Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African Cornmeal Assessed in Healthy Young Men Using the Indicator Amino Acid Oxidation Technique

Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African... Abstract Background Maize is a staple food in many regions of the world, particularly in Africa and Latin America. However, maize protein is limiting in the indispensable amino acids lysine and tryptophan, making its protein of poor quality. Objective The main objective of this study was to determine the protein quality of white African cornmeal by determining the metabolic availability (MA) of lysine and tryptophan. Methods To determine the MA of lysine, 4 amounts of l-lysine (10, 13, 16, and 18 mg · kg−1 · d−1 totaling 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement of 35 mg · kg−1 · d−1, respectively) were studied in 6 healthy young men in a repeated-measures design. To determine the MA of tryptophan, 4 amounts of l-tryptophan (0.5, 1, 1.5, and 2 mg · kg−1 · d−1 totaling 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement of 4 mg · kg−1 · d−1, respectively) were studied in 7 healthy young men in a repeated-measures design. The MAs of lysine and tryptophan were estimated by comparing the indicator amino acid oxidation (IAAO) response with varying intakes of lysine and tryptophan in cooked white cornmeal compared with the IAAO response to l-lysine and l-tryptophan intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) with the use of the slope ratio method. Results The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively. Conclusion Our study provides a robust estimate of the availability of lysine and tryptophan in African white maize to healthy young men. This estimate provides a basis for postproduction fortification or supplementation of maize-based diets. This trial was registered at www.clinicaltrials.gov as NCT02402179. metabolic availability, protein quality, indicator amino acid oxidation, stable isotope, phenylalanine oxidation, cereal grains, maize, corn Introduction Maize is the most abundant crop produced in the world, and although the United States is the largest producer, Mexico and Africa are the largest consumers (1). Globally, maize is the cheapest cereal grain, hence its importance as a staple food for the poor. In Mexico, it provides 45% of the daily calorie intake compared with only 10% for all other grains combined (2). Africa alone consumes 30% of the world’s food maize, with average per capita consumption of 50 kg (1). In sub-Saharan countries such as Malawi, Lesotho, Zambia, and Kenya, maize provide 80% of the calories in the diet, with per capita consumption ranging from 90 to 180 kg (1). However, in such countries in which the poor depend largely on maize as the main staple and where there is little access to a diverse diet, there is growth faltering in children in the absence of biofortification of maize with lysine and tryptophan (3). Protein is the key determinant of growth and bodily function, and the quality of food proteins depends on their amino acid (AA) composition and bioavailability. Maize protein is limiting in the essential AAs lysine and tryptophan, which limits its potential as a sole protein source. In addition, the lysine and tryptophan contents of maize could be affected by heat and the presence of antinutritional factors, such as tannins and phytates, that render them unavailable for protein synthesis after absorption (4, 5). Recently, the FAO Expert Consultation on Protein Quality identified the need to evaluate the protein quality of all foods consumed by humans, beginning with commonly consumed foods, especially by vulnerable populations in low-income countries (6). The FAO recommends that dietary AAs be treated as individual nutrients for the purposes of protein quality determination, and that results be provided on an individual AA basis (7). Due to the increasing importance of maize as a staple food for the poor, knowledge of its protein quality is critical to understanding its ability to provide the required AA content in the diet. In addition, its relative deficiency in lysine and tryptophan makes these individual AAs premier candidates for the evaluation of the protein quality of maize. The traditional method of protein quality evaluation—the protein digestibility-corrected AA score—was recently replaced by the digestible indispensable AA score method (7). For the digestible indispensable AA score method, bioavailability is measured as ileal digestibility. Ileal digestibility is very invasive, which limits its viability in humans, particularly those who are more vulnerable. Our laboratory adapted the minimally invasive indicator amino acid oxidation (IAAO) method to directly determine protein quality in humans (8, 9) by measuring AA bioavailability termed “metabolic availability” (MA) in whole foods. MA accounts for the effect of digestion, absorption, and metabolic utilization of the AAs provided by the protein source, whereas ileal digestibility only measures the total amount of AAs absorbed at the site of the terminal ileum (10). Because some AAs are absorbed in a form unavailable for protein synthesis, ileal digestibility has the potential to overestimate MA and hence protein quality. The present study aimed to apply the minimally invasive IAAO method to evaluate the protein quality of white African cornmeal (maize) by studying the MA of the most limiting AAs, lysine and tryptophan. Methods Two separate studies were completed with different participants for the determination of the MA of lysine and tryptophan in white African cornmeal. Assumptions and variables The IAAO method was applied in this study to determine the availability of lysine and tryptophan from the dietary protein of cornmeal for protein synthesis. It is based on the premise that when 1 AA is limiting for protein synthesis, all other AAs are in relative excess and must be oxidized. This oxidation is monitored by the use of l-[1-13C]phenylalanine, which indicates the rate of whole-body protein synthesis as driven by the limiting AAs, lysine and tryptophan in this study. We have previously shown that the appearance of 14CO2 from l-[1-14C]phenylalanine at isotopic steady state reflects whole-body protein synthesis (11). Therefore, changes in the oxidation of l-[1-13C]phenylalanine in response to graded intakes of lysine or tryptophan reflects whole-body bioavailability of these 2 AAs and accounts for their losses during digestion and cellular metabolism (10, 12). Thus, the higher the oxidation of the indicator AA (l-[1-13C]phenylalanine), the lower the MA of the test AAs (lysine and tryptophan) for protein synthesis. To determine the MA of an AA in a food protein, the following key conditions are necessary: The test AA must be first limiting to drive indicator oxidation rates. The response in oxidation rates of the indicator AA l-[1-13C]phenylalanine to changes in the test AA must be linear to allow calculation of bioavailability according to the principles of the slope ratio assay (13). The bioavailability of the test AA in the food protein is calculated relative to a standard/reference, which is free AA assumed to be 100% bioavailable (14, 15). It is assumed that the responses of the 2 substances are the same at a base value of x, making as = at (13). That is, the 2 lines intersect at a base value so that the common value of the intercepts can be denoted simply as a, giving regression equations y = a + btx for the test substance and y = a + bsx for the reference/standard. Letting xs and xt denote amounts of the standard and test substances required to produce equivalent value of the response y, then a + bsxs = a + btxt. Solving gives a relative bioavailability = xt/xs = bt/bs, the ratio of slopes of the regression lines (13). The indicator oxidation rate must show good repeatability to allow accurate determination of MA (8, 12, 16). Participants A total of 13 healthy young men participated in these 2 studies: 6 in the lysine study and 7 in the tryptophan study. Health was assessed by medical history and history of weight loss. Participant characteristics, body composition, and energy requirements were assessed at study entry (Table 1). Fat-free mass was assessed by BIA (Imp SFB7, ImpediMed Ltd, Qld, Australia) bioelectrical impedance analysis and Bod Pod (Cosmed USA); fat mass was assessed by skinfold thickness. Resting energy expenditure (REE) was measured by Indirect calorimetry (Vmax Encore, metabolic cart; Viasys) indirect calorimetry. BMI was calculated as weight (kilograms) divided by height in meters squared. The study protocol was explained to each participant, and informed written consent was obtained. Participants received financial compensation for their inconvenience. The study was approved by the Research Ethics Board of the Hospital for Sick Children, Toronto, Ontario, Canada. The trial was registered at www.clinicalrials.gov as NCT02402179. TABLE 1 Characteristics of healthy young men1 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 1Values are means ± SEMs, n = 6 for the lysine study and n = 7 for the tryptophan study. With the use of ANOVA, no differences in FFM or percentage body fat measured by BIA or SF or BP (P = 0.40 and 0.18, respectively) for the lysine study were shown. BIA, bioelectrical impedance analysis; BP, Bod Pod; FFM, fat-free mass; NA, not available; REE, resting energy expenditure; SF, skinfold. 2Determined by BIA. 3Determined by SF analysis. 4Determined by BP. 5Determined by open-circuit indirect calorimetry. View Large TABLE 1 Characteristics of healthy young men1 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 1Values are means ± SEMs, n = 6 for the lysine study and n = 7 for the tryptophan study. With the use of ANOVA, no differences in FFM or percentage body fat measured by BIA or SF or BP (P = 0.40 and 0.18, respectively) for the lysine study were shown. BIA, bioelectrical impedance analysis; BP, Bod Pod; FFM, fat-free mass; NA, not available; REE, resting energy expenditure; SF, skinfold. 2Determined by BIA. 3Determined by SF analysis. 4Determined by BP. 5Determined by open-circuit indirect calorimetry. View Large Study design and dietary intervention The first experiment was conducted to determine the MA of lysine and the second experiment determined the MA of tryptophan. The MAs of lysine and tryptophan in cornmeal were assessed by comparing the slopes of IAAO response following the graded-intake contents of lysine and tryptophan in cornmeal compared with the reference protein with the use of the slope ratio method. A reference slope was constructed from the IAAO response measured after the feeding of graded intakes of lysine from a reference protein (crystalline AA mixture patterned after egg protein) (Table 2). The slope was constructed from 4 graded intakes of lysine and tryptophan studied in random order. The amounts of lysine studied were 10, 13, 16, and 18 mg · kg−1 · d−1, representing 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement (35 mg · kg−1 · d−1), respectively (17). The amounts of tryptophan studied were 0.5, 1.0, 1.5, and 2.0 mg · kg−1 · d−1, representing 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement (4.0 mg · kg−1 · d−1), respectively (18). TABLE 2 AA composition of reference and test protein consumed by healthy young men who participated in the IAAO study on MA of lysine and tryptophan in cornmeal1 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 1The reference protein was a crystalline AA mixture patterned after the AA composition of egg protein. AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2AAs that were lower in the cornmeal than in the reference AA mixture were adjusted to the level of the AA mixture by using free AA to ensure no other AAs other than lysine and tryptophan were deficient in the diet. 3Actual concentrations of AAs in HCl form: in AA mixture: l-arginine, 62.4 mg/g, l-lysine 60.6 mg/g View Large TABLE 2 AA composition of reference and test protein consumed by healthy young men who participated in the IAAO study on MA of lysine and tryptophan in cornmeal1 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 1The reference protein was a crystalline AA mixture patterned after the AA composition of egg protein. AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2AAs that were lower in the cornmeal than in the reference AA mixture were adjusted to the level of the AA mixture by using free AA to ensure no other AAs other than lysine and tryptophan were deficient in the diet. 3Actual concentrations of AAs in HCl form: in AA mixture: l-arginine, 62.4 mg/g, l-lysine 60.6 mg/g View Large The MA of lysine in cornmeal was determined by substituting a portion of the AA-based diet with the white African cornmeal (IWISA no.1 White Super Maize Meal; Premier Foods). Three amounts of lysine intake were studied in the cornmeal in random order: 13, 16, and 18 mg · kg−1 · d−1, with 10 mg · kg−1 · d−1 serving as the base lysine intake provided by the AA mixture, which constituted the “test slope lysine” (Table 3). The MA of tryptophan in cornmeal was determined by substituting a portion of the AA-based diet with white African cornmeal (IWISA no.1 White Super Maize Meal; Premier Foods). Three amounts of tryptophan intake were studied in the cornmeal in random order: 1.0, 1.5, and 2.0 mg · kg−1 · d−1, with 0.5 mg · kg−1 · d−1 serving as the base tryptophan intake provided by the AA mixture, which constituted the “test slope tryptophan” (Table 4). TABLE 3 Lysine distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of lysine in white African cornmeal1 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 1AA, amino acid; MA, metabolic availability. 2Amount of lysine above the base diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 10 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of lysine in a free AA diet in random order and was chosen to be <60% of the lysine requirement (17) of 35 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of lysine coming from the cornmeal above the base AA diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. View Large TABLE 3 Lysine distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of lysine in white African cornmeal1 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 1AA, amino acid; MA, metabolic availability. 2Amount of lysine above the base diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 10 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of lysine in a free AA diet in random order and was chosen to be <60% of the lysine requirement (17) of 35 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of lysine coming from the cornmeal above the base AA diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. View Large TABLE 4 Tryptophan distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of tryptophan in white African cornmeal1 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 1AA, amino acid; MA, metabolic availability. 2Amount of tryptophan above the base diet of 0.5 mg tryptophan ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 0.5 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of tryptophan in free AA diet in random order and was chosen to be <60% of the tryptophan requirement (18) of 4 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of tryptophan coming from the cornmeal above the base AA diet of 0.5 mg trytophan ⋅ kg−1 ⋅ d−1. View Large TABLE 4 Tryptophan distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of tryptophan in white African cornmeal1 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 1AA, amino acid; MA, metabolic availability. 2Amount of tryptophan above the base diet of 0.5 mg tryptophan ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 0.5 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of tryptophan in free AA diet in random order and was chosen to be <60% of the tryptophan requirement (18) of 4 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of tryptophan coming from the cornmeal above the base AA diet of 0.5 mg trytophan ⋅ kg−1 ⋅ d−1. View Large Two batches of cornmeal were purchased in bulk, 1 for each study. Each batch was mixed thoroughly, after which a sample was taken for analysis. The AA and protein composition of each cornmeal batch was analyzed by Evonik Degussa Canada Ltd. (Burlington, Ontario, Canada). The carbohydrate and fat contents were based on the composition of 20320 cornmeal taken from the USDA database (19). The AA composition of the cornmeal was matched to that of the reference protein by adding individual crystalline AA to the cooked cornmeal. This cornmeal was imported from Africa and chosen because of its importance as a main staple for people in that part of the world. Study protocol Reference slope Oxidation studies were performed on day 3 after a 2-d adaptation to the test diet (Figure 1) (20), providing energy, with REE measured by open-circuit indirect calorimetry (Vmax Encore; Viasys Healthcare) × 1.7, and protein of 1.0 g ⋅ kg−1 ⋅ d−1. The nonprotein calories in the diet were provided as protein-free powder (PFD1; Mead Johnson), flavored with Tang and Fresh Plus crystals (Lynch Foods), grapeseed oil, and protein-free cookies (21). FIGURE 1 View largeDownload slide Typical protocol for determining phenylalanine oxidation in healthy young men on each indicator amino acid oxidation study day. The experimental diet was a liquid formula providing lysine or tryptophan in the form of a free amino acid or from white African cornmeal. The diet was provided hourly for 9 h. Each meal was isocaloric and isonitrogenous and represented 1/12th of each participant’s requirement. Priming doses of l-[1-13C]phenylalanine and NaH13CO3 were started at the fifth meal and then a simultaneous continuous dose of l-[1-13C]phenylalanine was commenced simultaneously and continued hourly throughout the remaining 4 h of the study. Four baseline breath samples were collected every 15 min before the start of the isotope protocol at the fifth meal. Eight plateau breath samples were collected at isotopic steady state during the period from 150 to 270 min after initiation of the isotope protocol. VCO2 was measured by indirect calorimeter 4 h after consuming the experimental diet. VCO2, carbon dioxide production rate. FIGURE 1 View largeDownload slide Typical protocol for determining phenylalanine oxidation in healthy young men on each indicator amino acid oxidation study day. The experimental diet was a liquid formula providing lysine or tryptophan in the form of a free amino acid or from white African cornmeal. The diet was provided hourly for 9 h. Each meal was isocaloric and isonitrogenous and represented 1/12th of each participant’s requirement. Priming doses of l-[1-13C]phenylalanine and NaH13CO3 were started at the fifth meal and then a simultaneous continuous dose of l-[1-13C]phenylalanine was commenced simultaneously and continued hourly throughout the remaining 4 h of the study. Four baseline breath samples were collected every 15 min before the start of the isotope protocol at the fifth meal. Eight plateau breath samples were collected at isotopic steady state during the period from 150 to 270 min after initiation of the isotope protocol. VCO2 was measured by indirect calorimeter 4 h after consuming the experimental diet. VCO2, carbon dioxide production rate. The adaptation diet was consumed as 4 equal meals, with a mean of 52%, 37%, and 11% of energy from carbohydrates, fat, and protein, respectively. On the study day, after a 10-h overnight fast, participants arrived at the Clinical Research Center at The Hospital for Sick Children, Toronto, Canada, for a period of 8.5 h. They were randomly assigned to receive 1 of 4 test amounts of lysine or tryptophan on each of the study days. The study day diet content was similar to the adaptation diet. It was consumed as 9 isonitrogenous and isocaloric hourly meals, with each meal representing 1/12th of the participant’s total daily protein (1 g ⋅ kg−1 ⋅ d−1) and energy requirement (1.5 × REE). For the duration of all experiments, participants consumed a daily multivitamin supplement (Centrum Forte; Wyeth Consumer Health Care) to ensure adequate vitamin intake. Test cornmeal The MA of lysine and tryptophan in cooked white cornmeal was determined by substituting the cooked cornmeal (in the form of a porridge) for a portion of the lysine, tryptophan, protein, and carbohydrate intake. The cornmeal used for the experimental diets was weighed, and cooked in the form of a porridge (“pap”) as is done in Africa. The participants were studied in a repeated-measures design. Tracer protocol The oral tracer protocol started on day 3 of each experiment (study day) with the fifth meal, by administering 2.07 µmol NaH13CO3/kg and l-[1-13C]phenylalanine [99 atom percent excess (APE), 3.99 µmol/kg; Cambridge Isotope Laboratories] as prime and 7.99 µmol · kg−1 · h−1 given hourly until the ninth meal. The amount of phenylalanine provided as tracer was subtracted from the dietary provision, such that the total intake of phenylalanine was 30 mg · kg−1 · d−1 in the lysine study and 34.5 mg · kg−1 · d−1 in the tryptophan study. The total phenylalanine intake was higher in the tryptophan study because the cornmeal used in the tryptophan study had a slightly higher phenylalanine content than the cornmeal used in the lysine study (Table 2). A phenylalanine intake of 34.5 mg · kg−1 · d−1 was therefore the lowest phenylalanine intake possible in order to provide tryptophan at the highest intake of 2.0 mg · kg−1 · d−1 in the cornmeal. Tyrosine intake was set at the level of 160% (40 mg · kg−1 · d−1) of the total estimated aromatic AA requirement (22) to ensure no labeled phenylalanine was used to meet the demand for tyrosine and to facilitate the channeling toward oxidation of any tyrosine formed from phenylalanine (23). Because phenylalanine intake was kept constant within each study, this ensured that the changes in phenylalanine oxidation observed were due to changes in lysine and tryptophan intakes when supplied as free AA or as protein bound from the cornmeal. The decrease in phenylalanine oxidation with increasing intakes of the AAs thus reflects increased uptake for protein synthesis. Sampling of breath and analysis On each study day, breath samples were collected and later analyzed for 13CO2 enrichment. For each study day, 4 baseline breath samples were collected between the fourth and fifth hourly meals (before tracer intake) and 8 breath samples were collected every 15 min beginning 2.5 h after the start of the tracer and up to 0.5 h after the ninth meal at isotopic steady state. After the fifth meal, open-circuit indirect calorimetry was performed for 20 min to measure the rate of carbon dioxide production. Enrichment of 13C in breath was analyzed by continuous flow isotope ratio mass spectrometer (20/20 Isotope Analyzer; PDZ Europa). Calculations The difference between the mean breath isotopic enrichment values of the baseline and 8 plateau samples was expressed as APE above baseline at isotopic steady state. The rate of 13CO2 released by l-[1-13C]phenylalanine tracer oxidation was calculated by using the following equation (24): \begin{equation} {F^{13}}{\rm{C}}{{\rm{O}}_2} = {{\rm{FC}}{{\rm{O}}_2}} \times {{\rm{EC}}{{\rm{O}}_2}} \times {44.6} \times {60} {/} W \times 0.82 \times {100} \end{equation} (1) where FCO2 is the CO2 production rate (expressed as mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state (APE), and W is the weight (kilograms) of the participant. The constants 44.6 (μmol/mL) and 60 (min/h) were used to convert FCO2 to micromoles per hour. The factor of 0.82 is the correction for CO2 retained in the body because of bicarbonate fixation (25), and the factor 100 changes the APE to a fraction. Two slopes were constructed: 1 from the slope of the oxidation obtained with the crystalline form of the test AA (representing the maximal unit increase in net protein synthesis) (6) and the other from the increment of the AA intake (lysine or tryptophan) from the white African cornmeal. The MAs of lysine and tryptophan were calculated by the slope ratio method as oxidation change per gram of protein-bound AA (lysine or tryptophan) divided by oxidation change per gram of free AA by using the following equation: \begin{equation} {{Y}} = {{a}} + {{bSxS}} + {{bTxT}} \end{equation} (2) where xS and xT represent the amounts of intake for the reference and test sources of lysine and tryptophan, respectively, and bS and bT are the slopes for the reference and test sources, respectively (6, 26). The MAs of lysine and tryptophan were calculated as bT/bS. Statistical analysis Lysine and tryptophan intakes were expressed as the intake above that provided by the base diet from the crystalline AA mixture (10 and 0.5 mg · kg−1 · d−1 for lysine and tryptophan, respectively). The effect of adding lysine in the lysine study and that of tryptophan in the tryptophan study by protein source (crystalline AA or cooked cornmeal) on phenylalanine oxidation was tested with the use of the procedure “MIXED,” with subject as a random variable and oxidation day as a repeated measure. Nesting lysine or tryptophan intake within type of lysine or tryptophan addition (l-lysine or l-tryptophan or lysine or tryptophan in cooked white cornmeal) gave the change in the slope in phenylalanine oxidation per milligram of lysine or tryptophan for each type of lysine or tryptophan addition (free AAs or from cooked cornmeal). The repeatability of oxidation measurements was assessed with the use of the mean CV within participants and treatments. Differences in percentage of fat and fat-free mass measured by the different methods were assessed by using ANOVA. Results were expressed as means ± SEMs. P < 0.05 was regarded as significant. All of the statistical analyses were conducted with the use of using SAS version 9.3 for Windows (SAS Institute Inc.). Results For the determination of the MA of lysine in white African cornmeal, a total of 37 experiments were conducted in the 6 participants. For the reference slope, a total of 24 experiments were conducted. For the test slope, a total of 13 of 18 experiments were completed. For the determination of the MA of tryptophan in white African cornmeal, a total of 32 experiments were conducted in 7 participants. Six participants completed all 3 experiments for the reference slope, whereas 1 participant completed 2 experiments for a total of 20 of 21 experiments for the reference slope. Six participants each completed 2 test tryptophan experiments for a total of 12 of 14 experiments. Linearity of response to lysine intake from free AA diet As lysine intake from the AA mix increased from 10 to 13 to 16 to 18 mg · kg−1 · d−1 (28.6–51.4% of lysine requirement of 35 mg · kg−1 · d−1), phenylalanine oxidation decreased linearly. The application of linear regression determined a negative slope of the best-fit line of −0.00961 (SEM = 0.0033; P = 0.006). Metabolic availability of lysine in cooked white cornmeal Lysine intake (3–8 mg · kg−1 · d−1 above base) from cooked white cornmeal resulted in a significant decrease in phenylalanine oxidation (slope = −0.00686; SEM = 0.0034; P = 0.048). The ratio of the response to additional lysine intake from the cooked white cornmeal compared with that of lysine from the AA mix was 0.71. Thus, the MA of lysine from cooked white African cornmeal was 71% (Table 5, Figure 2A). FIGURE 2 View largeDownload slide Linearity of l-[1-13C]phenylalanine oxidation (F13CO2) in response to graded intakes of l-lysine (A) and l-tryptophan (B) as free amino acid (reference curves depicted as circles) and protein-bound lysine and tryptophan from white African cornmeal (test curves depicted as squares) in healthy young men provided with graded intakes of lysine and tryptophan. Values are means ± SDs, n = 6 (A) or n = 7 (B). The dotted lines around the equation represent the IAAO response to graded intakes of lysine from the cornmeal. The dots are used to show that this equation pertains to the slope with the dots below. FIGURE 2 View largeDownload slide Linearity of l-[1-13C]phenylalanine oxidation (F13CO2) in response to graded intakes of l-lysine (A) and l-tryptophan (B) as free amino acid (reference curves depicted as circles) and protein-bound lysine and tryptophan from white African cornmeal (test curves depicted as squares) in healthy young men provided with graded intakes of lysine and tryptophan. Values are means ± SDs, n = 6 (A) or n = 7 (B). The dotted lines around the equation represent the IAAO response to graded intakes of lysine from the cornmeal. The dots are used to show that this equation pertains to the slope with the dots below. TABLE 5 MA of l-lysine and l-tryptophan in cooked cornmeal based on the IAAO of l-[1-13C]phenylalanine1 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 1AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2MA from the AA mixture was assumed to be 100% (14). View Large TABLE 5 MA of l-lysine and l-tryptophan in cooked cornmeal based on the IAAO of l-[1-13C]phenylalanine1 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 1AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2MA from the AA mixture was assumed to be 100% (14). View Large Linearity of response to tryptophan intake from free AA diet As tryptophan intake from the AA mix increased from 0.5 to 1 to 1.5 to 2 mg · kg−1 · d−1 (12.5–50% of tryptophan requirement of 4 mg · kg−1 · d−1), phenylalanine oxidation decreased linearly. The application of linear regression determined a negative slope of the best-fit line of −0.00696 (SEM = 0.0026; P = 0.0116). Metabolic availability of tryptophan in cooked white cornmeal Tryptophan intake (0.5–1.5 mg · kg−1 · d−1 above base) from cooked white cornmeal had a significant effect on phenylalanine oxidation (slope = −0.00558; SEM = 0.0023; P = 0.0462). The ratio of the response to additional tryptophan intake from the cooked white cornmeal compared with that of tryptophan from the AA mix was 0.8017. Thus, the MA of tryptophan from cooked white cornmeal was 80% (Table 5, Figure 2B). Discussion Maize is the main staple in the diet of many native African populations, with 16 of the top 22 countries in the world in which maize provides the highest percentage of calories in the diet coming from that continent (27). The most commonly consumed maize in these countries is the white African cornmeal used in the current study. In addition, maize is the most important staple in the Latin American diet, where the typical adult and child consume ≤560 and 150 g/d, respectively (28). The heavy reliance on maize has led to a deficiency of lysine and tryptophan in the diet, resulting in poor growth in children (3, 29). The poor quality of maize protein, the heavy reliance of so many of the world’s food-insecure population on its calories, and current understanding that protein requirement recommendations are severely underestimated (30–33) point to maize as a food for which the protein quality requires urgent evaluation. With the use of the noninvasive IAAO method, which our group recently adapted for the study of protein quality in humans (8), we evaluated the protein quality of white African maize by studying the MA of the 2 most limiting AAs: lysine and tryptophan. The results of the current study show that 71% and 80% of the lysine and tryptophan in white African cornmeal are available for protein synthesis when the cornmeal is prepared in the traditional moist cooking method to make “pap.” This is the first study to our knowledge to assess the protein quality of white African cornmeal directly in humans and shows that the MA of lysine is lower than that of tryptophan and is the dominant AA, which limits the protein quality of white African cornmeal. The ileal AA digestibilities of lysine and tryptophan in Indian maize flour were determined in growing rats and found to be 92% and 84%, respectively (34). Although the digestibility of tryptophan is similar to 80% MA found in the current study, the 92% digestibility of lysine is very different from the 71% found in the current study. There are a number of possible reasons why this might have occurred: The rat is an inferior model for the study of protein quality of foods for human consumption (35), and although true ileal digestibility was used, the correction factor was based on endogenous AA flows from growing rats reported in a different study. It is possible that different processing methods were used on the maize kernels between India and Africa. There are 2 main methods of processing maize for human consumption: dry milling or alkaline processing (nixtamalization) (36). Alkaline processing changes the zein protein matrix, which makes AAs (particularly lysine) more available to the body (37). Maize that is processed by nixtamalization is softer and usually ground into flour, whereas maize that is dry milled is grounded into a coarser texture (37). It is quite possible therefore that the Indian maize flour used in the rat study (34) was processed by nixtamalization—hence, the higher lysine digestibility. The beneficial effect of nixtamalization on protein quality was previously shown when rats fed maize dough and tortillas made from alkaline processed maize showed greater weight gain and protein efficiency ratio than rats fed unprocessed maize (28). The variety of maize used in our study was likely very different from the variety used to prepare the Indian maize flour in the rat study (34). Although white maize is the preferred variety in Africa, yellow maize is very common in many other parts of the world (27). The difference in the MA of lysine and tryptophan in our study and the previous study (34) highlights the importance of studying different varieties of the same foods from different countries. This will allow for a more accurate classification of the protein quality of all foods and a better understanding of the foods needed to meet the requirement for appropriate nourishment of the human populations around the world. The results of the current study provide evidence against the use of the protein digestibility-corrected AA score, in which a single digestibility factor is used to predict AA digestibility in general. The current results show that the MAs of different AAs vary within the same food (lysine and tryptophan in this case) and validate the FAO/WHO decision to use AAs as individual nutrients for the study of protein quality (6). MA as measured in the current study is based on the principle of the slope ratio assay, in which changes in a measured response (usually growth) as a result of varying the intakes of a test protein are compared with changes in the same measured response to changes in the response to a reference protein (10, 38). Slope ratio assays are considered the standard for measuring AA bioavailability (38). The advantages of the slope ratio as applied in the current study is that the carbon dioxide output measured in the IAAO studies represents an end product of AA metabolism during digestion, absorption, and cellular metabolism (10). These constitute all of the components affecting bioavailability. Hence, MA is more representative of the bioavailability of AAs than digestibility, which only covers the losses of AAs during digestion. The main limitation of the slope ratio assay is that only 1 AA can be examined at a time (10). Nevertheless, the short adaptation time (<2 d) (39) of the IAAO technique allows for the evaluation of multiple AAs over a relatively short time (10). The IAAO technique represents the partitioning of the indicator AA between protein synthesis and oxidation where decreasing oxidation represents an increase in the test AA for protein synthesis. To achieve the criterion of linearity required for the slope ratio assay, the test AAs (lysine and tryptophan in this study), need to be kept below the level of the requirement (at most, 60%). Because the indicator AA was kept constant throughout each individual study, decreases in oxidation in response to graded intakes of lysine or tryptophan are indicative of an increase in uptake for protein synthesis (12). The white African cornmeal used in the current study contains 151 mg lysine, 7.46 g protein, and 372 kcal/100 g raw cornmeal. With the use of the FAO-proposed lysine requirement of 30 mg/kg (40), a man in Africa aged 30–40 y and weighing 71 kg will require 2130 mg lysine/d. If his diet consisted of cornmeal only, he would need 797 g cornmeal/d to meet approximate energy needs of 2960 kcal/d on the basis of an estimated energy need of 42 kcal ⋅ kg−1 ⋅ d−1 for a moderate to active lifestyle (41). This amount of cornmeal supplies 59 g protein and 1203 mg lysine, with a net lysine content of 854 mg based on the MA as determined in the current study. The protein and net lysine contents represent only 59% and 40% of the protein and lysine needs, respectively. White African cornmeal should therefore be combined with a legume such as lentils or chickpeas in order to increase the lysine and protein contents. Legumes, however, are expensive, so knowledge of the MA of the lysine in those foods is needed to guide recommendations on appropriate ratios to be combined to meet lysine needs. In summary, this is the first study to our knowledge to determine directly in humans the MA of lysine and tryptophan in white African cornmeal. With the use of the minimally invasive IAAO method, we found that the MAs of lysine and tryptophan were 71% and 80%, respectively. This methodology has the greatest potential to advance knowledge on protein quality of foods for human consumption because it can be applied to evaluate the protein quality of all whole foods. Acknowledgments We thank Jasmine Donohue, Department of Nutrition and Food Services, The Hospital for Sick Children, for preparing the protein-free cookies. The authors’ responsibilities were as follows—RE, ROB, PBP, and GC-M: designed the research; MR: conducted the research; MR and GC-M: analyzed the data and wrote the manuscript; GC-M: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by the Canadian Institute of Health Research (grant MT 10321). Pfizer Consumer Healthcare (Mississauga, Ontario) donated the multivitamins. Mead Johnson Nutritionals donated the protein-free powder for experimental diets. Author disclosures: MR, RE, ROB, PBP, and GC-M, no conflicts of interest. 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Indicator amino acid oxidation is not affected by period of adaptation to a wide range of lysine intake in healthy young men . J Nutr 2009 ; 139 : 1082 – 7 . Google Scholar CrossRef Search ADS PubMed 40. WHO/FAO/UN University . Protein and amino acid requirements in human nutrition . Report of a joint WHO/FAO/UNU expert consultation . World Health Organ Tech Rep Ser 2007 . 41. FAO, Food and Nutrition Technical Report Series ; Human energy requirements, Report of a joint FAO/WHO/UNU Expert Consultation , Rome October 17–24 2001 . © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Nutrition Oxford University Press

Metabolic Availability of the Limiting Amino Acids Lysine and Tryptophan in Cooked White African Cornmeal Assessed in Healthy Young Men Using the Indicator Amino Acid Oxidation Technique

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Oxford University Press
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© 2018 American Society for Nutrition.
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0022-3166
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1541-6100
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10.1093/jn/nxy039
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Abstract

Abstract Background Maize is a staple food in many regions of the world, particularly in Africa and Latin America. However, maize protein is limiting in the indispensable amino acids lysine and tryptophan, making its protein of poor quality. Objective The main objective of this study was to determine the protein quality of white African cornmeal by determining the metabolic availability (MA) of lysine and tryptophan. Methods To determine the MA of lysine, 4 amounts of l-lysine (10, 13, 16, and 18 mg · kg−1 · d−1 totaling 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement of 35 mg · kg−1 · d−1, respectively) were studied in 6 healthy young men in a repeated-measures design. To determine the MA of tryptophan, 4 amounts of l-tryptophan (0.5, 1, 1.5, and 2 mg · kg−1 · d−1 totaling 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement of 4 mg · kg−1 · d−1, respectively) were studied in 7 healthy young men in a repeated-measures design. The MAs of lysine and tryptophan were estimated by comparing the indicator amino acid oxidation (IAAO) response with varying intakes of lysine and tryptophan in cooked white cornmeal compared with the IAAO response to l-lysine and l-tryptophan intakes in the reference protein (crystalline amino acid mixture patterned after egg protein) with the use of the slope ratio method. Results The MAs of lysine and tryptophan from African cooked white cornmeal were 71% and 80%, respectively. Conclusion Our study provides a robust estimate of the availability of lysine and tryptophan in African white maize to healthy young men. This estimate provides a basis for postproduction fortification or supplementation of maize-based diets. This trial was registered at www.clinicaltrials.gov as NCT02402179. metabolic availability, protein quality, indicator amino acid oxidation, stable isotope, phenylalanine oxidation, cereal grains, maize, corn Introduction Maize is the most abundant crop produced in the world, and although the United States is the largest producer, Mexico and Africa are the largest consumers (1). Globally, maize is the cheapest cereal grain, hence its importance as a staple food for the poor. In Mexico, it provides 45% of the daily calorie intake compared with only 10% for all other grains combined (2). Africa alone consumes 30% of the world’s food maize, with average per capita consumption of 50 kg (1). In sub-Saharan countries such as Malawi, Lesotho, Zambia, and Kenya, maize provide 80% of the calories in the diet, with per capita consumption ranging from 90 to 180 kg (1). However, in such countries in which the poor depend largely on maize as the main staple and where there is little access to a diverse diet, there is growth faltering in children in the absence of biofortification of maize with lysine and tryptophan (3). Protein is the key determinant of growth and bodily function, and the quality of food proteins depends on their amino acid (AA) composition and bioavailability. Maize protein is limiting in the essential AAs lysine and tryptophan, which limits its potential as a sole protein source. In addition, the lysine and tryptophan contents of maize could be affected by heat and the presence of antinutritional factors, such as tannins and phytates, that render them unavailable for protein synthesis after absorption (4, 5). Recently, the FAO Expert Consultation on Protein Quality identified the need to evaluate the protein quality of all foods consumed by humans, beginning with commonly consumed foods, especially by vulnerable populations in low-income countries (6). The FAO recommends that dietary AAs be treated as individual nutrients for the purposes of protein quality determination, and that results be provided on an individual AA basis (7). Due to the increasing importance of maize as a staple food for the poor, knowledge of its protein quality is critical to understanding its ability to provide the required AA content in the diet. In addition, its relative deficiency in lysine and tryptophan makes these individual AAs premier candidates for the evaluation of the protein quality of maize. The traditional method of protein quality evaluation—the protein digestibility-corrected AA score—was recently replaced by the digestible indispensable AA score method (7). For the digestible indispensable AA score method, bioavailability is measured as ileal digestibility. Ileal digestibility is very invasive, which limits its viability in humans, particularly those who are more vulnerable. Our laboratory adapted the minimally invasive indicator amino acid oxidation (IAAO) method to directly determine protein quality in humans (8, 9) by measuring AA bioavailability termed “metabolic availability” (MA) in whole foods. MA accounts for the effect of digestion, absorption, and metabolic utilization of the AAs provided by the protein source, whereas ileal digestibility only measures the total amount of AAs absorbed at the site of the terminal ileum (10). Because some AAs are absorbed in a form unavailable for protein synthesis, ileal digestibility has the potential to overestimate MA and hence protein quality. The present study aimed to apply the minimally invasive IAAO method to evaluate the protein quality of white African cornmeal (maize) by studying the MA of the most limiting AAs, lysine and tryptophan. Methods Two separate studies were completed with different participants for the determination of the MA of lysine and tryptophan in white African cornmeal. Assumptions and variables The IAAO method was applied in this study to determine the availability of lysine and tryptophan from the dietary protein of cornmeal for protein synthesis. It is based on the premise that when 1 AA is limiting for protein synthesis, all other AAs are in relative excess and must be oxidized. This oxidation is monitored by the use of l-[1-13C]phenylalanine, which indicates the rate of whole-body protein synthesis as driven by the limiting AAs, lysine and tryptophan in this study. We have previously shown that the appearance of 14CO2 from l-[1-14C]phenylalanine at isotopic steady state reflects whole-body protein synthesis (11). Therefore, changes in the oxidation of l-[1-13C]phenylalanine in response to graded intakes of lysine or tryptophan reflects whole-body bioavailability of these 2 AAs and accounts for their losses during digestion and cellular metabolism (10, 12). Thus, the higher the oxidation of the indicator AA (l-[1-13C]phenylalanine), the lower the MA of the test AAs (lysine and tryptophan) for protein synthesis. To determine the MA of an AA in a food protein, the following key conditions are necessary: The test AA must be first limiting to drive indicator oxidation rates. The response in oxidation rates of the indicator AA l-[1-13C]phenylalanine to changes in the test AA must be linear to allow calculation of bioavailability according to the principles of the slope ratio assay (13). The bioavailability of the test AA in the food protein is calculated relative to a standard/reference, which is free AA assumed to be 100% bioavailable (14, 15). It is assumed that the responses of the 2 substances are the same at a base value of x, making as = at (13). That is, the 2 lines intersect at a base value so that the common value of the intercepts can be denoted simply as a, giving regression equations y = a + btx for the test substance and y = a + bsx for the reference/standard. Letting xs and xt denote amounts of the standard and test substances required to produce equivalent value of the response y, then a + bsxs = a + btxt. Solving gives a relative bioavailability = xt/xs = bt/bs, the ratio of slopes of the regression lines (13). The indicator oxidation rate must show good repeatability to allow accurate determination of MA (8, 12, 16). Participants A total of 13 healthy young men participated in these 2 studies: 6 in the lysine study and 7 in the tryptophan study. Health was assessed by medical history and history of weight loss. Participant characteristics, body composition, and energy requirements were assessed at study entry (Table 1). Fat-free mass was assessed by BIA (Imp SFB7, ImpediMed Ltd, Qld, Australia) bioelectrical impedance analysis and Bod Pod (Cosmed USA); fat mass was assessed by skinfold thickness. Resting energy expenditure (REE) was measured by Indirect calorimetry (Vmax Encore, metabolic cart; Viasys) indirect calorimetry. BMI was calculated as weight (kilograms) divided by height in meters squared. The study protocol was explained to each participant, and informed written consent was obtained. Participants received financial compensation for their inconvenience. The study was approved by the Research Ethics Board of the Hospital for Sick Children, Toronto, Ontario, Canada. The trial was registered at www.clinicalrials.gov as NCT02402179. TABLE 1 Characteristics of healthy young men1 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 1Values are means ± SEMs, n = 6 for the lysine study and n = 7 for the tryptophan study. With the use of ANOVA, no differences in FFM or percentage body fat measured by BIA or SF or BP (P = 0.40 and 0.18, respectively) for the lysine study were shown. BIA, bioelectrical impedance analysis; BP, Bod Pod; FFM, fat-free mass; NA, not available; REE, resting energy expenditure; SF, skinfold. 2Determined by BIA. 3Determined by SF analysis. 4Determined by BP. 5Determined by open-circuit indirect calorimetry. View Large TABLE 1 Characteristics of healthy young men1 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 Characteristics Lysine study Tryptophan study Age, y 23.7 ± 1.1 23.7 ± 1.4 Weight, kg 66.9 ± 3.2 68.7 ± 3.1 Height, cm 175 ± 2.5 174 ± 3.6 BMI, kg/m2 22.0 ± 1.3 22.7 ± 0.7 FFM-BIA,2 kg 51.2 ± 1. 6 55.6 ± 1.9 FFM-SF,3 kg 49.9 ± 1.7 NA FFM-BP,4 kg 53.3 ± 1.7 59.6 ± 3.1 Fat-BIA,2 % 23.0 ± 2.2 18.7 ± 1.9 Fat-SF,3 % 26.7 ± 2.9 NA Fat-BP,4 % 18.4 ± 3.3 13.5 ± 1.1 REE,5 kcal/d 1490 ± 59.4 1645 ± 56.2 1Values are means ± SEMs, n = 6 for the lysine study and n = 7 for the tryptophan study. With the use of ANOVA, no differences in FFM or percentage body fat measured by BIA or SF or BP (P = 0.40 and 0.18, respectively) for the lysine study were shown. BIA, bioelectrical impedance analysis; BP, Bod Pod; FFM, fat-free mass; NA, not available; REE, resting energy expenditure; SF, skinfold. 2Determined by BIA. 3Determined by SF analysis. 4Determined by BP. 5Determined by open-circuit indirect calorimetry. View Large Study design and dietary intervention The first experiment was conducted to determine the MA of lysine and the second experiment determined the MA of tryptophan. The MAs of lysine and tryptophan in cornmeal were assessed by comparing the slopes of IAAO response following the graded-intake contents of lysine and tryptophan in cornmeal compared with the reference protein with the use of the slope ratio method. A reference slope was constructed from the IAAO response measured after the feeding of graded intakes of lysine from a reference protein (crystalline AA mixture patterned after egg protein) (Table 2). The slope was constructed from 4 graded intakes of lysine and tryptophan studied in random order. The amounts of lysine studied were 10, 13, 16, and 18 mg · kg−1 · d−1, representing 28.6%, 37.1%, 45.7%, and 51.4% of the mean lysine requirement (35 mg · kg−1 · d−1), respectively (17). The amounts of tryptophan studied were 0.5, 1.0, 1.5, and 2.0 mg · kg−1 · d−1, representing 12.5%, 25.0%, 37.5%, and 50.0% of the mean tryptophan requirement (4.0 mg · kg−1 · d−1), respectively (18). TABLE 2 AA composition of reference and test protein consumed by healthy young men who participated in the IAAO study on MA of lysine and tryptophan in cornmeal1 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 1The reference protein was a crystalline AA mixture patterned after the AA composition of egg protein. AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2AAs that were lower in the cornmeal than in the reference AA mixture were adjusted to the level of the AA mixture by using free AA to ensure no other AAs other than lysine and tryptophan were deficient in the diet. 3Actual concentrations of AAs in HCl form: in AA mixture: l-arginine, 62.4 mg/g, l-lysine 60.6 mg/g View Large TABLE 2 AA composition of reference and test protein consumed by healthy young men who participated in the IAAO study on MA of lysine and tryptophan in cornmeal1 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 Cornmeal,2 mg/g protein AA AA mixture, mg/g protein Lysine study Tryptophan study l-Alanine 61.5 82.3 80.4 l-Arginine · HCl3 74.5 43.2 39.2 l-Asparagine 33.0 30.4 29.3 l-Aspartic acid 33.0 30.4 29.3 l-Cysteine 21.9 24.3 21.6 l-Glutamine 56.2 106 105 l-Glutamic acid 56.2 106 105 l-Glycine 33.0 32.0 29.3 l-Histidine 22.5 28.4 27.9 l-Isoleucine 62.4 37.1 36.7 l-Leucine 82.6 150 151 l-Lysine · HCl3 75.1 25.3 21.9 l-Methionine 29.5 30.6 20.2 l-Phenylalanine 54.2 44.6 47.2 l-Proline 41.6 103 101 l-Serine 83.2 50.1 49.2 l-Threonine 46.7 34.6 33.2 l-Tryptophan 15.5 7.00 5.69 l-Tyrosine 40.4 32.1 32.1 l-Valine 69.7 47.7 46.4 Total 993 1040 1010 1The reference protein was a crystalline AA mixture patterned after the AA composition of egg protein. AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2AAs that were lower in the cornmeal than in the reference AA mixture were adjusted to the level of the AA mixture by using free AA to ensure no other AAs other than lysine and tryptophan were deficient in the diet. 3Actual concentrations of AAs in HCl form: in AA mixture: l-arginine, 62.4 mg/g, l-lysine 60.6 mg/g View Large The MA of lysine in cornmeal was determined by substituting a portion of the AA-based diet with the white African cornmeal (IWISA no.1 White Super Maize Meal; Premier Foods). Three amounts of lysine intake were studied in the cornmeal in random order: 13, 16, and 18 mg · kg−1 · d−1, with 10 mg · kg−1 · d−1 serving as the base lysine intake provided by the AA mixture, which constituted the “test slope lysine” (Table 3). The MA of tryptophan in cornmeal was determined by substituting a portion of the AA-based diet with white African cornmeal (IWISA no.1 White Super Maize Meal; Premier Foods). Three amounts of tryptophan intake were studied in the cornmeal in random order: 1.0, 1.5, and 2.0 mg · kg−1 · d−1, with 0.5 mg · kg−1 · d−1 serving as the base tryptophan intake provided by the AA mixture, which constituted the “test slope tryptophan” (Table 4). TABLE 3 Lysine distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of lysine in white African cornmeal1 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 1AA, amino acid; MA, metabolic availability. 2Amount of lysine above the base diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 10 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of lysine in a free AA diet in random order and was chosen to be <60% of the lysine requirement (17) of 35 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of lysine coming from the cornmeal above the base AA diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. View Large TABLE 3 Lysine distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of lysine in white African cornmeal1 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Source of lysine, mg ⋅ kg−1 ⋅ d−1 Total test amounts of lysine intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Lysine addition2 Reference slope3  10 (28.6) 10 0  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 Cornmeal slope4  13 (37.1) 10 3  16 (45.7) 10 6  19 (51.4) 10 9 1AA, amino acid; MA, metabolic availability. 2Amount of lysine above the base diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 10 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of lysine in a free AA diet in random order and was chosen to be <60% of the lysine requirement (17) of 35 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of lysine coming from the cornmeal above the base AA diet of 10 mg lysine ⋅ kg−1 ⋅ d−1. View Large TABLE 4 Tryptophan distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of tryptophan in white African cornmeal1 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 1AA, amino acid; MA, metabolic availability. 2Amount of tryptophan above the base diet of 0.5 mg tryptophan ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 0.5 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of tryptophan in free AA diet in random order and was chosen to be <60% of the tryptophan requirement (18) of 4 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of tryptophan coming from the cornmeal above the base AA diet of 0.5 mg trytophan ⋅ kg−1 ⋅ d−1. View Large TABLE 4 Tryptophan distribution from free AA and from cornmeal in the diets of healthy young men who participated in the studies to determine the MA of tryptophan in white African cornmeal1 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Source of tryptophan, mg ⋅ kg−1 ⋅ d−1 Total test amounts of tryptophan intake, mg ⋅ kg−1 ⋅ d−1 (% of requirement) Free AAs Tryptophan addition2 Reference slope3  0.5 (12.5) 0.5 0  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 Cornmeal slope4  1.0 (25.0) 0.5 0.5  1.5 (37.5) 0.5 1.0  2.0 (50.0) 0.5 1.5 1AA, amino acid; MA, metabolic availability. 2Amount of tryptophan above the base diet of 0.5 mg tryptophan ⋅ kg−1 ⋅ d−1. The amount above base was provided as free AA in the reference slope and from the cornmeal in the test/cornmeal slope. The base amount of 0.5 mg ⋅ kg−1 ⋅ d−1 was provided at each intake amount to ensure both reference and test slopes have a common intercept. 3The reference slope for each participant was created from 4 graded intakes of tryptophan in free AA diet in random order and was chosen to be <60% of the tryptophan requirement (18) of 4 mg ⋅ kg−1 ⋅ d−1. 4The cornmeal slope for each participant was created from 3 graded intakes of tryptophan coming from the cornmeal above the base AA diet of 0.5 mg trytophan ⋅ kg−1 ⋅ d−1. View Large Two batches of cornmeal were purchased in bulk, 1 for each study. Each batch was mixed thoroughly, after which a sample was taken for analysis. The AA and protein composition of each cornmeal batch was analyzed by Evonik Degussa Canada Ltd. (Burlington, Ontario, Canada). The carbohydrate and fat contents were based on the composition of 20320 cornmeal taken from the USDA database (19). The AA composition of the cornmeal was matched to that of the reference protein by adding individual crystalline AA to the cooked cornmeal. This cornmeal was imported from Africa and chosen because of its importance as a main staple for people in that part of the world. Study protocol Reference slope Oxidation studies were performed on day 3 after a 2-d adaptation to the test diet (Figure 1) (20), providing energy, with REE measured by open-circuit indirect calorimetry (Vmax Encore; Viasys Healthcare) × 1.7, and protein of 1.0 g ⋅ kg−1 ⋅ d−1. The nonprotein calories in the diet were provided as protein-free powder (PFD1; Mead Johnson), flavored with Tang and Fresh Plus crystals (Lynch Foods), grapeseed oil, and protein-free cookies (21). FIGURE 1 View largeDownload slide Typical protocol for determining phenylalanine oxidation in healthy young men on each indicator amino acid oxidation study day. The experimental diet was a liquid formula providing lysine or tryptophan in the form of a free amino acid or from white African cornmeal. The diet was provided hourly for 9 h. Each meal was isocaloric and isonitrogenous and represented 1/12th of each participant’s requirement. Priming doses of l-[1-13C]phenylalanine and NaH13CO3 were started at the fifth meal and then a simultaneous continuous dose of l-[1-13C]phenylalanine was commenced simultaneously and continued hourly throughout the remaining 4 h of the study. Four baseline breath samples were collected every 15 min before the start of the isotope protocol at the fifth meal. Eight plateau breath samples were collected at isotopic steady state during the period from 150 to 270 min after initiation of the isotope protocol. VCO2 was measured by indirect calorimeter 4 h after consuming the experimental diet. VCO2, carbon dioxide production rate. FIGURE 1 View largeDownload slide Typical protocol for determining phenylalanine oxidation in healthy young men on each indicator amino acid oxidation study day. The experimental diet was a liquid formula providing lysine or tryptophan in the form of a free amino acid or from white African cornmeal. The diet was provided hourly for 9 h. Each meal was isocaloric and isonitrogenous and represented 1/12th of each participant’s requirement. Priming doses of l-[1-13C]phenylalanine and NaH13CO3 were started at the fifth meal and then a simultaneous continuous dose of l-[1-13C]phenylalanine was commenced simultaneously and continued hourly throughout the remaining 4 h of the study. Four baseline breath samples were collected every 15 min before the start of the isotope protocol at the fifth meal. Eight plateau breath samples were collected at isotopic steady state during the period from 150 to 270 min after initiation of the isotope protocol. VCO2 was measured by indirect calorimeter 4 h after consuming the experimental diet. VCO2, carbon dioxide production rate. The adaptation diet was consumed as 4 equal meals, with a mean of 52%, 37%, and 11% of energy from carbohydrates, fat, and protein, respectively. On the study day, after a 10-h overnight fast, participants arrived at the Clinical Research Center at The Hospital for Sick Children, Toronto, Canada, for a period of 8.5 h. They were randomly assigned to receive 1 of 4 test amounts of lysine or tryptophan on each of the study days. The study day diet content was similar to the adaptation diet. It was consumed as 9 isonitrogenous and isocaloric hourly meals, with each meal representing 1/12th of the participant’s total daily protein (1 g ⋅ kg−1 ⋅ d−1) and energy requirement (1.5 × REE). For the duration of all experiments, participants consumed a daily multivitamin supplement (Centrum Forte; Wyeth Consumer Health Care) to ensure adequate vitamin intake. Test cornmeal The MA of lysine and tryptophan in cooked white cornmeal was determined by substituting the cooked cornmeal (in the form of a porridge) for a portion of the lysine, tryptophan, protein, and carbohydrate intake. The cornmeal used for the experimental diets was weighed, and cooked in the form of a porridge (“pap”) as is done in Africa. The participants were studied in a repeated-measures design. Tracer protocol The oral tracer protocol started on day 3 of each experiment (study day) with the fifth meal, by administering 2.07 µmol NaH13CO3/kg and l-[1-13C]phenylalanine [99 atom percent excess (APE), 3.99 µmol/kg; Cambridge Isotope Laboratories] as prime and 7.99 µmol · kg−1 · h−1 given hourly until the ninth meal. The amount of phenylalanine provided as tracer was subtracted from the dietary provision, such that the total intake of phenylalanine was 30 mg · kg−1 · d−1 in the lysine study and 34.5 mg · kg−1 · d−1 in the tryptophan study. The total phenylalanine intake was higher in the tryptophan study because the cornmeal used in the tryptophan study had a slightly higher phenylalanine content than the cornmeal used in the lysine study (Table 2). A phenylalanine intake of 34.5 mg · kg−1 · d−1 was therefore the lowest phenylalanine intake possible in order to provide tryptophan at the highest intake of 2.0 mg · kg−1 · d−1 in the cornmeal. Tyrosine intake was set at the level of 160% (40 mg · kg−1 · d−1) of the total estimated aromatic AA requirement (22) to ensure no labeled phenylalanine was used to meet the demand for tyrosine and to facilitate the channeling toward oxidation of any tyrosine formed from phenylalanine (23). Because phenylalanine intake was kept constant within each study, this ensured that the changes in phenylalanine oxidation observed were due to changes in lysine and tryptophan intakes when supplied as free AA or as protein bound from the cornmeal. The decrease in phenylalanine oxidation with increasing intakes of the AAs thus reflects increased uptake for protein synthesis. Sampling of breath and analysis On each study day, breath samples were collected and later analyzed for 13CO2 enrichment. For each study day, 4 baseline breath samples were collected between the fourth and fifth hourly meals (before tracer intake) and 8 breath samples were collected every 15 min beginning 2.5 h after the start of the tracer and up to 0.5 h after the ninth meal at isotopic steady state. After the fifth meal, open-circuit indirect calorimetry was performed for 20 min to measure the rate of carbon dioxide production. Enrichment of 13C in breath was analyzed by continuous flow isotope ratio mass spectrometer (20/20 Isotope Analyzer; PDZ Europa). Calculations The difference between the mean breath isotopic enrichment values of the baseline and 8 plateau samples was expressed as APE above baseline at isotopic steady state. The rate of 13CO2 released by l-[1-13C]phenylalanine tracer oxidation was calculated by using the following equation (24): \begin{equation} {F^{13}}{\rm{C}}{{\rm{O}}_2} = {{\rm{FC}}{{\rm{O}}_2}} \times {{\rm{EC}}{{\rm{O}}_2}} \times {44.6} \times {60} {/} W \times 0.82 \times {100} \end{equation} (1) where FCO2 is the CO2 production rate (expressed as mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state (APE), and W is the weight (kilograms) of the participant. The constants 44.6 (μmol/mL) and 60 (min/h) were used to convert FCO2 to micromoles per hour. The factor of 0.82 is the correction for CO2 retained in the body because of bicarbonate fixation (25), and the factor 100 changes the APE to a fraction. Two slopes were constructed: 1 from the slope of the oxidation obtained with the crystalline form of the test AA (representing the maximal unit increase in net protein synthesis) (6) and the other from the increment of the AA intake (lysine or tryptophan) from the white African cornmeal. The MAs of lysine and tryptophan were calculated by the slope ratio method as oxidation change per gram of protein-bound AA (lysine or tryptophan) divided by oxidation change per gram of free AA by using the following equation: \begin{equation} {{Y}} = {{a}} + {{bSxS}} + {{bTxT}} \end{equation} (2) where xS and xT represent the amounts of intake for the reference and test sources of lysine and tryptophan, respectively, and bS and bT are the slopes for the reference and test sources, respectively (6, 26). The MAs of lysine and tryptophan were calculated as bT/bS. Statistical analysis Lysine and tryptophan intakes were expressed as the intake above that provided by the base diet from the crystalline AA mixture (10 and 0.5 mg · kg−1 · d−1 for lysine and tryptophan, respectively). The effect of adding lysine in the lysine study and that of tryptophan in the tryptophan study by protein source (crystalline AA or cooked cornmeal) on phenylalanine oxidation was tested with the use of the procedure “MIXED,” with subject as a random variable and oxidation day as a repeated measure. Nesting lysine or tryptophan intake within type of lysine or tryptophan addition (l-lysine or l-tryptophan or lysine or tryptophan in cooked white cornmeal) gave the change in the slope in phenylalanine oxidation per milligram of lysine or tryptophan for each type of lysine or tryptophan addition (free AAs or from cooked cornmeal). The repeatability of oxidation measurements was assessed with the use of the mean CV within participants and treatments. Differences in percentage of fat and fat-free mass measured by the different methods were assessed by using ANOVA. Results were expressed as means ± SEMs. P < 0.05 was regarded as significant. All of the statistical analyses were conducted with the use of using SAS version 9.3 for Windows (SAS Institute Inc.). Results For the determination of the MA of lysine in white African cornmeal, a total of 37 experiments were conducted in the 6 participants. For the reference slope, a total of 24 experiments were conducted. For the test slope, a total of 13 of 18 experiments were completed. For the determination of the MA of tryptophan in white African cornmeal, a total of 32 experiments were conducted in 7 participants. Six participants completed all 3 experiments for the reference slope, whereas 1 participant completed 2 experiments for a total of 20 of 21 experiments for the reference slope. Six participants each completed 2 test tryptophan experiments for a total of 12 of 14 experiments. Linearity of response to lysine intake from free AA diet As lysine intake from the AA mix increased from 10 to 13 to 16 to 18 mg · kg−1 · d−1 (28.6–51.4% of lysine requirement of 35 mg · kg−1 · d−1), phenylalanine oxidation decreased linearly. The application of linear regression determined a negative slope of the best-fit line of −0.00961 (SEM = 0.0033; P = 0.006). Metabolic availability of lysine in cooked white cornmeal Lysine intake (3–8 mg · kg−1 · d−1 above base) from cooked white cornmeal resulted in a significant decrease in phenylalanine oxidation (slope = −0.00686; SEM = 0.0034; P = 0.048). The ratio of the response to additional lysine intake from the cooked white cornmeal compared with that of lysine from the AA mix was 0.71. Thus, the MA of lysine from cooked white African cornmeal was 71% (Table 5, Figure 2A). FIGURE 2 View largeDownload slide Linearity of l-[1-13C]phenylalanine oxidation (F13CO2) in response to graded intakes of l-lysine (A) and l-tryptophan (B) as free amino acid (reference curves depicted as circles) and protein-bound lysine and tryptophan from white African cornmeal (test curves depicted as squares) in healthy young men provided with graded intakes of lysine and tryptophan. Values are means ± SDs, n = 6 (A) or n = 7 (B). The dotted lines around the equation represent the IAAO response to graded intakes of lysine from the cornmeal. The dots are used to show that this equation pertains to the slope with the dots below. FIGURE 2 View largeDownload slide Linearity of l-[1-13C]phenylalanine oxidation (F13CO2) in response to graded intakes of l-lysine (A) and l-tryptophan (B) as free amino acid (reference curves depicted as circles) and protein-bound lysine and tryptophan from white African cornmeal (test curves depicted as squares) in healthy young men provided with graded intakes of lysine and tryptophan. Values are means ± SDs, n = 6 (A) or n = 7 (B). The dotted lines around the equation represent the IAAO response to graded intakes of lysine from the cornmeal. The dots are used to show that this equation pertains to the slope with the dots below. TABLE 5 MA of l-lysine and l-tryptophan in cooked cornmeal based on the IAAO of l-[1-13C]phenylalanine1 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 1AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2MA from the AA mixture was assumed to be 100% (14). View Large TABLE 5 MA of l-lysine and l-tryptophan in cooked cornmeal based on the IAAO of l-[1-13C]phenylalanine1 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 n Slope equation MA P Lysine source  AA mix 7 −0.00961x + 1.35 1002 0.0062  Cooked cornmeal 6 −0.00686x + 1.35 71 0.048 Tryptophan source  AA mix 7 −0.00696x + 1.51 1002 0.012  Cooked cornmeal 6 −0.00558x + 1.51 80 0.046 1AA, amino acid; IAAO, indicator amino acid oxidation; MA, metabolic availability. 2MA from the AA mixture was assumed to be 100% (14). View Large Linearity of response to tryptophan intake from free AA diet As tryptophan intake from the AA mix increased from 0.5 to 1 to 1.5 to 2 mg · kg−1 · d−1 (12.5–50% of tryptophan requirement of 4 mg · kg−1 · d−1), phenylalanine oxidation decreased linearly. The application of linear regression determined a negative slope of the best-fit line of −0.00696 (SEM = 0.0026; P = 0.0116). Metabolic availability of tryptophan in cooked white cornmeal Tryptophan intake (0.5–1.5 mg · kg−1 · d−1 above base) from cooked white cornmeal had a significant effect on phenylalanine oxidation (slope = −0.00558; SEM = 0.0023; P = 0.0462). The ratio of the response to additional tryptophan intake from the cooked white cornmeal compared with that of tryptophan from the AA mix was 0.8017. Thus, the MA of tryptophan from cooked white cornmeal was 80% (Table 5, Figure 2B). Discussion Maize is the main staple in the diet of many native African populations, with 16 of the top 22 countries in the world in which maize provides the highest percentage of calories in the diet coming from that continent (27). The most commonly consumed maize in these countries is the white African cornmeal used in the current study. In addition, maize is the most important staple in the Latin American diet, where the typical adult and child consume ≤560 and 150 g/d, respectively (28). The heavy reliance on maize has led to a deficiency of lysine and tryptophan in the diet, resulting in poor growth in children (3, 29). The poor quality of maize protein, the heavy reliance of so many of the world’s food-insecure population on its calories, and current understanding that protein requirement recommendations are severely underestimated (30–33) point to maize as a food for which the protein quality requires urgent evaluation. With the use of the noninvasive IAAO method, which our group recently adapted for the study of protein quality in humans (8), we evaluated the protein quality of white African maize by studying the MA of the 2 most limiting AAs: lysine and tryptophan. The results of the current study show that 71% and 80% of the lysine and tryptophan in white African cornmeal are available for protein synthesis when the cornmeal is prepared in the traditional moist cooking method to make “pap.” This is the first study to our knowledge to assess the protein quality of white African cornmeal directly in humans and shows that the MA of lysine is lower than that of tryptophan and is the dominant AA, which limits the protein quality of white African cornmeal. The ileal AA digestibilities of lysine and tryptophan in Indian maize flour were determined in growing rats and found to be 92% and 84%, respectively (34). Although the digestibility of tryptophan is similar to 80% MA found in the current study, the 92% digestibility of lysine is very different from the 71% found in the current study. There are a number of possible reasons why this might have occurred: The rat is an inferior model for the study of protein quality of foods for human consumption (35), and although true ileal digestibility was used, the correction factor was based on endogenous AA flows from growing rats reported in a different study. It is possible that different processing methods were used on the maize kernels between India and Africa. There are 2 main methods of processing maize for human consumption: dry milling or alkaline processing (nixtamalization) (36). Alkaline processing changes the zein protein matrix, which makes AAs (particularly lysine) more available to the body (37). Maize that is processed by nixtamalization is softer and usually ground into flour, whereas maize that is dry milled is grounded into a coarser texture (37). It is quite possible therefore that the Indian maize flour used in the rat study (34) was processed by nixtamalization—hence, the higher lysine digestibility. The beneficial effect of nixtamalization on protein quality was previously shown when rats fed maize dough and tortillas made from alkaline processed maize showed greater weight gain and protein efficiency ratio than rats fed unprocessed maize (28). The variety of maize used in our study was likely very different from the variety used to prepare the Indian maize flour in the rat study (34). Although white maize is the preferred variety in Africa, yellow maize is very common in many other parts of the world (27). The difference in the MA of lysine and tryptophan in our study and the previous study (34) highlights the importance of studying different varieties of the same foods from different countries. This will allow for a more accurate classification of the protein quality of all foods and a better understanding of the foods needed to meet the requirement for appropriate nourishment of the human populations around the world. The results of the current study provide evidence against the use of the protein digestibility-corrected AA score, in which a single digestibility factor is used to predict AA digestibility in general. The current results show that the MAs of different AAs vary within the same food (lysine and tryptophan in this case) and validate the FAO/WHO decision to use AAs as individual nutrients for the study of protein quality (6). MA as measured in the current study is based on the principle of the slope ratio assay, in which changes in a measured response (usually growth) as a result of varying the intakes of a test protein are compared with changes in the same measured response to changes in the response to a reference protein (10, 38). Slope ratio assays are considered the standard for measuring AA bioavailability (38). The advantages of the slope ratio as applied in the current study is that the carbon dioxide output measured in the IAAO studies represents an end product of AA metabolism during digestion, absorption, and cellular metabolism (10). These constitute all of the components affecting bioavailability. Hence, MA is more representative of the bioavailability of AAs than digestibility, which only covers the losses of AAs during digestion. The main limitation of the slope ratio assay is that only 1 AA can be examined at a time (10). Nevertheless, the short adaptation time (<2 d) (39) of the IAAO technique allows for the evaluation of multiple AAs over a relatively short time (10). The IAAO technique represents the partitioning of the indicator AA between protein synthesis and oxidation where decreasing oxidation represents an increase in the test AA for protein synthesis. To achieve the criterion of linearity required for the slope ratio assay, the test AAs (lysine and tryptophan in this study), need to be kept below the level of the requirement (at most, 60%). Because the indicator AA was kept constant throughout each individual study, decreases in oxidation in response to graded intakes of lysine or tryptophan are indicative of an increase in uptake for protein synthesis (12). The white African cornmeal used in the current study contains 151 mg lysine, 7.46 g protein, and 372 kcal/100 g raw cornmeal. With the use of the FAO-proposed lysine requirement of 30 mg/kg (40), a man in Africa aged 30–40 y and weighing 71 kg will require 2130 mg lysine/d. If his diet consisted of cornmeal only, he would need 797 g cornmeal/d to meet approximate energy needs of 2960 kcal/d on the basis of an estimated energy need of 42 kcal ⋅ kg−1 ⋅ d−1 for a moderate to active lifestyle (41). This amount of cornmeal supplies 59 g protein and 1203 mg lysine, with a net lysine content of 854 mg based on the MA as determined in the current study. The protein and net lysine contents represent only 59% and 40% of the protein and lysine needs, respectively. White African cornmeal should therefore be combined with a legume such as lentils or chickpeas in order to increase the lysine and protein contents. Legumes, however, are expensive, so knowledge of the MA of the lysine in those foods is needed to guide recommendations on appropriate ratios to be combined to meet lysine needs. In summary, this is the first study to our knowledge to determine directly in humans the MA of lysine and tryptophan in white African cornmeal. With the use of the minimally invasive IAAO method, we found that the MAs of lysine and tryptophan were 71% and 80%, respectively. This methodology has the greatest potential to advance knowledge on protein quality of foods for human consumption because it can be applied to evaluate the protein quality of all whole foods. Acknowledgments We thank Jasmine Donohue, Department of Nutrition and Food Services, The Hospital for Sick Children, for preparing the protein-free cookies. The authors’ responsibilities were as follows—RE, ROB, PBP, and GC-M: designed the research; MR: conducted the research; MR and GC-M: analyzed the data and wrote the manuscript; GC-M: had primary responsibility for the final content; and all authors: read and approved the final manuscript. Notes Supported by the Canadian Institute of Health Research (grant MT 10321). Pfizer Consumer Healthcare (Mississauga, Ontario) donated the multivitamins. Mead Johnson Nutritionals donated the protein-free powder for experimental diets. Author disclosures: MR, RE, ROB, PBP, and GC-M, no conflicts of interest. 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Journal of NutritionOxford University Press

Published: May 7, 2018

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