Background: Ready-to-use therapeutic food (RUTF) is used to treat children suffering from severe acute malnutrition (SAM). Standard RUTF uses milk as the primary protein source, which makes the product expensive, and given the high worldwide SAM burden, having a less expensive effective alternative is a public health priority. Objective: The objective of this study was to evaluate whether newly developed amino acid-enriched milk-free RUTF (FSMS-RUTF) or amino acid-enriched low-milk RUTF (MSMS-RUTF) treatment could replenish plasma amino acids to levels comparable to those following standard peanut-milk RUTF (PM-RUTF) treatment and to improve understanding of the effects of treatment on anthropometric measurements. A secondary analysis was performed to test the noninferiority hypothesis of plasma essential amino acid (EAA) levels. Methods: Plasma EAA levels were measured in a nonblinded, 3-arm, parallel-group simple randomized controlled trial conducted in Malawi to examine the efficacy of FSMS-RUTF, MSMS-RUTF and PM-RUTF in the treatment of SAM in 2 groups of children aged 6–23 and 24–59 months (mo). Sample size calculations were performed based on the previous our study. A noninferiority margin was set at -25% of the PM-RUTF arm at discharge. Results: The relative values of the differences (95% CI) in plasma EAA levels between PM-RUTF treatment and FSMS-RUTF and MSMS-RUTF treatments at discharge were -7.9% (-18.6, 2.8) and 9.8% (0.2, 19.5), respectively, in children aged 6–23 mo, while in those aged 24–59 mo, the difference values were 17.8% (1.6, 34.1) and 13.6% (-2.8, 29.9), respectively. Conclusion: At discharge, the plasma EAA concentrations in 6-59-mo-old SAM children treated with FSMS-RUTF and MSMS-RUTF were not less than those of children treated with PM-RUTF. These findings indicate that treatment with either of the 3 RUTFs was associated with adequate protein synthesis and that all the formulations provided sufficient functional metabolites of plasma amino acids to support nutritional recovery from SAM.
Research Article; Physical sciences; Chemistry; Chemical compounds; Organic compounds; Amino acids; Sulfur containing amino acids; Methionine; Organic chemistry; Biology and life sciences; Biochemistry; Proteins; Molecular biology; Molecular biology techniques; Molecular biology assays and analysis techniques; Amino acid analysis; Research and analysis methods; Aliphatic amino acids; Leucine; Nutrition; Diet; Beverages; Milk; Medicine and health sciences; Anatomy; Body fluids; Physiology; Blood; Blood plasma; Malnutrition; Chemical synthesis; Biosynthetic techniques; Protein synthesis; Experimental organism systems; Model organisms; Maize; Organisms; Eukaryota; Plants; Grasses; Plant and algal models
Severe acute malnutrition (SAM) is classified by weight-for-height measurements being 3 standard deviations (SDs) below the median of the WHO growth curves, by the presence of bilateral pitting edema or by a mid-upper-arm circumference (MUAC) of less than 115 mm in children 6–59 months (mo) of age [[
The most widely used RUTF is peanut-milk RUTF (PM-RUTF) [[
Many efficacy trials for RUTFs have been implemented in the past, with positive outcomes in recovery of SAM and in recovery of anthropometric measurements [[
Recent studies have shown that changes in plasma amino acid profiles correlate with physiological variables such as dietary protein intake and malnutrition [[
Since the newly developed RUTFs are based on using plant proteins and supplementation with amino acids, comparing the protein nutrition of the treated children with that of PM-RUTF-treated children is important for understanding the biological mechanism by which the newly developed product affects SAM recovery. In a previous efficacy study of SMS-RUTF treatment conducted in DRC, we measured plasma amino acid levels in a subgroup of participating children [[
This study aimed to evaluate whether FSMS or MSMS-RUTF treatment could maintain plasma EAA, leucine, methionine and cystine to levels comparable to those in children treated with PM-RUTF and provide further understanding of the primary outcomes (FSMS- and MSMS-RUTF treatments showed noninferiority for recovery rate, program length of stay, and weight gain compared to PM-RUTF treatment) of the previously reported study [[
This study is a secondary analysis of a nonblinded, 3-arm, parallel-group simple randomized controlled trial conducted in the central region of Malawi to examine the efficacy of FSMS-RUTF, MSMS-RUTF and PM-RUTF for the treatment of SAM in 2 groups of children aged 6–23 and 24–59 mo. This trial was registered at the Pan African Clinical Trials Registry as trial no. PACTR201505001101224 (
Before data collection began, we obtained permission to conduct the study from the National Ethics Committee of the Malawi Ministry of Health and from the Ajinomoto Institutional Review Board. At the time of admission, each child’s parent or caregiver was informed about the nature and purpose of the study and asked for verbal and written consent for their child to be included and for their child’s medical information to be used for research purposes. Given the short duration of the study, no interim analysis was planned, and no stopping rule was predefined. No serious side effects were detected, and no reasons for interrupting the study were identified.
In mainstream treatments for SAM, children with no medical complications access treatment as outpatients while staying in their homes (community-based management of acute malnutrition; CMAM) rather than in therapeutic centers [[
Children with any medical or nutritional complications during follow-up were referred to the participating inpatient facility for appropriate treatment, after which they were readmitted into the daycare program and remained in their original study group. Children meeting referral criteria whose caregivers refused transfer and chose to remain in daycare were excluded from the study. Medical complications were defined using the WHO CMAM and Integrated Management of Childhood Illnesses standard definitions [[
Study participants were selected from all children aged 6–59 mo who had been diagnosed with SAM and admitted into the CMAM programs operated by the Ministry of Health. SAM was defined as a MUAC<115 mm or bilateral pitting edema of any degree. Children with an MUAC<115 mm and those with grade 1 or 2 bilateral pitting edema with good appetite and no medical complications were admitted directly into the daycare program and enrolled in the study.
There, they were re-examined by senior supervisors to confirm the SAM diagnosis. Children admitted into the CMAM program were excluded from the study if senior supervisors did not confirm the presence of SAM. Children with congenital or acquired disorders affecting growth, any history of any food allergy or intolerance or a history of treatment for SAM in the previous 3 mo and those from visiting families were also excluded.
This study used simple randomization, with each of the 21 sites recruiting subjects into each of the 3 arms at a ratio of 1:1:1. After confirming the subjects’ eligibility for study inclusion, we used a closed envelope method to randomly assign children to receive the FSMS-RUTF, MSMS-RUTF, or PM-RUTF treatment. The trial statistician prepared a computer-generated sequentially numbered randomization list that contained the allocations and codes for each site. These data were sent to the national study coordinator, who then assigned participants to groups at the time of enrollment. The required sample size was achieved in Jun 2018.
Sample size calculations were performed based on the DRC study [[
On the basis of the DRC study [[
The treatment was conducted based on a general CMAM program. Children staying within 1 h walking distance from feeding points in the study community were enrolled and followed-up from admission to nutrition recovery using a daycare approach. Enrolled children attended the site daily from 9 a.m. to 3 p.m. Children were offered 200 kcal/kg/day of one of the study RUTFs. The nutritional compositions of the study RUTFs (FSMS, MSMS and PM) are shown in S1 and S2 Tables. Children were declared nonrecovered if they did not meet the discharge criteria (MUAC≥ 12.5 cm and no edema) after 3 consecutive mo of treatment. Apart from the difference in RUTF administered, children of all three study groups were treated and given medical treatment generally following Malawi national guidelines, with the exception of the daycare approach.
To date, there are no official clinical values for the concentrations of plasma amino acids. Therefore, in order to define a margin for the noninferiority analysis, we used circadian values of plasma amino acids in healthy adult humans since plasma amino acid levels are known to exhibit diurnal variations. Studies have shown that the concentrations of most plasma amino acids oscillate more than 25% during the day [[
Blood sampling for amino acid analysis was performed on the subjects from whom blood samples could be collected within 48 h after enrollment, and collections continued until the planned sample number was reached (n = 499). The flow diagram of the participants is indicated in Fig 1. Trained pediatric phlebotomists collected venous blood and obtained plasma using ethylenediaminetetraacetic acid disodium dehydrate (CAS: 6381-92-6) as an anticoagulant. All blood specimens were collected in the morning, immediately stored in a CubeCooler (Forte Grow Medical Co. Ltd., Tochigi, Japan) and kept at 4°C [[
Amino acid (methionine, leucine, valine, isoleucine, lysine, phenylalanine, tryptophan, threonine, histidine and cystine) concentrations were measured by an automatic amino acid analyzer (L-8800; Hitachi High-Technologies Corporation., Tokyo, Japan). Briefly, amino acids, separated by cation-exchange chromatography, were detected spectrophotometrically after a postcolumn reaction with ninhydrin reagent.
Total BCAA (leucine, valine and isoleucine) and EAA concentrations were calculated as the summation of the concentration of each BCAA and each EAA. Plasma amino acid concentrations are given in units of micromolar (μM).
All analyses were performed on an intention-to-treat basis. Means and SDs or standard errors (SEs) or proportions and 95% confidence intervals (CIs) were used to describe the admission, discharge and difference parameters, as appropriate. To adjust for population heterogeneity, we divided the children into two subgroups, a 6-23-mo-old subgroup and a 24-59-mo-old subgroup, as described the study protocol (S1 Study Protocol). Tukey’s multiple comparisons tests or Fisher’s exact test were used for baseline characteristic comparisons.
Plasma amino acids analysis considered the CMAM program by using mixed models that recognized the multilevel structure of the data where individual patients were nested within one of the 21 clusters. A model-based approach can be efficient and effective for handling missing data [[
Since intervention had not yet started at admission, the “intervention” variable included the allocation effect of each arm (FSMS arm, MSMS arm and PM arm); the “sampling point” variable included the effect of a treatment period common to the arms. Interaction of “intervention” and “sampling point” included the effect of each RUTF (FSMS, MSMS and PM). The “admission value” variable included the effect of interindividual differences in plasma amino acid concentrations. To determine the model coefficients, the model was fitted by a residual maximum likelihood estimation using age subgroup data with the “lme4” (ver. 1.1.13) package for R [[
The calculated predicted plasma amino acid concentrations at discharge were tested in a noninferiority hypothesis that those of FSMS-RUTF and MSMS-RUTF would not be less than those of PM-RUTF. Our generic models were adjusted for individual admission value; therefore, predicted discharge values were controlled by each subject admission value, and comparison of predicted discharge values between arms was equal to comparison of actual differences between admission and discharge between arms. For the noninferiority test, the 2-sided 95% CI of the differences between the FSMS-RUTF arm and the PM-RUTF arm and between the MSMS-RUTF arm and the PM-RUTF arm were estimated by simultaneous inference procedures in the LMM with the “multcomp” (ver. 1.4.6) package for R [[
All statistical analyses were performed within the R (ver. 3.3.0) platform (https://
Table 1 presents the baseline characteristics of the children included in the analyses for each age subgroup. In the study, marasmus (without edema) was the dominant form of SAM among children aged 6–23 mo, and kwashiorkor (with edema) was the dominant form of SAM among children aged 24–59 mo. Percentage of sex significantly differed between the MSMS-RUTF arm and PM-RUTF arm for children aged 24–59 mo. MUAC and height were significantly different between the FSMS-RUTF arm and PM-RUTF arm for children aged 24–59 mo. There were no significant differences (p>0.05) between the PM-RUTF arm and the MSMS-RUTF and FSMS-RUTF arms in the other baseline parameters considered in either of the 2 age subgroups. Breastfeeding rate was not significantly different between arms in either of the 2 age subgroups (data not shown).
Table 1: Baseline characteristics for children1.
6–23 mo of age 24–59 mo of age Criteria FSMS-RUTF MSMS-RUTF PM-RUTF FSMS-RUTF MSMS-RUTF PM-RUTF Participants, n 91 112 114 52 56 41 All Male sex, n 45 ( 49.5% ) 50 ( 44.6% ) 51 ( 44.7% ) 27 ( 51.9% ) 25 ( 44.6% ) 30 ( 73.2% ) Age, mo 13.5 ± 4.6 14.2 ± 5.9 13.9 ± 4.9 30.4 ± 5.6 31.9 ± 7.2 33.7 ± 8.3 Midupper arm circumference, mm 111 ± 9 114 ± 9 113 ± 9 120 ± 14 128 ± 16 128 ± 16 Weight, kg 6.3 ± 1.1 6.6 ± 1.4 6.5 ± 1.2 8.8 ± 2.0 9.8 ± 1.9 9.6 ± 2.4 Height, cm 67.3 ± 6.1 68.3 ± 6.3 67.7 ± 5.9 78.1 ± 6.1 81.9 ± 6.5 81.5 ± 6.6 Bilateral pitting edema 30 ( 33.0% ) 42 ( 37.5% ) 33 ( 28.9% ) 30 ( 57.7% ) 44 ( 78.6% ) 30 ( 73.2% ) Weight-for-age z score -3.5 ± 1.3 -3.2 ± 1.2 -3.4 ± 1.2 -3.4 ± 1.5 -2.6 ± 1.5 -3.1 ± 1.6 Height-for-age z score -3.4 ± 1.7 -3.2 ± 1.4 -3.4 ± 1.6 -3.8 ± 1.4 -2.9 ± 1.7 -3.4 ± 1.5 Weight-for-height z score -2.3 ± 1.3 -2.0 ± 1.2 -2.1 ± 1.2 -1.9 ± 1.5 -1.4 ± 1.4 -1.9 ± 1.8 Children without edema, n 61 70 81 22 12 11 Male sex, n 31 ( 50.8% ) 35 ( 50.0% ) 36 ( 44.4% ) 9 ( 40.9% ) 4 ( 33.3% ) 9 ( 81.8% ) Age, mo 12.2 ± 4.7 12.8 ± 5.6 13.1 ± 4.5 29.1 ± 5.6 31.9 ± 6.3 32.0 ± 7.4 Midupper arm circumference, mm 108 ± 7 109 ± 6 110 ± 5 110 ± 4 108 ± 7 110 ± 4 Weight, kg 5.9 ± 0.9 6.0 ± 1.0 6.1 ± 1.0 7.3 ± 0.9 8.0 ± 1.0 7.7 ± 1.2 Height, cm 65.4 ± 5.9 66.0 ± 5.4 66.6 ± 5.6 74.6 ± 4.5 78.7 ± 5.0 77.5 ± 5.3 Weight-for-age z score -3.8 ± 1.2 -3.8 ± 1.0 -3.6 ± 1.1 -4.5 ± 0.7 -4.0 ± 0.9 -4.5 ± 0.9 Height-for-age z score -3.6 ± 1.9 -3.6 ± 1.4 -3.5 ± 1.6 -4.6 ± 1.1 -3.8 ± 1.3 -4.3 ± 1.4 Weight-for-height z score -2.4 ± 1.2 -2.4 ± 1.1 -2.3 ± 1.1 -3.0 ± 1.0 -2.8 ± 0.9 -3.4 ± 0.9 Children with edema, n 30 42 33 30 44 30 Male sex, n 14 ( 46.7% ) 15 ( 35.7% ) 15 ( 45.5% ) 18 ( 60.0% ) 21 ( 47.7% ) 21 ( 70.0% ) Age, mo 16.1 ± 3.2 16.4 ± 5.7 15.9 ± 5.1 31.3 ± 5.5 32.0 ± 7.5 34.4 ± 8.7 Midupper arm circumference, mm 118 ± 10 121 ± 9 120 ± 11 128 ± 13 134 ± 13 134 ± 14 Weight, kg 7.2 ± 1.2 7.7 ± 1.2 7.2 ± 1.3 9.8 ± 1.9 10.2 ± 1.8 10.3 ± 2.4 Height, cm 71.2 ± 4.3 72.1 ± 6.0 70.3 ± 5.8 80.7 ± 5.8 82.7 ± 6.6 83.0 ± 6.5 Weight-for-age z score -3.1 ± 1.3 -2.4 ± 1.0 -2.9 ± 1.3 -2.6 ± 1.4 -2.2 ± 1.4 -2.6 ± 1.5 Height-for-age z score -3.0 ± 1.1 -2.6 ± 1.1 -3.1 ± 1.5 -3.3 ± 1.3 -2.7 ± 1.8 -3.1 ± 1.4 Weight-for-height z score -2.1 ± 1.4 -1.4 ± 1.1 -1.7 ± 1.1 -1.1 ± 1.3 -1.0 ± 1.3 -1.3 ± 1.7
1 1Values are expressed as n (%) or means ± SDs unless otherwise indicated.
2 FSMS, milk-free, soy, maize, and sorghum; MSMS, milk, soy, maize, and sorghum; PM, peanut and milk; RUTF, ready-to-use therapeutic food.
Table 2 presents the predicted plasma amino acid concentrations at admission and discharge in each age subgroup. For all amino acids, the β
Table 2: Predicted plasma amino acid concentrations in the age subgroup1.
6–23 mo of age 24–59 mo of age FSMS-RUTF MSMS-RUTF PM-RUTF FSMS-RUTF MSMS-RUTF PM-RUTF Amino acids Admission* Discharge Admission* Discharge Admission* Discharge Admission* Discharge Admission* Discharge Admission* Discharge Methionine 22.5 ± 1.0 23.5 ± 1.2 23.3 ± 0.9 26.6 ± 1.0 22.8 ± 0.9 26.2 ± 1.0 22.9 ± 1.3 24.6 ± 1.5 21.6 ± 1.2 28.6 ± 1.5 22.2 ± 1.5 23.9 ± 1.6 Leucine 112.4 ± 3.7 119.5 ± 4.7 112.2 ± 3.3 137.3 ± 3.9 116.0 ± 3.3 125.4 ± 3.9 109.2 ± 5.7 153.3 ± 6.8 104.2 ± 5.5 146.0 ± 6.6 108.5 ± 6.4 114.5 ± 7.0 Valine 175.3 ± 6.2 179.7 ± 7.9 174.5 ± 5.6 215.2 ± 6.5 183.0 ± 5.6 210.6 ± 6.6 168.5 ± 9.4 230.7 ± 11.2 158.0 ± 9.1 224.6 ± 10.8 167.5 ± 10.5 185.3 ± 11.6 Isoleucine 74.8 ± 2.7 70.8 ± 3.4 73.4 ± 2.4 84.6 ± 2.8 74.0 ± 2.4 77.8 ± 2.9 75.9 ± 4.2 90.9 ± 5.0 68.9 ± 4.0 88.2 ± 4.7 71.3 ± 4.6 69.8 ± 5.1 Lysine 188.2 ± 8.0 179.4 ± 10.0 186.7 ± 7.3 224.2 ± 8.4 175.6 ± 7.2 191.4 ± 8.4 187.9 ± 10.4 223.9 ± 12.4 175.7 ± 9.9 224.6 ± 11.8 172.3 ± 11.7 184.8 ± 12.8 Phenylalanine 74.4 ± 2.2 66.1 ± 2.8 73.0 ± 2.0 76.7 ± 2.4 74.3 ± 2.0 77.4 ± 2.4 80.1 ± 3.6 82.5 ± 4.3 74.7 ± 3.5 89.2 ± 4.1 74.9 ± 4.1 76.6 ± 4.5 Tryptophan 24.4 ± 1.1 25.6 ± 1.4 23.8 ± 1.0 31.9 ± 1.2 25.3 ± 1.0 29.7 ± 1.2 17.9 ± 1.6 27.4 ± 1.9 15.8 ± 1.6 28.9 ± 1.9 15.9 ± 1.8 26.9 ± 2.1 Threonine 87.1 ± 3.2 82.9 ± 4.0 87.0 ± 2.9 91.7 ± 3.3 91.6 ± 2.8 95.2 ± 3.4 84.1 ± 4.3 94.5 ± 5.2 77.6 ± 4.2 88.4 ± 5.0 84.5 ± 4.9 85.2 ± 5.4 Histidine 70.6 ± 1.7 69.0 ± 2.1 70.6 ± 1.5 73.8 ± 1.8 71.8 ± 1.5 73.2 ± 1.8 75.8 ± 2.4 80.6 ± 2.9 74.5 ± 2.4 76.2 ± 2.8 78.2 ± 2.8 65.9 ± 3.0 BCAA 362.6 ± 12.4 370.0 ± 15.6 360.1 ± 11.1 437.1 ± 12.9 373.0 ± 11.1 413.7 ± 13.1 353.4 ± 18.7 474.9 ± 22.4 331.2 ± 18.1 459.1 ± 21.5 347.1 ± 21.0 369.4 ± 23.0 EAA 841.5 ± 26.3 823.1 ± 32.9 834.7 ± 23.3 981.6 ± 27.2 848.4 ± 23.2 893.6 ± 27.6 864.6 ± 37.4 1030.4 ± 43.6 812.5 ± 36.5 993.7 ± 44.6 825.2 ± 41.2 874.8 ± 47.2 Cystine 22.6 ± 0.6 26.9 ± 0.8 22.5 ± 0.6 27.9 ± 0.7 24.1 ± 0.6 29.5 ± 0.7 20.5 ± 0.9 29.7 ± 1.1 19.7 ± 0.9 28.5 ± 1.0 20.7 ± 1.0 30.2 ± 1.1
- 4 1 Plasma amino acid concentrations were predicted from the mixed model by using the age subgroup data. The fixed effects were intervention (RUTF) and sampling point (admission or discharge). The model was adjusted for admission value and random effect of subadministrative areas. Predicted concentrations were expressed as the means ± SEs [μM].
- 3 * There were no significant differences between the plasma amino acid concentrations of the FSMS-RUTF or MSMS-RUTF arms and those of the PM-RUTF arm at admission.
- 5 FSMS, milk-free, soy, maize, and sorghum; MSMS, milk, soy, maize, and sorghum; PM, peanut and milk; RUTF, ready-to-use therapeutic food; BCAA, branched-chain amino acid; EAA, essential amino acid.
For children aged 6–23 mo, the relative values of the difference (95% CI) in plasma EAA concentration based on the PM-RUTF arm at discharge were -7.9% (-18.6, 2.8) and 9.8% (0.2, 19.5) for the FSMS-RUTF and MSMS-RUTF arms, respectively (Fig 2A, Table 3). In those aged 24–59 mo, the relative values of the difference (95% CI) in the EAA concentration based on PM-RUTF arm at discharge were 17.8% (1.6, 34.1) and 13.6% (-2.8, 29.9) for the FSMS-RUTF and MSMS-RUTF arms, respectively (Fig 2A, Table 3). Absolute values are shown in S3 Table. These results indicated that the plasma EAA concentrations of the FSMS-RUTF and MSMS-RUTF arms were not less than those of the PM-RUTF arm at discharge in both age groups (Fig 2A, Table 3).
Table 3: Testing noninferiority of the plasma amino acid concentrations at discharge between the PM-RUTF arm and the FSMS-RUTF and MSMS-RUTF arms in the age subgroup1.
6–23 mo of age 24–59 mo of age Amino acids Comparison of treatment arm Difference[%] (95% CI)2 Difference[%] (95% CI)2 Methionine FSMS-RUTF vs PM-RUTF -10.1 ( -23.6 , 3.3 ) 3.1 ( -17.3 , 23.6 ) MSMS-RUTF vs PM-RUTF 1.7 ( -10.5 , 13.8 ) 19.7 ( -0.3 , 39.7 ) Leucine FSMS-RUTF vs PM-RUTF -4.7 ( -15.5 , 6.0 ) 33.9 ( 14.9 , 52.9 ) MSMS-RUTF vs PM-RUTF 9.5 ( -0.3 , 19.2 ) 27.6 ( 9.0 , 46.2 ) Valine FSMS-RUTF vs PM-RUTF -14.7 ( -25.5 , -3.8 ) 24.5 ( 5.3 , 43.7 ) MSMS-RUTF vs PM-RUTF 2.2 ( -7.6 , 12.0 ) 21.3 ( 2.3 , 40.2 ) Isoleucine FSMS-RUTF vs PM-RUTF -9.0 ( -21.7 , 3.7 ) 30.2 ( 7.7 , 52.7 ) MSMS-RUTF vs PM-RUTF 8.8 ( -2.7 , 20.3 ) 26.4 ( 4.4 , 48.3 ) Lysine FSMS-RUTF vs PM-RUTF -6.3 ( -21.2 , 8.6 ) 21.2 ( -0.2 , 42.5 ) MSMS-RUTF vs PM-RUTF 17.1 ( 3.7 , 30.6 ) 21.5 ( 0.8 , 42.3 ) Phenylalanine FSMS-RUTF vs PM-RUTF -14.7 ( -25.1 , -4.3 ) 7.7 ( -10.2 , 25.6 ) MSMS-RUTF vs PM-RUTF -0.9 ( -10.3 , 8.4 ) 16.4 ( -1.0 , 33.9 ) Tryptophan FSMS-RUTF vs PM-RUTF -13.6 ( -27.6 , 0.4 ) 1.9 ( -20.7 , 24.4 ) MSMS-RUTF vs PM-RUTF 7.5 ( -5.3 , 20.2 ) 7.6 ( -15.1 , 30.3 ) Threonine FSMS-RUTF vs PM-RUTF -12.9 ( -25.1 , -0.8 ) 11.0 ( -8.3 , 30.4 ) MSMS-RUTF vs PM-RUTF -3.7 ( -14.7 , 7.3 ) 3.8 ( -15.2 , 22.8 ) Histidine FSMS-RUTF vs PM-RUTF -5.7 ( -14.1 , 2.7 ) 22.3 ( 8.2 , 36.4 ) MSMS-RUTF vs PM-RUTF 0.9 ( -6.7 , 8.5 ) 15.6 ( 1.8 , 29.6 ) BCAA FSMS-RUTF vs PM-RUTF -10.6 ( -21.5 , 0.3 ) 28.5 ( 9.4 , 47.6 ) MSMS-RUTF vs PM-RUTF 5.6 ( -4.2 , 15.5 ) 24.3 ( 5.4 , 43.0 ) EAA FSMS-RUTF vs PM-RUTF -7.9 ( -18.6 , 2.8 ) 17.8 ( 1.6 , 34.1 ) MSMS-RUTF vs PM-RUTF 9.8 ( 0.2 , 19.5 ) 13.6 ( -2.8 , 29.9 ) Cystine FSMS-RUTF vs PM-RUTF -9.0 ( -16.9 , -1.1 ) -1.8 ( -12.8 , 9.2 ) MSMS-RUTF vs PM-RUTF -5.7 ( -12.9 , 1.5 ) -5.7 ( -16.5 , 5.1 )
- 7 1 The point estimate and 95% CI of the difference in plasma amino acid concentrations at discharge between the FSMS-RUTF and PM-RUTF arms and between the MSMS-RUTF and PM-RUTF arms by using the age subgroup data. The differences are shown as relative values based on the plasma amino acid concentration of the PM-RUTF arm at discharge.
- 6 2 CIs were estimated by simultaneous inference procedures in mixed model. The noninferiority margin was -25% of the plasma amino acid concentration of the PM-RUTF arm at discharge. Therefore, the lower limit of 95% CI larger than -25% indicated noninferiority.
- 8 FSMS, milk-free, soy, maize, and sorghum; MSMS, milk, soy, maize, and sorghum; PM, peanut and milk; RUTF, ready-to-use therapeutic food; CI, confidence interval; BCAA, branched-chain amino acid; EAA, essential amino acid.
Similarly, the plasma concentrations of leucine in the FSMS-RUTF and MSMS-RUTF arms were also not less than the concentration in the PM-RUTF arm in both age groups (Fig 2B, Table 3).
For children aged 6–23 mo, the relative values of the difference (95% CI) in plasma methionine concentration based on the PM-RUTF arm at discharge were -10.1% (-23.6, 3.3) and 1.7% (-10.5, 13.8) for the FSMS-RUTF and MSMS-RUTF arms, respectively (Fig 2C, Table 3). In those aged 24–59 mo, the relative values of the difference (95% CI) in the methionine concentration based on the PM-RUTF arm at discharge was 3.1% (-17.3, 23.6) and 19.7% (-0.3, 39.7) for the FSMS-RUTF and MSMS-RUTF arms, respectively (Fig 2C, Table 3). Absolute values are shown in S3 Table. These results indicated that the plasma methionine concentrations in the FSMS-RUTF and MSMS-RUTF arms were not less than those in the PM-RUTF arm at discharge in both age groups (Fig 2C, Table 3).
Similarly, the plasma concentrations of cystine in the FSMS-RUTF and MSMS-RUTF arms were also not less than those in the PM-RUTF arm in both age groups (Fig 2D, Table 3).
In this report, we focused on assessing both EAAs and leucine to compare the efficacy of FSMS-RUTF and MSMS-RUTF with that of PM-RUTF in the noninferiority test since they EAAs and leucine are the key amino acids in protein synthesis. Our hypothesis was that maintaining an adequate concentration of plasma EAAs is important for protein synthesis. The noninferiority of the differences in plasma EAA and leucine concentrations in the FSMS–RUTF arm and the MSMS–RUTF arm at discharge (Fig 2, Table 3) demonstrated comparable levels of protein synthesis, which explains the reported SAM recovery rates (percentage of children who were discharged as recovered from the study divided by the total number of children who exited the study) [[
The protein synthesis rate is higher in infants and young children and decreases with aging [[
Our second aim was to verify whether the enriched concentrations of sulfur-containing amino acids in plasma in the FSMS-RUTF and MSMS-RUTF treatments were fully maintained compared to those in the PM-RUTF treatment. The results from this study revealed that plasma methionine and cystine concentrations in the FSMS–RUTF arm and the MSMS–RUTF arm were not less than those in the PM-RUTF arm at discharge (Fig 2, Table 3). This finding strongly suggests that the FSMS-RUTF treatment and MSMS-RUTF treatment could supply adequate amounts of methionine and cystine for children recovering from SAM.
Glutathione (GSH) is thought to play an important role in recovery from malnutrition, especially in kwashiorkor. GSH is considered to be the most abundant molecule among the endogenous antioxidants, and the GSH redox cycle is a major component of the body’s overall antioxidant defenses. GSH synthesis is largely limited by the availability of its precursor L-cysteine, and a portion of it is derived from methionine [[
One of the major limitations of this study is that we could not control for the mealtime of the subjects upon blood draw. Since concentrations of plasma amino acids are known to be influenced by nutrient intake during meals, the ideal blood draw for detecting baseline levels of plasma amino acids would be after a sufficient time intervals after the last meal [[
Another major limitation of the study was the unavailability of samples at the time of discharge. Data at discharge were missing for children who defaulted or had died. The proportion of missing data due to default was large, and that due to death was very small, as previously described [[
The present clinical trial in Malawi showed a noninferiority of FSMS-RUTF and MSMS-RUTF compared to PM-RUTF for children aged 6–59 mo in the recovery rate and weight gain reported in a previously published paper [[
In conclusion, the plasma EAA concentrations following FSMS-RUTF and MSMS-RUTF treatments were not less than those following PM-RUTF treatment in 6-59-mo-old children who were diagnosed with SAM. We believe that the requirements for adequate protein synthesis and a sufficient supply of functional metabolites of amino acids such as GSH were met and contributed to the SAM recovery rates observed in all arms [[
S1 CONSORT Checklist. CONSORT checklist for the trial.(DOC)
S1 Table. Nutritional composition of the study RUTFs*.
S2 Table. Comparison of the amino acid profiles of the studied RUTFs obtained by laboratory analysis*.
S3 Table. Testing noninferiority of the plasma amino acid concentrations at discharge between the FSMS-RUTF arm and the MSMS-RUTF and PM-RUTF arms in the age subgroup1.
S1 Study Protocol. Study protocol of acceptability and efficacy of an innovative soya based RUTF for the treatment of severe acute malnutrition.(PDF)
S1 Dataset. Dataset of plasma amino acid concentrations in study participants.(XLSX)
The authors would like to thank Michihiro Takada for expert advice on the statistical analysis, Tetsuya Takimoto for a critical reading of the manuscript, and Emi Maekawa and Akiko Onozuka for assistance in the amino acid analysis.
DIAGRAM: Fig 1: Study participant flow diagram. CHW, community health worker; MOH, Ministry of Health; OPD, outpatient department; OTP, outpatient program; SAM, severe acute malnutrition; MUAC, mid-upper arm circumference; FSMS, milk-free, soy, maize, and sorghum; RUTF, ready-to-use therapeutic food; MSMS, milk, soy, maize, and sorghum; PM, peanut and milk; Met, methionine; Leu, leucine; Val, valine; Ile, isoleucine; Lys, lysine; Phe, phenylalanine; Trp, tryptophan; Thr, threonine; His, histidine; BCAAs, branched-chain amino acids; EAAs, essential amino acids; Cys2, cystine.
DIAGRAM: Fig 2: Comparison of the difference in plasma amino acid concentrations at discharge between the FSMS-RUTF arm and PM-RUTF arm and between the MSMS-RUTF arm and PM-RUTF arm by using the age subgroup data. The differences are shown as relative values based on the plasma amino acid concentration of the PM-RUTF arm at discharge. 95% CIs were estimated by simultaneous inference procedures in the mixed model. The filled circle and error bar indicate a point estimate of the difference and 95% CI. The dotted line shows the noninferiority margin (-25%). A: Relative difference in plasma EAA concentration between arms. B: Relative difference in plasma leucine concentration between arms. C: Relative difference in plasma methionine concentration between arms. D: Relative difference in plasma cystine concentration between arms.
By Wataru Sato, Writing – original draft; Chie Furuta, Writing – original draft; Keiko Matsunaga, Data curation; Paluku Bahwere, Writing – review & editing; Steve Collins, Conceptualization; Kate Sadler, Conceptualization; Peter Akomo, Conceptualization; Chrissy Banda, Data curation; Elizabeth Maganga, Data curation; Sylvester Kathumba, Data curation and Hitoshi Murakami, Writing – review & editing