malnutrition. The aim of this study was to investigate whether consumption of biofortified fresh maize can supply nutritionally meaningful amounts of provitamin A carotenoids (PVA), zinc, lysine, and tryptophan. The accumulation patterns for PVA and tocochromanol compounds in developing grain of 23 PVA hybrids was studied, and nutritionally meaningful amounts of those compounds were found in grain by milk stage, when fresh maize is eaten. The highest PVA and tocochromanol accumulation occurred by physiological maturity. The percent apparent retention in boiled fresh maize was 92%, 117%, 99%, and 66% for PVA, zinc, lysine, and tryptophan, respectively. Consumption of 0.5 to 2 ears of fresh maize daily could supply 33-62.2%, 11-24% and more than 85% of the estimated average requirement of PVA, tryptophan, and zinc, respectively. The results indicate that eating biofortified fresh maize can contribute to improved micronutrient nutrition.

Harnessing genetic potential of wheat germplasm banks through impact-oriented-prebreeding for future food and nutritional security.

The value of exotic wheat genetic resources for accelerating grain yield gains is largely unproven and unrealized. We used next-generation sequencing, together with multi-environment phenotyping, to study the contribution of exotic genomes to 984 three-way-cross-derived (exotic/elite1//elite2) pre-breeding lines (PBLs). Genomic characterization of these lines with haplotype map-based and SNP marker approaches revealed exotic specific imprints of 16.1 to 25.1%, which compares to theoretical expectation of 25%. A rare and favorable haplotype (GT) with 0.4% frequency in gene bank identified on chromosome 6D minimized grain yield (GY) loss under heat stress without GY penalty under irrigated conditions. More specifically, the 'T' allele of the haplotype GT originated in Aegilops tauschii and was absent in all elite lines used in study. In silico analysis of the SNP showed hits with a candidate gene coding for isoflavone reductase IRL-like protein in Ae. tauschii. Rare haplotypes were also identified on chromosomes 1A, 6A and 2B effective against abiotic/biotic stresses. Results demonstrate positive contributions of exotic germplasm to PBLs derived from crosses of exotics with CIMMYT's best elite lines. This is a major impact-oriented pre-breeding effort at CIMMYT, resulting in large-scale development of PBLs for deployment in breeding programs addressing food security under climate change scenarios.

The research and implementation continuum of biofortified sweet potato and maize in Africa.

The enhancement of sweet potato and maize with provitamin A carotenoids has been part of HarvestPlus's research continuum since the formation of the biofortification project. This review includes case studies of biofortification strategies used for sweet potato in Uganda and orange maize in Zambia. The current status of the science and release of biofortified varieties was reviewed by three scientists who were part of the HarvestPlus program for more than a decade with input from a scientist who experienced orange maize dissemination in Zambia. High ß-carotene varieties of sweet potato were introduced into South Africa and Mozambique, and efficacy and effectiveness studies, respectively, showed promise to improve vitamin A status, followed by dissemination efforts in Uganda. A randomized, controlled effectiveness trial tested extension models to promote sweet potato and assessed vitamin A intake among Ugandans. Orange maize breeding was initially a challenge, but considering that the carotenoid biosynthetic pathway was present in maize germplasm, breeders quickly bred higher amounts of provitamin A into the maize that was ultimately released in Zambia. Initial resistance occurred because orange maize was associated with yellow maize, which had negative connotations associated with food aid and animal feed, and consumers preferred white maize. Currently, both orange crops are available on the market.

Maize genotype and food matrix affect the provitamin A carotenoid bioefficacy from staple and carrot-fortified feeds in Mongolian gerbils (Meriones unguiculatus).

Biofortification to increase provitamin A carotenoids is an agronomic approach to alleviate vitamin A deficiency. Two studies compared biofortified foods using in vitro and in vivo methods. Study 1 screened maize genotypes (n = 44) using in vitro analysis, which demonstrated decreasing micellarization with increasing provitamin A. Thereafter, seven 50% biofortified maize feeds that hypothesized a one-to-one equivalency between ß-cryptoxanthin and ß-carotene were fed to Mongolian gerbils. Total liver retinol differed among the maize groups (P = 0.0043). Study 2 assessed provitamin A bioefficacy from 0.5% high-carotene carrots added to 60% staple-food feeds, followed by in vitro screening. Liver retinol was highest in the potato and banana groups, maize group retinol did not differ from baseline, and all treatments differed from control (P < 0.0001). In conclusion, ß-cryptoxanthin and ß-carotene have similar bioefficacy; meal matrix effects influence provitamin A absorption from carrot; and in vitro micellarization does not predict bioefficacy.

Reliable and inexpensive colorimetric method for determining protein-bound tryptophan in maize kernels.

Biofortification programs in maize have led to the development of quality protein maize (QPM) with increased contents of the essential amino acids lysine and tryptophan, and increased nutritional value for protein deficient populations where maize is a staple food. Because multiple genetic systems control and modify the protein quality of QPM, tryptophan or lysine monitoring is required to maximize genetic gain in breeding programs. The objective of this work was to develop an accurate, reliable, and inexpensive method for tryptophan analysis in whole-grain maize flour to support QPM research efforts around the world. Tryptophan reacts with glyoxylic acid in the presence of sulfuric acid and ferric chloride, producing a colored compound that absorbs at 560 nm. A series of experiments varying the reagent concentrations, hydrolysis time, and length of the colorimetric reaction resulted in an optimized protocol which uses 0.1 M glyoxylic acid in 7 N sulfuric acid and 1.8 mM ferric chloride, and 30 min reaction time. This method produced stable and reproducible results for tryptophan concentration in whole-grain maize flour and was validated by comparison with data obtained using an acetic acid-based colorimetric procedure (r(2) = 0.80) and high pressure liquid chromatography (HPLC) (r(2) = 0.71). We describe adaptations that permit high throughput application of this tryptophan analysis method using a microplate platform.