# Changes in polyunsaturated fatty acids during treatment of malnourished children may be insufficient to reach required essential fatty acid levels – A randomised controlled trial

Sanne Sigh, Lotte Lauritzen, Frank Wieringa, Arnaud Laillou, Chhoun Chamnan, Ken Stark, Nanna Roos

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## To cite this version:

Sanne Sigh, Lotte Lauritzen, Frank Wieringa, Arnaud Laillou, Chhoun Chamnan, et al.. Changes in polyunsaturated fatty acids during treatment of malnourished children may be insufficient to reach required essential fatty acid levels – A randomised controlled trial. Clinical Nutrition, 2023, 42 (9), pp.1778-1787. 10.1016/j.clnu.2023.08.003. hal-05408617

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Randomized Control Trials

# Changes in polyunsaturated fatty acids during treatment of malnourished children may be insufficient to reach required essential fatty acid levels — A randomised controlled trial

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Sanne Sigh a, b, *, Lotte Lauritzen a, Frank T. Wieringa c, d, Arnaud Laillou e, Chhoun Chamnan b, Ken D. Stark f, Nanna Roos a

## ARTICLE INFO

**Article history:**
Received 21 March 2023
Accepted 2 August 2023

**Keywords:**
Changes in fatty acids
Polyunsaturated fatty acids
Whole-blood
Treatment
Severe acutely malnourished children
Cambodia

## SUMMARY

**Background & aims:** Severe acute malnutrition (SAM) is a global concern. Studies on the impact of ready-to-use therapeutic foods (RUTFs) on polyunsaturated fatty acids (PUFA) are almost non-existent. The aim was to investigate the change in whole-blood PUFA and nutrition and health markers among Cambodian children with SAM after treatment with RUTFs.

**Methods:** The trial was an 8-week randomised clinical trial of the effectiveness of locally produced fish-based RUTF (L-RUTF) vs standard milk-based RUFT (S-RUTF). Whole-blood fatty acids were analysed using dried blood spots. Nutrition and health markers were assessed using anthropometric assessment and blood samples for markers of inflammation. The trial was conducted at the National Pediatric Hospital, Phnom Penh, Cambodia, with one hundred and twenty-one 6-59-month-old children in treatment for SAM.

**Results:** L-RUTF had a higher content of n-3 PUFA and a higher content of arachidonic acid (AA) and docosahexaenoic acid (DHA), while S-RUTF had the highest content of n-6 PUFA. At baseline, the children presented with a Mead acid level in whole-blood of around 0.08% of total fatty acids (FA%) and an omega-3 index of ~0.91 ± 0.44. After eight weeks of S-RUTF treatment, linoleic acid (LA), AA, n-6/n-3 PUFA ratio, and Mead acid levels were increased. The L-RUTF intervention did not change the whole-blood PUFAs from baseline. At discharge, the children in the L-RUTF group had a lower n-6/n-3 PUFA ratio than the children in the S-RUTF group, driven by a lower alpha-linolenic acid (ALA) (0.20 vs 0.27 FA%, p = 0.004) and lower LA (15.77 vs 14.21 FA%, p = 0.018) with no significant differences in AA or DHA levels. Weight-for-height z-score at discharge was negatively associated with total PUFA (β = 1.4 FA%, 95%CI. -2.7; -0.1), n-6 LCPUFA (β = 1.3 FA%, 95%CI. -1.3; -0.3), and AA (β = 0.6 FA%, 95%CI. -1.0; -0.2). Age-adjusted height was negatively associated with the Mead acid:AA ratio (β = 1.2 FA%, 95%CI. -2.2; -0.2). No significant change was seen in inflammation markers within groups or between groups during treatment, and n-3 and n-6 PUFAs were not associated with markers of inflammation or haemoglobin status at discharge.

**Conclusion:** The trial found that whole-blood markers of PUFA status were low in children at admission and discharge from SAM treatment, indicating that the currently recommended composition of RUTFs are not able to correct their compromised essential fatty acid status. The higher content of DHA and AA in

**Abbreviations:** AA, Arachidonic acid (20:4n-6); AGP, α₁-acid glycoprotein; ALA, α-linolenic acid (18:3n-3); CRP, C-reactive protein; DHA, Docosahexaenoic acid (22:6n-3); DPA, Docosapentaenoic acid (22:5n-6); EPA, Eicosapentanoic acid (20:5n-3); E%, Energy percent; FA%, the relative percentage of all fatty acids; HAZ, Height-for-age z-score; LA, Linolenic acid (18:2n-6); LCPUFA, Long-chain polyunsaturated fatty acids; MUAC, Mid-upper-arm circumference; MUFA, monounsaturated fatty acids; PUFA, Polyunsaturated; RUTF, Ready-to-use therapeutic foods; SAM, severe acute malnutrition; SD, Standard deviations; SFA, saturated fatty acids; WHO, World Health Organization; WHZ, Weight-for-height z-score.

* Corresponding author. Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark.

**E-mail addresses:** sigh@nexs.ku.dk (S. Sigh), ll@nexs.ku.dk (L. Lauritzen), franck.wieringa@ird.fr (F.T. Wieringa), alaillou@unicef.org (A. Laillou), chhounchamnan@gmail.com (C. Chamnan), stkark@uwaterloo.ca (K.D. Stark), nro@nexs.ku.dk (N. Roos).

<footnote>a Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark</footnote>

<footnote>b Department of Fisheries Post-Harvest Technologies and Quality Control, Fisheries Administration, #186 Preah Norodom Boulevard, 12101 Phnom Penh, Cambodia</footnote>

<footnote>c UMR QualiSud, Institut de Recherche Pour le Développement (IRD), 34394 Montpellier, France</footnote>

<footnote>d Qualisud, University of Montpellier, Avignon University, CIRAD, Institut Agro, IRD, Université de la Réunion, 34394 Montpellier, France</footnote>

<footnote>e Nutrition Section, UNICEF West and Central Africa Region, Dakar 29720, Senegal</footnote>

<footnote>f University of Waterloo, Department of Kinesiology and Health Sciences, 200 University Avenue, Waterloo, ON N2L3G1, Canada</footnote>
L-RUTF did not give rise to any improvement in PUFA status. No changes in health markers or associations between PUFA and health markers were found.

Trial registration: ClinicalTrials.gov: NCT02907424.

© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

# 1. Introduction

An estimated 49.5 million children under the age of five suffer from acute malnutrition globally [1]. Undernutrition was estimated to be the underlying cause of 45% of all child deaths [2]. Children with severe acute malnutrition (SAM) are at high risk of mortality and morbidity [2]. One of the most life-threatening consequences of undernutrition in children is increased susceptibility to infections. In addition, the risk of infectious death increases with the severity of undernutrition among children [3]. Children hospitalised with complicated SAM have shown a high burden of infections [4–6], which persisted during the recovery phase after they had been discharged [5,7–9].

SAM is caused by insufficient energy and nutrients for the body to maintain essential bodily functions, but also a consequence of a high load of diseases. Maintenance of a healthy immune response depends on an adequate and appropriate nutritious diet [10], and markers of inflammation are associated with reduced food intake [11]. However, diarrhoea, frequent among malnourished children from low-and-middle-income countries, causes decreased nutrient absorption [12]. Thus, children with SAM are vulnerable to essential nutrient deficiencies caused by insufficient food intake and disturbed absorption and metabolism [10,13,14] due to undernutrition.

Essential fatty acids, linoleic acid (18:2n-6, LA), α-linolenic acid (18:3n-3, ALA), and the long-chain polyunsaturated fatty acids (LCPUFA with >18 carbons and >3 double bonds) that are metabolised in the liver [15] are critical for growth, and brain development [16–18]. As the conversion of ALA to n-3 LCPUFA can be low do to competition with dietary LA [19], the dietary intake of preformed n-3 LCPUFA may be necessary to fulfil the body's requirements.

The nutritional status of essential fatty acids depends not only on intake but is also influenced by inflammation, as they are used to form signalling molecules in regulating various immune responses, allergy reactions, and blood coagulation [20,21].

Children with uncomplicated SAM are treated with ready-to-use-therapeutic foods (RUTFs). RUTFs focus on the energy, protein, and micronutrient requirements of young children with SAM. Still, they are not developed to optimise the long-term status of micronutrients or fatty acids. Fat is predominantly used in RUTFs to provide energy due to its high energy density, and the essential fatty acid composition has been given little attention. Studies have observed associations between SAM and low essential fatty acid status at admission [14,19,20]. Still, there is limited data on changes in blood levels of LA, ALA and LCPUFA during the treatment of SAM. A study in Ugandan children with SAM [22] found that the current therapeutic foods did not correct whole-blood LCPUFA levels in the inpatient or outpatient settings. However, a study from Pakistan saw improved essential fatty acid status and higher levels of biologically active LCPUFA during RUTF treatment in children with SAM [23].

Given the importance of essential fatty acids in children's development and their impact on immune function and health, this study used data from an 8-week randomised clinical trial evaluating the effectiveness of a locally produced fish-based RUTF

(L-RUTF) compared to a standard milk-based RUTF (S-RUTF). This article examines changes in PUFA markers and markers of nutrition and health status among Cambodian children during treatment for SAM with RUTFs.

# 2. Materials & methods

## 2.1. Trial design and setting

The trial used data from admission and discharge in an 8-week randomised controlled interventional trial that evaluated the effectiveness of an L-RUTF compared to an S-RUTF during home treatment for SAM among Cambodian children [24,25]. The trial was conducted between September 7, 2015, and January 7, 2017. Further details on the trial setting can be found in Sigh et al. [26].

## 2.2. Randomisation

The trial was a single-blinded, randomised, controlled interventional trial with active control to ensure children's recovery from SAM. The allocation ratio was 1:1 (the ratio of the intended number of children in each RUTF group). After recruitment, each child was randomised to one of the two interventions: 1) the milk-based S-RUTF, active control or 2) the fish-based L-RUTF, the intervention. Product codes X and Z replaced the names of the two RUTFs in all data registration. Binding was of the principal investigator, as the products were visibly different. Further details are described in Sigh et al. [24].

## 2.3. Eligibility criteria

Children 6–59 months of age with non-complicated SAM were eligible. SAM was defined as weight-for-height z-score (WHZ) $\le -2.8$, mid-upper-arm circumference (MUAC) $\le 115$ mm and/or the presence of nutritional oedema at admission to outpatient SAM treatment [24].

Children with SAM and complications who required hospitalisation were not eligible for the trial. In addition, non-eligibility was participation in other studies or any complications affecting dietary intake.

## 2.4. Sample size

The sample size was based on the superiority in weight gain (g/kg/day) [24]. We expected an average increase of 4 g/kg/day and a standard deviation (SD) of 0.7 g/kg/day based on the WHO recommendations for a minimum average increase in body weight [27,28] and an observed mean weight gain of 4.0 ± 4.6 g/day in the home-based treatment of SAM from Cambodia in a previous study [29]. To detect a 10% difference, which was regarded as biologically relevant, the required sample size was 49 children per RUTF group (80% power; $\alpha = 0.05$; two-sided). To account for dropouts, the aim was to recruit at least 60 children in each group.
## 2.5. Ethical consideration

Trial approval was obtained from the National Ethical Committee for Health Research by the Ministry of Health, Kingdom of Cambodia (April 2015 Version N2). This trial was conducted according to the guidelines laid down in the declaration of Helsinki. Written and oral consent to all procedures was obtained by a parent or legal guardian before collecting data or samples. All children admitted for SAM treatment received similar routine medical and nutritional treatment regardless of participation in the trial. The trial was registered at [ClinicalTrials.gov](https://ClinicalTrials.gov) ("Comparison of a Locally Produced RUTF With a Commercial RUTF in the Treatment of SAM" (FLNS_SAM). Trial registration; NCT02907424, URL: [https://clinicaltrials.gov/ct2/show/NCT02907424](https://clinicaltrials.gov/ct2/show/NCT02907424) (accessed on March 03, 2023).

## 2.6. RUTF composition and distribution

The S-RUTF is a bar whose main protein source is skimmed milk powder and milk protein. Carbohydrates are mainly driven from baked wheat, oat flour, and icing sugar, while fats are supplied from vegetable oils (palm, rapeseed and soybean oil). The L-RUTF was a fish-based paste wrapped into a crispy wafer. The main protein source was whole small, indigenous, freshwater fish made into a fish powder. Mung beans, rice and soybeans added protein and carbohydrates. Carbohydrates were mainly added from icing sugar and maltodextrin, while fat was from canola oil and palm vegetable shortening. The wafer was made from water, sugar, salt, rice flour, eggs and coconut. Further details on the composition and development are available in Sigh et al., 2018 [25].

Children received their daily food ration based on their weight. The total fatty intake was calculated based on the daily dose per treatment arm described (Table 1).

## 2.7. Data collection

Using a structured questionnaire, the caregiver was asked regarding socio-demographic and personal information. Children's age was obtained from growth monitoring ('yellow') cards, which the health authorities distributed at birth. If the card was unavailable, the data collector asked the caregiver (self-reported). At the same time, the current breastfeeding status was also obtained.

## 2.8. Anthropometric

Weight, length/height, and MUAC were used to assess nutritional status using the equipment previously described in Sigh et al. [24]. The World Health Organization (WHO) Growth Standards 2006 for 6–59 months [30] to calculate anthropometric z-score weight-for-height (WHZ, wasting) and height-for-age (HAZ, stunting).

<table><caption>**Table 1**

Daily food ration and total lipid intake per RUTFs by child's weight.</caption><thead><tr><th rowspan="2">Weight (kg)</th><th colspan="3">Standard RUTF</th><th colspan="3">Locally RUTF</th></tr><tr><th>RUTF quantity/day (bar)</th><th>RUTF quantity/day (gram)</th><th>Fatty acids (g)<sup>a</sup></th><th>RUTF quantity/day (wafer)</th><th>RUTF quantity/day (gram)</th><th>Fatty acids (g)<sup>a</sup></th></tr></thead><tbody><tr><td>3.0–3.4</td><td>2</td><td>~114</td><td>35.4</td><td>17</td><td>~170</td><td>49.3</td></tr><tr><td>3.5–4.9</td><td>2.5</td><td>~142.5</td><td>44.2</td><td>20</td><td>~200</td><td>58.0</td></tr><tr><td>5.0–6.9</td><td>4</td><td>~228</td><td>70.7</td><td>27</td><td>~270</td><td>78.3</td></tr><tr><td>7.0–9.9</td><td>5</td><td>~285</td><td>88.4</td><td>40</td><td>~400</td><td>116</td></tr><tr><td>10.0–14.0</td><td>6</td><td>~342</td><td>106.0</td><td>53</td><td>~530</td><td>153.7</td></tr></tbody></table>

Abbreviations: RUTF, ready-to-use therapeutic foods; kcal, calories; kg, kilogram.

## 2.9. Blood sampling

At admission to outpatient treatment and discharge after eight weeks of treatment, four millilitres of whole venous blood were drawn from the cubital vein. The four millilitres of whole-blood were collected in a 2 × 2 mL ethylene diamine tetra acetic coated vacutainer and a 6-mL trace element sodium NH heparin vacutainer (VACUETTE®, Greiner Bio-One International GmbH, Germany). Blood from an ethylene diamine tetra acetic vacutainer was used to perform dried blood spot tests to measure fatty acids at admission and discharge from the trial.

## 2.10. Fatty acids analysis of RUTF

The final RUTF products' fatty acid composition was analysed by Célia Moustriés and as described by Moutstiés et al., 2022 [31]. In short, each product was analysed six times. The S-RUTF was analysed 41 months (shelf-life 48 months), and the L-RUTF 6 months (shelf-life 12 months) after production. Each RUTF was tested rapidly at the reception after 3, 6, or 11 months of storage in the field and then stored in the laboratory at ~20 °C for further characterisation [31]. A dose of each sample RUTF was mixed to reach 200 g, to obtain a representative sample for analysis.

Total lipid content was determined in triplicate gravimetrically. After the RUTFs had been thoroughly homogenised, lipids were extracted using chloroform and methanol (2:1) [32]. Each extract was analysed in duplicate. Neutral lipids were separated by one-dimensional migrations using hexane/diethylether/acetic acid (65:35:1, v/v/v). From each sample, 4 µL and 8 µL lipid solution were applied on a thin layer of chromatography (pre using a Camag automatic sampler 4 (Muttenz, Switzerland), which is being controlled by the Wincats 2008 software system. After application, plates were dried for 2 min (70 °C), followed by revealing lipids using a 15.9% copper sulfate in orthophosphoric acid/water (92:8). A laser UV detection system (Camag TLC scanner 3, Muttenz, Switzerland) scanned the plates. The maximum absorption of neutral lipid for wavelength was 500 nm, quantified by spot intensity integration using Wincats software 2008. Standards were used for fatty acids calibration. Beyond fatty acids, vitamin profiles, dry matter and water activity were analysed for each product [31].

## 2.11. Dried blood spots and fatty acid analysis

The dried blood spots protocol and fatty acid analyses have been described in detail previously [26]. In summary, dried blood spot tests were collected using laboratory facilities at the National Pediatric Hospital, following validated procedures [33]. A total of 40 µL whole-blood was applied on chromatography paper (Grade 3 MM, Whatman Ltd.) pre-treated with butylated hydroxytoluene in methanol and deferoxamine mesylate salt in distilled water, dried at room temperature, and stored at 4 °C in a polypropylene

<footnote>a Total lipid content is based on estimate from the manufacturer.</footnote>
'Ziplock' bag for up to 2 months. Samples were shipped to the University of Waterloo in Canada for fatty acid analysis by gas chromatography with flame ionization detection [33–38]. Briefly, fatty acid methyl esters were prepared by direct transesterification by 14% boron-triflouride in methanol with hexane containing 22:3n-3 ethyl ester (internal standard, Nu-CheckPrep, Elysian, MN, USA). Fatty acids results were expressed as a relative percentage of all fatty acids (FA%) and total fatty acid concentration (µg per 100 µL whole-blood).

The inter- and intra-assays for the dried blood spot fatty acid analyses were similar to values previously reported for wet whole-blood analyses [39]. All reported fatty acids results had inter-assay CVs <7%. The intra-assay CVs were <3%, with the higher CVs being associated with the fatty acids with relatively low abundance (i.e. 22:5n-6 and Mead acid).

The omega-3 index, or the sum of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) FA% in erythrocytes [40] was calculated using a conversion factor by Stark et al. [41].

## 2.12. Health markers

Health markers assessed were the presence of anaemia (haemoglobin concentration), α₁-acid glycoprotein (AGP) (progressed inflammation) and C-reactive protein (CRP) (acute inflammation). A Hematology analyser (Sysmex Kx-21, Japan, ID. No. NP021 and NP023) with ~300 µL ethylene diamine tetra acetic was used to assess haemoglobin concentrations. Haemoglobin concentration was used to diagnose anaemia according to cut-off values recommended by WHO [42].

To analyse CRP and AGP, a duplicated combined sandwich ELISA method was used [43]. Control samples' (n=40) inter-assay coefficient of variations were 5.8% (CRP) and 8.1% (AGP). Inflammation was defined by a CRP concentration >5 mg/L and/or AGP concentration >1 g/L [44].

## 2.13. Statistical analysis

All non-biological data collected were directly entered using a tablet and uploaded to an online software system (Kobotoolbox). Biological data were double-entered into EpiData version 3.1 (The EpiData Association, Odense, Denmark). R studio (Windows, version 1.4.1106), a statistical software program, was used for statistical analysis. Descriptive statistics are presented as mean, standard deviations, or percentages and numbers (n). Data were analysed using the children's actual treatment as a consequence of the treatment switch during the trial.

A paired t-test was used to analyse changes in fatty acids % at admission and discharge. An ANCOVA model was used for changes in mean values from admission, identify PUFAs predictors of changes on nutritional markers with adjustment for age, gender, baseline value, compliance to RUTF and breastfeeding at discharge, due to the well-known effect of breastfeeding on especially PUFAs [45–48]. The results are reported as regression coefficients (β) and 95% confidence intervals.

Associations between nutrition and health status markers and PUFAs, a simple or multiple linear regression adjusted for breastfeeding, age and gender was applied. The results are reported as regressions coefficients (β) with 95% confidence intervals and p-values. Only discharge data were used as admission associations have been reported elsewhere [26].

Statistical significance was considered as *p* < 0.05. The association between PUFAs and markers of nutrition status was consistent across variables. The p-values would be overcorrected if adjusted using the Bonferroni method, resulting in a lower association factor

and increased risk of type 2 errors. Therefore, p-values were not adjusted for false discovery rates.

# 3. Results

## 3.1. Trial population

The trial screened 526 children, of which 121 were eligible for enrolment (Fig. 1). The children's characteristics are shown in Table 2. The mean age across the two RUTF groups was 21.2 months. The children in the L-RUTF group were slightly older than children receiving the S-RUTF, which was reflected in a slightly higher proportion of the children still breastfed and marginally lower body weight and height.

## 3.2. Fatty acids analysis of RUTFs

The energy content of S-RUTF and L-RUTF are reported to be 526 kcal/100 g and 506 kcal/100 g, respectively. The estimated total lipid content of the standard RUTF was 31 g/100 g, equal to 52 E%, and that of the L-RUTF was 29 g/100 g and hence 50 E% [25]. The total lipid content L-RUTF was slightly higher in our assessment but also somewhat higher in the S-RUTF (Table 3).

The specific fatty acid comparison of the two RUTFs differed in several fatty acids (Table 3). The S-RUTF had the highest content of total SFA, and n-6 PUFA, while L-RUTF had the highest proportions of AA, ALA, and DHA and the highest n-3 PUFA content, and thus, a lower n-6/n-3 PUFA ratio.

## 3.3. Changes in whole-blood fatty acids during treatment

The children had a Mead acid (20:3n-9) level of around 0.08 FA% and an EPA + DHA in whole blood of ~0.9 ± 0.44 FA% at baseline. Children in the L-RUTF group had an EPA + DHA of 0.94 ± 0.44 FA%, while the S-RUTF group had 1.01 ± 0.43 FA%. The EPA + DHA in whole blood is equivalent to erythrocyte omega-3 index values of 1.64 at baseline and 1.68 in L-RUTF and 1.76 in S-RUTF.

The whole-blood fatty acid composition at discharge reflected the difference in the fatty acid content of the two RUTFs the children had received during the eight-week treatment period. The children given the S-RUTF had higher levels of n-6 PUFA, specifically LA. In contrast, those in the L-RUTFs group had the highest levels of ALA and a lower n-6/n-3 PUFA ratio, but there were no significant group differences for AA or DHA (Table 4). The L-RUTF did not give rise to any within-group changes in whole-blood fatty acid composition, apart from a slight increase in saturated fatty acids (SFA). The S-RUTF group, on the other hand, showed a decrease in MUFA and an increase in n-6 PUFA, mainly LA and AA, during the treatment period. Surprisingly, despite the increase in n-6 PUFA, Mead acid's relative content slightly increased in the S-RUTF group only.

## 3.4. The implication of PUFA status on nutrition and health markers

There were no significant changes in markers of inflammation between admission and discharge in any of the RUTF groups. No significant differences were found in inflammation markers from admission to discharge, although a slight reduction was found (Table 4).

This trial found that total PUFA, n-6 LCPUFA, and AA in whole-blood at discharge were negatively associated with WHZ after treatment (Table 5). The estimate indicates a −0.0014 (−1.4 × 10⁻³) FA%-point decrease in total PUFA was associated with an increase of 1 SD in WHZ. The association seems to be driven by a −0.0008 FA%-point decrease in n-6 LCPUFA, mainly a 0.0006 FA%-point
<figure><img src="image_4.png" /><figcaption>Fig. 1. Flowchart of children enrolment into the trial.</figcaption></figure>

<table><caption>**Table 2**

Characteristics of children enrolled for treatment of SAM who were allocated to the S-RUTF and the L-RUTF group.</caption><thead><tr><th>Characteristics</th><th>S-RUTF (n = 60)</th><th>L-RUTF (n = 61)</th></tr></thead><tbody><tr><td>Age (months)</td><td>20.5 (13.2)</td><td>21.9 (14.3)</td></tr><tr><td>Gender (% (n) female)</td><td>33 (20)</td><td>49 (30)</td></tr><tr><td>Currently breastfeed (% (n))</td><td>50 (30)</td><td>44 (27)</td></tr><tr><td>Nutritional status</td><td></td><td></td></tr><tr><td>WHZ (z-score)<sup>a</sup></td><td>-3.0 (0.7)</td><td>-3.0 (0.5)</td></tr><tr><td>Body weight (kg)</td><td>7.4 (1.9)</td><td>7.6 (1.8)</td></tr><tr><td>HAZ (z-score)</td><td>-2.3 (1.3)</td><td>-2.1 (1.4)</td></tr><tr><td>Height (cm)</td><td>75 (10)</td><td>77 (10)</td></tr><tr><td>MUAC (mm)</td><td>119 (9)</td><td>118 (7)</td></tr><tr><td>Health status</td><td></td><td></td></tr><tr><td>Oedema (% (n))</td><td>0 (0)</td><td>0 (0)</td></tr><tr><td>Haemoglobin (g/dl)</td><td>10.5 (1.2)</td><td>11.1 (1.7)</td></tr><tr><td>Anaemia (% (n))<sup>b</sup></td><td>62 (37)</td><td>48 (29)</td></tr><tr><td>Inflammation markers</td><td></td><td></td></tr><tr><td>Plasma CRP (mg/L)</td><td>4.1 (10.8)</td><td>3.0 (5.5)</td></tr><tr><td>Acute infection (% (n))<sup>c</sup></td><td>14 (8)<sup>b</sup></td><td>19 (10)<sup>b</sup></td></tr><tr><td>Plasma AGP (g/L)</td><td>1.1 (0.9)</td><td>1.0 (0.6)</td></tr><tr><td>Chronic infection (% (n))<sup>c</sup></td><td>40 (23)<sup>b</sup></td><td>40 (21)<sup>b</sup></td></tr><tr><td>Acute and chronic infection (% (n))<sup>c</sup></td><td>11 (6)<sup>b</sup></td><td>17 (9)<sup>b</sup></td></tr></tbody></table>

Data are given as mean with SD or % and the number of children. Abbreviation: RUTF, ready-to-use therapeutic foods; WHZ, weight-for-height z-score; HAZ, height-for-age z-score; MUAC, mid-upper-arm circumference; CRP, C-reactive protein; AGP, α₁-acid glycoprotein.

<table><caption>**Table 3**

Fatty acid comparison of the two ready-to-use therapeutic foods (RUTF).</caption><thead><tr><th>Fatty acids (g/100 g)</th><th>Standard RUTF<sup>a</sup></th><th>Local RUTF<sup>a</sup></th><th>Difference<sup>b</sup></th></tr></thead><tbody><tr><td>Total SFA</td><td>13.6 (0.51)</td><td>10.3 (0.51)</td><td>3.22 (0.72)*</td></tr><tr><td>Total MUFA</td><td>13.0 (0.47)</td><td>12.3 (0.47)</td><td>0.66 (0.67)</td></tr><tr><td>Palmitoleic acid (16:1n-9)</td><td>0.03 (0.01)</td><td>0.14 (0.01)</td><td>-0.11 (0.01)*</td></tr><tr><td>Oleic acid (18:1n-9)</td><td>12.84 (0.48)</td><td>11.81 (0.48)</td><td>1.03 (0.78)</td></tr><tr><td>Gondoic acid (20:1n-9)</td><td>0.09 (0.05)</td><td>0.30 (0.05)</td><td>-0.21 (0.08)**</td></tr><tr><td>Total n-6 PUFA</td><td>5.06 (0.18)</td><td>4.30 (0.18)</td><td>0.76 (0.26)**</td></tr><tr><td>LA (18:2n-6)</td><td>5.02 (0.18)</td><td>4.09 (0.18)</td><td>0.93 (0.25)**</td></tr><tr><td>β-linoleic acid (18:3n-6)</td><td>0.03 (0.00)</td><td>0.04 (0.00)</td><td>-0.02 (0.00)*</td></tr><tr><td>AA (20:4n-6)</td><td>0.01 (0.03)</td><td>0.10 (0.02)</td><td>-0.08 (0.03)**</td></tr><tr><td>Total n-3 PUFA</td><td>0.52 (0.04)</td><td>1.19 (0.04)</td><td>-0.67 (0.06)*</td></tr><tr><td>ALA (18:3n-3)</td><td>0.49 (0.04)</td><td>1.08 (0.04)</td><td>-0.65 (0.05)*</td></tr><tr><td>EPA (20:5n-3)</td><td>0.04 (0.01)</td><td>0.04 (0.01)</td><td>0.01 (0.01)</td></tr><tr><td>DHA (22:6n-3)</td><td>n.d.</td><td>0.03 (0.00)</td><td>-0.02 (0.00)**</td></tr><tr><td>n-6/n-3 PUFA ratio</td><td>9.77 (0.16)</td><td>3.63 (0.16)</td><td>6.14 (0.22)*</td></tr><tr><td>Total fat content (g/100 g)</td><td>37 (1)</td><td>33 (1)</td><td>4 (2)</td></tr></tbody></table>

Values are reported as mean and SD. Abbreviation: RUTF, ready-to-use therapeutic foods; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; LA, Linoleic acid; AA, Arachidonic acid; ALA, α-linoleic acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; n.d., not detected.

<footnote>a Anaemia are based on age-dependent cut-off.</footnote>

<footnote>b Based on available data from n = 57 S-RUTF, n = 53 L-RUTF, and all children n = 110.</footnote>

<footnote>c Acute infection defined as CRP >5 mg/L and chronic infection AGP >1 g/L.</footnote>

<footnote>*p* < 0.05, **p* < 0.001.</footnote>

<footnote>a Average values from 6 samples.</footnote>

<footnote>b Difference with the standard RUTF as the reference product.</footnote>
<table><caption>**Table 4**
Whole-blood fatty acid composition at admission and discharge of children with SAM.</caption><thead><tr><th rowspan="2">Fatty acid (FA%)</th><th colspan="3">L-RUTF</th><th colspan="3">S-RUTF</th><th colspan="2">Difference in change between RUTFs</th></tr><tr><th>Admission (59)</th><th>Discharge (37)</th><th>p-value</th><th>Admission (59)</th><th>Discharge (38)</th><th>p-value</th><th>Estimated<br>difference (SE)</th><th>p-value</th></tr></thead><tbody><tr><td>Total SFA</td><td>45.32 (2.98)</td><td>47.31 (2.81)</td><td><strong>0.001</strong></td><td>45.58 (2.87)</td><td>46.21 (2.81)</td><td>0.256</td><td>1.34 (0.63)</td><td><strong>0.038</strong></td></tr><tr><td>Total MUFA</td><td>29.81 (3.46)</td><td>28.97 (3.44)</td><td>0.898</td><td>30.54 (3.37)</td><td>28.41 (3.45)</td><td><strong>0.015</strong></td><td>0.29 (0.75)</td><td>0.702</td></tr><tr><td>Mead acid (20:3n-9)</td><td>0.07 (0.04)</td><td>0.08 (0.07)</td><td>0.374</td><td>0.08 (0.08)</td><td>0.09 (0.06)</td><td><strong>0.021</strong></td><td>-0.01 (0.02)</td><td>0.654</td></tr><tr><td>LA (18:2n-6)</td><td>14.22 (2.61)</td><td>14.21 (2.44)</td><td>0.727</td><td>13.46 (2.45)</td><td>15.77 (2.87)</td><td><strong>0.006</strong></td><td>-1.54 (0.63)</td><td><strong>0.018</strong></td></tr><tr><td>AA (20:4n-6)</td><td>3.92 (1.29)</td><td>4.01 (1.46)</td><td>0.593</td><td>3.64 (1.26)</td><td>4.29 (1.45)</td><td><strong>0.043</strong></td><td>-0.21 (0.33)</td><td>0.533</td></tr><tr><td>DPA (22:5n-6)</td><td>0.25 (0.10)</td><td>0.24 (0.12)</td><td>0.153</td><td>0.23 (0.09)</td><td>0.24 (0.10)</td><td>0.196</td><td>-0.02 (0.02)</td><td>0.440</td></tr><tr><td>Total n-6 PUFA</td><td>19.83 (3.29)</td><td>19.86 (3.51)</td><td>0.871</td><td>18.75 (2.97)</td><td>21.88 (3.70)</td><td><strong>0.004</strong></td><td>-1.88 (0.85)</td><td><strong>0.031</strong></td></tr><tr><td>ALA (18:3n-3)</td><td>0.22 (0.11)</td><td>0.27 (0.15)</td><td>0.069</td><td>0.21 (0.11)</td><td>0.20 (0.09)</td><td>0.073</td><td>0.08 (0.03)</td><td><strong>0.004</strong></td></tr><tr><td>EPA (20:5n-3)</td><td>0.09 (0.05)</td><td>0.08 (0.05)</td><td>0.094</td><td>0.09 (0.06)</td><td>0.07 (0.05)</td><td><strong>0.036</strong></td><td>0.02 (0.01)</td><td>0.267</td></tr><tr><td>DHA (22:6n-3)</td><td>0.85 (0.41)</td><td>0.83 (0.40)</td><td>0.469</td><td>0.79 (0.38)</td><td>0.93 (0.46)</td><td>0.240</td><td>-0.03 (0.01)</td><td>0.755</td></tr><tr><td>Total n-3 PUFA</td><td>1.59 (0.48)</td><td>1.63 (0.46)</td><td>0.915</td><td>1.51 (0.45)</td><td>1.66 (0.56)</td><td>0.414</td><td>0.03 (0.09)</td><td>0.784</td></tr><tr><td>Mead acid/AA</td><td>0.02 (0.01)</td><td>0.02 (0.02)</td><td>0.176</td><td>0.02 (0.03)</td><td>0.02 (0.01)</td><td>0.648</td><td>0.00 (0.00)</td><td>0.809</td></tr><tr><td>DPA n-6/DHA</td><td>0.34 (0.15)</td><td>0.32 (0.15)</td><td><strong>0.055</strong></td><td>0.34 (0.19)</td><td>0.30 (0.15)</td><td>0.402</td><td>-0.00 (0.02)</td><td>0.854</td></tr><tr><td>n-6/n-3 PUFA ratio</td><td>13.51 (3.76)</td><td>12.80 (2.40)</td><td>0.450</td><td>13.29 (3.07)</td><td>14.43 (3.95)</td><td><strong>0.018</strong></td><td>-1.85 (0.70)</td><td><strong>0.010</strong></td></tr><tr><td>EPA + DHA</td><td>0.94 (0.44)</td><td>0.91 (0.43)</td><td>0.402</td><td>0.87 (0.43)</td><td>1.01 (0.49)</td><td>0.427</td><td>-0.10 (0.15)</td><td>0.510</td></tr><tr><td>Total concentration (µg/100 µL)</td><td>347 (119)</td><td>315 (77)</td><td>0.224</td><td>322 (74)</td><td>323 (82)</td><td>0.672</td><td>-6.55 (18.90)</td><td>0.730</td></tr><tr><td colspan="9">Inflammation markers</td></tr><tr><td>CRP, mg/L</td><td>3.01 (5.45)</td><td>2.81 (5.59)</td><td>0.841</td><td>4.14 (10.78)</td><td>2.11 (5.50)</td><td>0.385</td><td>2.09 (1.22)</td><td>0.091</td></tr><tr><td>AGP, g/L</td><td>0.98 (0.62)</td><td>0.73 (0.47)</td><td>0.216</td><td>1.11 (0.92)</td><td>0.79 (0.63)</td><td>0.186</td><td>0.06 (0.15)</td><td>0.669</td></tr></tbody></table>

Values are reported as mean and SD analysed using paired t-test or estimated difference, SE, and p-values analysed using an ANCOVA model.

AbbreviationFA%, relative percentages of all fatty acids; SE, standard error; SD, standard deviation; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; LA, linoleic acid; AA, arachidonic acid; DPA, docosapentaenoic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; CRP, C-reactive protein; AGP, α₁-acid glycoprotein.

<table><caption>**Table 5**
Association between polyunsaturated fatty acids in whole-blood and markers of health and nutritional status in children after treatment for SAM.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Total PUFA</th><th colspan="6">n-6 polyunsaturated fatty acids</th></tr><tr><th>β</th><th>95% CI</th><th>LA</th><th>95% CI</th><th>AA</th><th>95% CI</th><th>n-6 LCPUFA</th><th>95% CI</th></tr></thead><tbody><tr><td>Nutritional status</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>WHZ, z-score (n 75)<sup>a</sup></td><td>-1.4</td><td>-2.7; -0.1*</td><td>-0.5</td><td>-1.4; 0.5</td><td>-0.6</td><td>-1.0; -0.2*</td><td>-0.8</td><td>-1.3; -0.3*</td><td>-0.25</td><td>-7.7; 2.8</td></tr><tr><td>HAZ, z-score (n 75)<sup>a</sup></td><td>-0.2</td><td>-1.0; 0.5</td><td>0.1</td><td>-0.4; 0.6</td><td>-0.2</td><td>-0.4; 0.1</td><td>-0.3</td><td>-0.6; 0.0</td><td>-2.3</td><td>-5.3; 0.6</td></tr><tr><td>Height, cm (n 75)</td><td>-0.1</td><td>-0.3; 0.2</td><td>0.01</td><td>-0.2; 0.2</td><td>-0.0</td><td>-0.1; 0.1</td><td>-0.1</td><td>-0.2; 0.1</td><td>-1.2</td><td>-2.2; -0.22*</td></tr><tr><td>Health status</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Haemoglobin, g/dl (n 75)</td><td>0.5</td><td>-0.3; 1.2</td><td>0.3</td><td>-0.3; 0.8</td><td>0.2</td><td>-0.1; 0.4</td><td>0.2</td><td>-0.1; 0.5</td><td>-0.8</td><td>-4.0; 2.4</td></tr><tr><td>Inflammation markers</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CRP, mg/L (× 10<sup>-4</sup>) (n 72)</td><td>-0.2</td><td>-0.4; 0.0</td><td>-0.1</td><td>-0.3; 0.0</td><td>-0.03</td><td>-0.1; 0.4</td><td>-0.1</td><td>-0.1; 0.0</td><td>0.0</td><td>-0.9; 0.9</td></tr><tr><td>AGP, g/L (× 10<sup>2</sup>) (n 72)</td><td>-0.8</td><td>-2.5; 0.9</td><td>-0.8</td><td>-1.9; 0.5</td><td>-0.2</td><td>-0.8; 0.4</td><td>-0.1</td><td>-0.9; 0.6</td><td>3.2</td><td>-3.9; 0.0</td></tr><tr><td colspan="11">n-3 polyunsaturated fatty acids</td></tr><tr><td></td><th>EPA<sup>b</sup></th><th>95% CI</th><th>DHA<sup>b</sup></th><th>95% CI</th><th>n-3 LCPUFA<sup>b</sup></th><th>95% CI</th><th>n-6 DPA:DHA<sup>b</sup></th><th>95% CI</th></tr><tr><td>Nutritional status</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>WHZ, z-score (n 75)<sup>a</sup></td><td>-3.2</td><td>-20.2; 13.7</td><td>-104</td><td>-220; 10</td><td>-133</td><td>-272; 5</td><td>7.7</td><td>-34.7; 50.2</td><td></td><td></td></tr><tr><td>HAZ, z-score (n 75)<sup>a</sup></td><td>-10.4</td><td>-19.7; 1.1</td><td>-28.9</td><td>-95.6; 37.8</td><td>-46.7</td><td>-127; 34</td><td>-8.4</td><td>-32.5; 15.8</td><td></td><td></td></tr><tr><td>Height, cm (n 75)</td><td>-2.1</td><td>-0.2; 1.3</td><td>-2.8</td><td>-26.7; 21.3</td><td>-11.0</td><td>-40.0; 18.1</td><td>-3.3</td><td>-12.0; 5.3</td><td></td><td></td></tr><tr><td>Health status</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Haemoglobin, g/dl (n 75)</td><td>-8.4</td><td>-18.7; 1.9</td><td>10.0</td><td>-63.5; 83.4</td><td>-16.3</td><td>-106; 73.0</td><td>13.2</td><td>-13.3; 39.6</td><td></td><td></td></tr><tr><td>Inflammation markers</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CRP, mg/L (× 10<sup>-4</sup>) (n 72)</td><td>-1.6</td><td>-4.4; 1.3</td><td>-14.5</td><td>-34.2; 5.3</td><td>-13.0</td><td>-37.1; 11.1</td><td>1.2</td><td>-6.1; 8.5</td><td></td><td></td></tr><tr><td>AGP, g/L (× 10<sup>2</sup>) (n 72)</td><td>5.9</td><td>-17.5; 29.3</td><td>9.3</td><td>-156; 174</td><td>29.3</td><td>-172; 230</td><td>-32.0</td><td>-87.2; 23.3</td><td></td><td></td></tr></tbody></table>

Data are given as estimated regression coefficients (β) with 95% confidence intervals based on an ANOVA analysis adjusted for age, gender, baseline value, compliance to RUTF and breastfeeding at discharge.

Abbreviations: PUFA, polyunsaturated fatty acids; LA, linoleic acid; AA, arachidonic acids; LCPUFA, long-chain polyunsaturated fatty acid; WHZ, weight-for-height z-score; HAZ, height-for-age z-score; CRP, C-reactive protein; AGP, AGP, α₁-acid glycoprotein; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid.

decrease in AA. There was no association between WHZ and Mead acid:AA ratio but a negative association between Mead acid:AA and age-adjusted height (cm). No statistically significant association was found between n-6 fatty acids and health status markers haemoglobin, CRP and AGP (Table 5). Lastly, no association was found between n-3 PUFAs, nutrition status, or health markers (Table 5).

## 4. Discussion

L-RUTF had a higher content of n-3 PUFA, AA and DHA, while S-RUTF had the highest n-6 PUFA. This resulted in a lower n-6/n-3 PUFA ratio of children in the L-RUTF vs S-RUTF group at discharge. Inflammation did not significantly change, nor was it associated

<footnote><sup>a</sup> Only adjusted for breastfeeding. *p < 0.05.</footnote>

<footnote><sup>b</sup> Results are presented × 10³ due to the low FA% of fatty acid or ratio.</footnote>
with n-6 and n-3 PUFA. Wasting was negatively associated with PUFA, AA, and n-6 LCUFAs.

## 4.1. Changes in PUFAs during treatment of SAM

No recommendations exist for the n-6:n-3 PUFA or LA/ALA ratio [49,50], intake of LCPUFA or in children with SAM. ALA's conversion to DHA is generally low [16]; hence consuming preformed LCPUFA is considered a more efficient way to cover the needs [49]. The L-RUTF in the current trial had higher levels of AA, ALA, and DHA as compared with the S-RUTF due to the use of small fish [47,48] and/or canola oil [51], but there was no significant change in DHA and AA blood levels after L-RUTF feed. The levels of DHA and AA were likely not high enough to change whole-blood levels.

Overall, low whole-blood PUFAs at admission and discharge, independent of RUTF consumed, were found, although the n-6/n-3 ratio was 9.8 (S-RUTF) and 3.6 (L-RUTF). A study among Ugandan children with SAM [22] has also shown that the current therapeutic foods did not correct whole-blood levels of LCPUFA after eight weeks of SAM treatment in an inpatient or outpatient setting. They also found that the children did not reach the same levels as either healthy control of similar age or improved children with SAM-compromised PUFA levels [22]. Standard RUTFs have previously been associated with declining DHA blood status during the nutrition rehabilitation of SAM [26,52,53], which was also the case in the study from Uganda. AA increased significantly during the treatment of children receiving the S-RUTF, whereas a non-significant increase was found for the L-RUTF, although this contained some preformed AA.

Generally, after treatment for SAM, Cambodian children had very low n-6 PUFA markers (Mead acid:AA ratio), like that of well-nourished Ugandan children, but a better status than children treated for SAM in Uganda. A very low n-3 PUFA was observed among all children treated for SAM (n-6 DPA:DHA ratio and n-6:n-3 PUFA ratio) which did not appear to reach the same level as well-nourished Ugandan children. This was also shown in a generally low whole-blood omega-3 index ranging from 0.91 ± 0.43 (L-RUTF discharge) to 1.01 ± 0.40 FA% (S-RUTF discharge), which is considerably low compared to whole-blood omega-3 index results from Europe, Scandinavia and North America ranging from 2.21 to 4.16 [54].

Generally, children with SAM in Cambodia and Uganda had lower total PUFA, n-6 PUFA and n-3 PUFA than well-nourished children, indicating a low impact of RUTFs during treatment in correcting PUFA levels.

Cambodian children had the lowest FA% of n-3 PUFAs and were about 0.9 FA% lower than well-nourished children, similar to Ugandan children treated for SAM. A low DHA primarily drove this after Cambodian children were treated with RUTF. This could be due to Cambodian children may consume lower quantities of preformed LCPUFAs (e.g. DHA). Lower n-3 PUFA's could also be contributed to a low conversion of LA and ALA to their LCPUFAs. This may be further limited by the competing fatty acid desaturase pathway, mineral deficiencies, or a high carbohydrate intake, resulting in a decreased activity of Δ6 desaturase [55,56] in Cambodian children compared to children from Uganda.

## 4.2. PUFA marker's potential implication on health status

Insufficient PUFA intakes during the treatment of SAM could impair linear growth and weight gain, which are critical for successful outcomes of SAM treatment. Therefore, studies assess the effectiveness of RUTFs on various indicators such as weight gain [24,57,58], recovery [59–65], or combined several indicators [66–73]. This research group has previously shown an association

between stunting, height and low n-6 PUFAs [26]. This trial found a negative association between height and Mead acid:AA ratio at discharge, indicating that a high n-6 PUFA could be important in preventing stunting.

At discharge, a consistent negative association was found between WHZ, the indicator for wasting, and total PUFA, AA, and n-6 LCPUFA indicating lower FA% of these fatty acids with higher WHZ. Similarly, no associations were found at admission [26]. A Ugandan study among 6-59-month-old children found no associations between WHZ and PUFAs [74]. Neither did a study among 2-6-year-old children from North Ghana [75] and South Ghana [76]. Like this trial, a study in Tanzania among 2-6-year-old children showed a negative association between WHZ and AA (−0.16, *p* = 0.009) [77].

In the current trial, the proportion of children with elevated CRP – acute infection decreased from 16% to 11% and chronic infection – elevated AGP decreased from 40% to 21% during the treatment of SAM. The decrease in whole-blood total PUFA and n-6 PUFAs among less wasted children may not be related to wasting per se but disease processes such as infections in children with SAM.

Whole-blood PUFAs reflect dietary intake and the metabolic processes involved in PUFA mobilisation and utilisation. Children treated for SAM progress from a catabolic to an anabolic state, thus requiring significant PUFA amounts for deposition in growing tissue and the eicosanoid biosynthesis, an essential regulatory function of our immune system [18]. Therefore, AA, n-6 LCPUFA and PUFAs may generally be mobilised to the cell membranes of tissues such as adipose, muscle, nervous and brain tissue as SAM subsides as recovery begins and growth of these tissues resumes during the treatment of SAM. Therefore, increases in PUFA intake may not be reflected in whole-blood during weight gain. An ad hoc analysis (Table S2 and S3, supplementary file) may indicate this might be the case. The analysis found a negative association between changes in weight gain from admission to discharge during treatment of SAM with changes in AA, n-6 DPA, EPA and DHA.

## 4.3. Implication on the nutritional composition of RUTFs

The conversion between LA and ALA to LCPUFA is complex [78–80]. In this trial, the estimated n-6 and n-3 E% for the S-RUTF were 8.7 E% and 0.9E%, respectively. For the L-RUTF, 7.2 E% was from n-6 fatty acids, and 2 E% was from n-3 fatty acids. Despite the higher E% of n-3 fatty acids in the L-RUTF, no statistically significant changes were observed between the two RUTFs after treatment. Contrary to the higher E% of n-6 in S-RUTF resulted in a significantly higher total PUFA at discharge (p=0.004), which was also considered a larger change compared with L-RUTF (p=0.031). The previous recommendation for 100 g RUTFs was that 3–10 E% and 0.3–2.5 E% should come from n-6 and n-3 fatty acids, respectively [50]. The specification was recently updated to 3–7 E% and 1-2.5E% for n-6 and n-3 fatty acids, respectively [81,82]. No larger changes were made in the fatty acid composition of RUTFs. Still, it allows a broad range of lipid compositions with high LA and low ALA levels without any requirements for preformed LCPUFA, particularly EPA and DHA.

The low impact of RUTFs in improving essential fatty acid levels calls for actions to ensure that health-compromised children can fully recover from undernourishment, sparing them from the potential long-term consequences of their development of having compromised essential fatty acid levels.

## 4.4. Limitations

The trial experienced a low attendance rate at discharge, resulting in a low sample size, although it was similar for both RUTFs [24]. The trial shares the limitation of observational studies,
which identify potential associations but not causality. Hence, it's not possible to determine the cause and effect between these nutritional markers and PUFAs. There is a risk of confounders that could impact the association, for example, children's diet, weight gain, or tissue build-up; hence, these associations must be interpreted with caution. It is important to note that the trial present secondary outcomes, hence the sample size calculation was not calculated to assess changes in PUFA status. The power might be too low especially considering the very small effect size. Another important note is the effect size and its relevance in practice where small observed effect sizes have limited practical implications. Therefore, there is a need to confirm the findings in a trial where changes in PUFA is the primary outcome.

## 5. Conclusion

The trial found that the whole-blood PUFA markers of the children was low at admission and discharged from SAM treatment. Thus, the currently recommended composition of RUTFs for rehabilitating children with SAM does not appear to correct their compromised contribution of whole-blood LCPUFAs especially combined with intake of other foods. The higher DHA and AA content in the L-RUTF did not improve PUFA status, being of concern due to their important role in the brain, immune system, and retina.

The FA% of LCPUFA at discharge may be a predictor of wasting, while no markers of inflammation neither changed during treatment nor are associated with PUFAs.

## Funding statement

The work was supported by UNICEF's national committees (Australia, the Republic of Korea, and Hong Kong, 2015–2016), the University of Copenhagen, Denmark (2015–2019), Neys-van Hoogstraten Foundation, Netherlands (Grant ID; CA271, 2016–2019), and the Institute de Recherché pour le Développement, France (2015–2019). The funding sources did not influence the trial design, data collection and analysis, or interpretation of the results.

## Author contribution

**Sanne Sigh:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Visualization, Project administration, Funding acquisition. **Lotte Lauritzen:** Methodology, Validation, Writing – original draft, Visualization, Project administration. **Frank T. Wieringa:** Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Supervisions, Project administration, Funding acquisition. **Arnaud Laillou:** Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Supervisions, Project administration, Funding acquisition. **Chhoun Chamnan:** Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Supervisions, Project administration. **Ken D. Stark:** Validation, Resources, Data curation, Writing – review & editing. **Nanna Roos:** Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Visualization, Supervision, Project administration, and Funding acquisition.

## Conflict of Interest

The authors declare no conflicts of interest. The authors' senti- ments do not reflect their organisations' official policies or opinions.

## Acknowledgements

The authors appreciate the National Pediatrics Hospital's collaboration and support in conducting the study. A deep and sincere thanks to all caregivers, participating children, and the data collection team for their participation in the study and efforts in collecting data. The research could not have been completed without them.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.clnu.2023.08.003.

## References

[1] United Nations Children's Fund (UNICEF). World health organization, international bank for reconstruction and development/the world bank. Levels and trends in child malnutrition: key findings of the 2019 edition of the joint child malnutrition estimates. Geneva: World Health Organization; 2019. Licence: CC BY-NC-SA 3.0 IGO.

[2] Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis M, et al. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet 2013;382(9890):427–51. https://doi.org/10.1016/S0140-6736(13)60937-X.

[3] Olofin I, McDonald CM, Ezzati M, Flaxman S, Black RE, Fawzi WW, et al. Associations of suboptimal growth with all-cause and cause-specific mortality in children under five years: a pooled analysis of ten prospective studies. PLoS One 2013;8(5):e64636. https://doi.org/10.1371/journal.pone.0064636.

[4] Page AL, de Rekeneire N, Sayadi S, Aberrane S, Janssens AC, Rieux C, et al. Infections in children admitted with complicated severe acute malnutrition in Niger. PLoS One 2013;8(7):e68699. https://doi.org/10.1371/journal.pone.0068699.

[5] Berkley JA, Ngari M, Thitiri J, Mwalekwa L, Timbwa M, Hamid F, et al. Daily co-trimoxazole prophylaxis to prevent mortality in children with complicated severe acute malnutrition: a multicentre, double-blind, randomised placebo-controlled trial. Lancet Global Health 2016;4:e464–73. https://doi.org/10.1016/S2214-109X(16)30096-1.

[6] Attia S, Verslott CJ, Voskuilj W, Vliet SJV, Giovanni VD, Zhang L, et al. Mortality in children with complicated severe acute malnutrition is related to intestinal and systemic inflammation: an observational cohort study. Am J Clin Nutr 2016;104(5):1441–9. https://doi.org/10.3945/ajcn.116.130518.

[7] Njunge JM, Gwela A, Kibinge NK, Ngari M, Nyamako L, Nyatichi E, et al. Biomarkers of post-discharge mortality among children with complicated severe acute malnutrition. Sci Rep 2019;9(1):5981. https://doi.org/10.1038/s41598-019-42436-y.

[8] Versloot CJ, Attia S, Bourdon C, Richardson SE, Potani I, Bandsma RHJ, et al. Intestinal pathogen clearance in children with severe acute malnutrition is unrelated to inpatient morbidity. Clin Nutr ESPEN 2018;24:109–13. https://doi.org/10.1016/j.clnesp.2018.01.004.

[9] Bourke CD, Berkley JA, Prendergast AJ. Immune dysfunction as a cause and consequence of malnutrition. Trends Immunol 2016;37(6):386–98. https://doi.org/10.1016/j.it.2016.04.003.

[10] Holman RT, Johnson SB, Mercuri O, Itarte HJ, Rodrigo MA, de Tomas ME. Essential fatty acid deficiency in malnourished children. Am J Clin Nutr 1981;34(8):1534–9. https://doi.org/10.1093/ajcn/34.8.1534.

[11] Pourhassan M, Cederholm T, Trampisch U, Volkert D, Wirth R. Inflammation as a diagnostic criterion in the GLIM definition of malnutrition—what CRP-threshold relates to reduced food intake in older patients with acute disease? Eur J Clin Nutr 2022;76:397–400. https://doi.org/10.1038/s41430-021-00977-4.

[12] Brown KH. Diarrhea and malnutrition. J Nutr 2003;133(1):328s–32s. https://doi.org/10.1093/jn/133.1.328s.

[13] Crane RJ, Jones K Dj, Berkley JA. Environmental enteric dysfunction: an overview. Food Nutr Bull 2015;36(1):S76–87. https://doi.org/10.1177/15648265150361S113.

[14] Smit EN, Muskie FAJ, Boersma ER. The possible role of essential fatty acids in the pathophysiology of malnutrition: a review. Prostaglandins Leukot Essent Fatty Acids 2004;71:241–50. https://doi.org/10.1016/j.plefa.2004.03.019.

[15] Friend A, Akomo P, Bahwere P, De Pee S, Dibari F, Golden MH, et al. Developing food supplements for moderately malnourished children: lessons learned from ready-to-use therapeutic foods. Food Nutr Bull 2015;36(1 Suppl):S53–8. https://doi.org/10.1177/15648265150361S109.

[16] Lauritzen L, Hansen HS, Jürgensen MH, Michaelsen KF. The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Prog Lipid Res 2001;40(1-2):1–94. https://doi.org/10.1016/s0163-7827(00)00017-5.

[17] Jacobson JL, Jacobson SW, Muckle G, Kaplan-Estrin M, Ayotte P, Dewailly E. Beneficial effects of a polyunsaturated fatty acid on infant development:
evidence from the inut of arctic quebec. J Pediatr 2008;152(3):356-64. https://doi.org/10.1016/j.jpeds.2007.07.008.

[18] Ratnayake WNM, Galli C. Fat and fatty acid terminology, methods of analysis and fat digestion and metabolism: a background review paper. Ann Nutr Metab 2009;55(1-3):8-43. https://doi.org/10.1159/00022894.

[19] Pawlosky RJ, Hibbeln JR, Novotny JA, Salem N. Physiological compartmental analysis of α-linolenic acid metabolism in adult humans. J Lipid Res 2001;42(8):1257-65. https://doi.org/10.1016/s0022-2275(20)31576-5.

[20] Raphael W, Sordillo LM. Dietary polyunsaturated fatty acids and inflammation: the role of phospholipid biosynthesis. Int J Mol Sci 2013;14(10): 21167-88. https://doi.org/10.3390/ijms141021167.

[21] Calder PC. N-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr 2006;83(6 Suppl):1505S-19S. https://doi.org/10.1093/ajcn/83.6.1505S. PMID: 16841861.

[22] Babirekere-Iriso E, Mortensen CG, Mupere E, Rytter MJH, Namusoke H, Michaelsen KF, et al. Changes in whole-blood PUFA and their predictors during recovery from severe acute malnutrition. Br J Nutr 2016;115(10): 1730-9. https://doi.org/10.1017/S0007114516000817.

[23] Shokry E, Sadiq K, Soofi S, Habib A, Bhutto N, Rizvi A, et al. Impact of treatment with RUTF on plasma lipid profiles of severely malnourished Pakistani children. Nutrients 2020;12(7):1-23. https://doi.org/10.3390/nu12072163.

[24] Sigh S, Roos N, Chamnan C, Laillou A, Prak S, Wieringa FT. Effectiveness of a locally produced, fish-based food product on weight gain among Cambodian children in the treatment of acute malnutrition: a randomised controlled trial. Nutrients 2018;10(7):909. https://doi.org/10.3390/nu10070909.

[25] Sigh S, Roos N, Sok D, Borg B, Chamnan C, Laillou A, et al. Development and acceptability of locally made fish-based, ready-to-use products for the prevention and treatment of malnutrition in Cambodia. Food Nutr Bull 2018;39(3):420-34. https://doi.org/10.1177/0379572118788266.

[26] Sigh S, Lauritzen L, Wieringa FT, Laillou A, Chamnan C, Angkeabos N, et al. Whole-blood PUFA and associations with markers of nutritional and health status in acutely malnourished children in Cambodia. Publ Health Nutr 2020;23(6):974-86. https://doi.org/10.1017/S1368980019003744.

[27] Prudhon C, Briand A, Weise Prinzo Z, Meg Daelmans B, WHO, UNICEF, and SCN informal consultation on community-based management of severe malnutrition in children management of severe malnutrition in children. Food Nutr Bull 2006:1-108.

[28] Briand A, Prinzo ZW. Dietary management of moderate malnutrition: time for a change. Food Nutr Bull 2009;30(3):s265-6. https://doi.org/10.1177/15648265090303S01.

[29] Harris S, Jack S. Home-based treatment of acute malnutrition in Cambodian urban poor communities. Food Nutr Bull 2011;32(4):333-9. https://doi.org/10.1177/156482651103200404.

[30] World Health Organization (WHO), United Nations Children's Funds (UNICEF). WHO child growth standards and the identification of severe acute malnutrition in infants and children. A Joint Statement by World Health Organization and the United Nations Children's Fund. Geneva: World Health Organization; 2009.

[31] Moustiés C, Bourlieu-Lacanal C, Hemery YM, Bareá B, Villeneuve P, Servent A, et al. Nutritional quality of Ready-to-Use Therapeutic Foods: focus on lipid composition and vitamin content. OCL - Oil Seeds and Fats, Crops and Lipids 2022;29(5):13. https://doi.org/10.1051/ocl/2022007.

[32] Folch J, Lees M, Sloane GH. A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226(1):497-509.

[33] Metherel AH, Aristizabal Henao JJ, Stark KD. EPA and DHA levels in whole blood decrease more rapidly when stored at -20 C as compared with room temperature, 4 and -75 C. Lipids 2013;48(11):1079-91. https://doi.org/10.1007/s11745-013-3827-x.

[34] Metherel AH, Stark KD. Cryopreservation prevents iron-initiated highly unsaturated fatty acid loss during storage of human blood on chromatography paper at 20°C. J Nutr 2015;145(3):654-60. https://doi.org/10.3945/jn.114.203679.

[35] Marangoni F, Colombo C, Galli C. A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies. Anal Biochem 2004;326(2):267-72. https://doi.org/10.1016/j.ab.2003.12.016.

[36] Metherel AH, Taha AY, Izadi H, Stark KD. The application of ultrasound energy to increase lipid extraction throughput of solid matrix samples (flaxseed). Prostaglandins Leukot Essent Fatty Acids 2009;81(5-6):417-23. https://doi.org/10.1016/j.plefa.2009.07.003.

[37] Metherel AH, Buzikievich LM, Charkhzarin P, Patterson AC, Peel AC, Howorth AM, et al. Omega-3 polyunsaturated fatty acid profiling using fingertip-prick whole blood does not require overnight fasting before blood collection. Nutr Res 2012;32(8):547-56. https://doi.org/10.1016/j.nutres.2012.06.016.

[38] Armstrong JM, Metherel AH, Stark KD. Direct microwave transesterification of fingertip prick blood samples for fatty acid determinations. Lipids 2008;43(2): 187-96. https://doi.org/10.1007/s11745-007-3141-6.

[39] Lauritzen L, Sørensen LB, Harsløf LB, Ritz C, Stark KD, Astrup A, et al. Mendelian randomisation shows sex-specific associations between long-chain PUFA-related genotypes and cognitive performance in Danish schoolchildren. Am J Clin Nutr 2017;106(1):88-95. https://doi.org/10.3945/ajcn.

[40] Harris WS. Omega-3 fatty acids and cardiovascular disease: a case for omega-3 index as a new risk factor. Pharmacol Res 2007;55(3):217-23. https://doi.org/10.1016/j.PHRS.2007.01.013.

[41] Stark KD, Aristizabal Henao JJ, Metherel AH, Pilote L. Translating plasma and whole blood fatty acid compositional data into the sum of eicosapentaenoic and docosahexaenoic acid in erythrocytes. Prostaglandins Leukot Essent Fatty Acids 2016;104:1-10. https://doi.org/10.1016/j.plefa.2015.11.002.

[42] World Health Organization. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and Mineral Nutrition Information System. Geneva: World Health Organization; 2011 (WHO/NMH/NHD/MNM/11.1).

[43] Erhardt JG, Estes JE, Pfeiffer CM, Biesalski HK, Craft NE. Nutritional methodology combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique. J Nutr 2004;134(11):3127-32.

[44] Thurnham DI, McCabe LD, Haldar S, Wieringa FT, Northrop-Clewes CA, McCabe GP. Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in the assessment of iron deficiency: a meta-analysis. Am J Clin Nutr 2010;92(3):546-55. https://doi.org/10.3945/ajcn.2010.29284.

[45] Yaméogo CW, Cichon B, Fabiansen C, Rytter MJH, Faurholt-Jepsen D, Stark KD, et al. Correlates of whole-blood polyunsaturated fatty acids among young children with moderate acute malnutrition. Nutr J 2017;16(1):44. https://doi.org/10.1186/s12937-017-0264-3.

[46] Dalmeijer GW, Wijga AH, Gehring U, Renders CM, Koppelman GH, Smit HA, et al. Fatty acid composition in breastfeeding and school performance in children aged 12 years. Eur J Nutr 2016;55(7):2199-207. https://doi.org/10.1007/s00394-015-1030-y.

[47] Thiel I, Koletzko Kinderpoliklinik der B, Berthold Koletzko P. Essential fatty acids in full term infants fed breast milk or formula. Arch Dis Child Fetal Neonatal Ed 1995;72(1):F23-8. https://doi.org/10.1136/fn.72.1.f23.

[48] Nurhasan M, Roos N, Skau JK, Wieringa FT, Friis H, Michaelsen KF, et al. Effect of complementary food with small amounts of freshwater fish on whole blood n-3 fatty acids in Cambodian infants age 6-15 months. Prostaglandins Leukot Essent Fatty Acids 2018;135:92-101. https://doi.org/10.1016/j.plefa.2018.07.002.

[49] Michaelsen KF, Dewey KG, Perez-Exposito AB, Nurhasan M, Lauritzen L, Roos N. Food sources and intake of n-6 and n-3 fatty acids in low-income countries with emphasis on infants, young children (6-24 months), and pregnant and lactating women. Matern Child Nutr 2011;7(Suppl 2):124-40. https://doi.org/10.1111/mc.1740-8709.2011.00302.x.

[50] World Health Organization; World Food Programme. United nation system standing committee on nutrition; the united nations children's fund. Community-based management of severe acute malnutrition. Geneva, Switzerland: World Health Organization; 2007. p. 1-8.

[51] Aukema H, Campbell L. Chapter 9 - oil nutrition and utilization. Canola Chemistry, Production, Processing, and Utilisation 2011;245-80.

[52] Jones KD, Ali R, Khasira MA, Odera D, West AL, Koster G, et al. Ready-to-use therapeutic food with elevated n-3 polyunsaturated fatty acid content, with or without fish oil, to treat severe acute malnutrition: a randomised controlled trial. BMC Med 2015;13:93. https://doi.org/10.1186/s12916-015-0315-6.

[53] Hsieh JC, Liu L, Zeilani M, Ickes S, Trehan I, Maleta K, et al. High-oleic ready-to-use therapeutic food maintains docosahexaenoic acid status in severe malnutrition. J Pediatr Gastroenterol Nutr 2015;61(1):138-43. https://doi.org/10.1097/MPG.0000000000000741.

[54] Stark KD, van Elswyk ME, Higgins MR, Weatherford CA, Salem N. Global survey of the omega-3 fatty acids, docosahexaenoic acid and eicosapentaenoic acid in the blood stream of healthy adults. Prog Lipid Res 2016;63:132-52. https://doi.org/10.1016/j.plipres.2016.05.001.

[55] Ayala A, Brenner R. Essential fatty acid status in zinc deficiency. Effect on lipid and fatty acid composition, desaturation activity and structure of miscrosomal membranes of rat liver and testes. Acta Physiol Lat Am 1983;33(3): 193-204.

[56] Koletzko B, Abiodun PO, Laryea MD, Bremer H. Fatty acid composition of plasma lipids in Nigerian children with protein-energy malnutrition. Eur J Pediat 1986;145(1-2):109-15. https://doi.org/10.1007/BF00441868.

[57] Hadji E, Diop I, Dossou NI, Ndou MM, Briand A, Wade C. Comparison of the efficacy of a solid ready-to-use food and a liquid, milk-based diet for the rehabilitation of severely malnourished children: a randomised trial. Am J Clin Nutr 2003;78(2):302-7. https://doi.org/10.1093/ajcn/78.2.302.

[58] Shewade HD, Patro BK, Bharti B, Soundappan K, Kaur A, Taneja N. Effectiveness of indigenous ready-to-use therapeutic food in community-based management of uncomplicated severe acute malnutrition: a randomised controlled trial from India. J Trop Pediatr 2013;59(5):393-8. https://doi.org/10.1093/tropej/fmt039.

[59] Bhandari N, Mohan SB, Bose A, Lyengar SD, Taneja S, Mazumder S, et al. Efficacy of three feeding regimens for home-based management of children with uncomplicated severe acute malnutrition: a randomised trial in India. BMJ Glob Health 2016;1(4):e000144. https://doi.org/10.1136/bmjgh-2016.

[60] Ciliberto MA, Sandige H, Ndekha MJ, Ashorn P, Briand A, Ciliberto HM, et al. Comparison of home-based therapy with ready-to-use therapeutic food with standard therapy in the treatment of malnourished Malawian children: a controlled, clinical effectiveness trial. Am J Clin Nutr 2005;81(4):864-70.

[61] Manary MJ, Ndekha MJ, Ashorn P, Maleta K, Briand A. Home based therapy for severe malnutrition with ready-to-use food. Arch Dis Child 2004;89(6): 557-61. https://doi.org/10.1136/adc.2003.034306.
[62] Manary M, Chang CY. Comparing milk fortified corn-soy blend (CSB++), soy ready-to-use supplementary food (RUSF), and soy/whey RUSF (supplementary Plumpy®) in the treatment of moderate acute malnutrition. FANTA project; 2012.

[63] LaGrone LN, Trehan I, Meuli GJ, Wang RJ, Thakwalakwa C, Maleta K, et al. A novel fortified blended flour, corn-soy blend "plus-plus," is not inferior to lipid-based ready-to-use supplementary foods for the treatment of moderate acute malnutrition in Malawian children. Am J Clin Nutr 2012;95(1):212-9. https://doi.org/10.3945/ajcn.111.022525.

[64] Sandige H, Ndekha MJ, Briend A, Ashorn P, Manary MJ. Home-based treatment of malnourished malawian children with locally produced or imported ready-to-use food. J Pediat Gastroenterol Nutr 2004;39(2):141-6. https://doi.org/10.1097/00005176-200408000-00003.

[65] Linneman Z, Matilsky D, Ndekha M, Manary MJ, Maleta K, Manary MJ. A large-scale operational study of home-based therapy with ready-to-use therapeutic food in childhood malnutrition in Malawi. Matern Child Nutr 2007;3(3):206-15. https://doi.org/10.1111/j.1740-8709.2007.00095.x.

[66] Bahwere P, Banda T, Sadler K, Nyirenda G, Owino V, Shaba B, et al. Efficacy of milk whey protein-based ready-to-use therapeutic food in treatment of severe acute malnutrition in Malawian under-5 children: a randomised, double-blind, controlled non-inferiority clinical trial. Matern Child Nutr 2014;10(3):436-51. https://doi.org/10.1111/mcn.12112.

[67] Nackers F, Broillet F, Oumarou D, Djibo A, Gabaulaud V, Guerin PJ, et al. Efficacy of ready-to-use therapeutic food compared to a corn/soy-blend-based pre-mix for the treatment of childhood moderate acute malnutrition in Niger. J Trop Pediatr 2010;56(6):407-13. https://doi.org/10.1093/tropej/fmq019.

[68] Gabaulaud V, Dan-Bouzoua N, Brasher C, Fedida G, Gergonne B, Brown V. Could nutritional rehabilitation at home complement or replace centre-based therapeutic feeding programmes for severe malnutrition? J Trop Pediatr 2007;53(1):49-51. https://doi.org/10.1093/tropej/fml052.

[69] Ashworth A. Efficacy and effectiveness of community-based treatment of severe malnutrition. Food Nutr Bull 2006;27(3 Suppl):S24-48. https://doi.org/10.1177/15648265060273S303.

[70] Tadesse AW, Tadesse E, Berhane Y, Ekström EC. Choosing anthropometric indicators to monitor the response to treatment for severe acute malnutrition in rural southern Ethiopia—empirical evidence. Nutrients 2017;9(12):1339. https://doi.org/10.3390/nu9121339.

[71] Amthor RE, Cole SM, Manary MJ. The use of home-based therapy with ready-to-Use Therapeutic food to treat malnutrition in a rural area during a food crisis. J Am Diet Assoc 2009;109(3):464-7. https://doi.org/10.1016/j.jada.2008.11.028.

[72] Schoonees A, Lombard M, Musekiwa A, Nel E, Volmink J. Ready-to-use therapeutic food for home-based treatment of severe acute malnutrition in children from six months to five years of age. Cochrane Database Syst Rev 2013;6. https://doi.org/10.1002/14651858.CD009000.pub2.

[73] Yebyo HG, Kendall C, Nigusse D, Lemma W. Outpatient therapeutic feeding program outcomes and determinants in treatment of severe acute malnutrition in tigray, northern Ethiopia: a retrospective cohort study. PLoS One 2013;8(6):e65840. https://doi.org/10.1371/journal.pone.0065840.

[74] Babirekere-Iriso E, Lauritzen L, Mortensen CG, Rytter MJH, Mupere E, Namusoke H, et al. Essential fatty acid composition and correlates in children with severe acute malnutrition. Clin Nutr ESPEN 2016;11:e40-6. https://doi.org/10.1016/j.clnesp.2015.12.001.

[75] Adjepong M, Austin Pickens C, Jain R, Harris WS, Annan RA, Fenton JI. Association of whole blood n-6 fatty acids with stunting in 2-to-6-year-old Northern Ghanaian children: a cross-sectional study. PLoS One 2018;13(3):e0193301.

[76] Adjepong M, Yakah W, Harris WS, Colecraft E, Marquis GS, Fenton JI. Association of whole blood fatty acids and growth in southern ghanaian children 2-6 years of age. Nutrients 2018;10(8):954. https://doi.org/10.3390/nu10080954.

[77] Jumbe T, Comstock SS, Hahn SL, Harris WS, Kinabo J, Fenton JI. Whole blood levels of the n-6 essential fatty acid linoleic acid are inversely associated with stunting in 2-to-6 year old Tanzanian children: a cross-sectional study. PLoS One 2016;11(5):e0154715. https://doi.org/10.1371/journal.pone.0154715.

[78] Jones PJH, Rideout T. Chapter 4: lipids, sterols, and their metabolites. In: Shils ME, Shike M, editors. Modern nutrition in health and disease. 11th ed. Ross AC, Caballero B, Cousins RJ: Lippincott Williams and Wilkins; 2014. p. 65-88.

[79] Innis SM. Essential fatty acid transfer and fetal development. Placenta 2005;26:70-5. https://doi.org/10.1016/j.placenta.2005.01.005.

[80] Pawlosky RJ, Hibbeln JR, Novotny JA, Salem N. Physiological compartmental analysis of α-linolenic acid metabolism in adult humans. J Lipid Res 2001;42(8):1257-65. https://doi.org/10.1016/s0022-2275(20)31576-5.

[81] Caron O, Fleet A. RUTF specification and codex updates. UNICEF & MSF presentation; 2022. at, https://www.unicef.org/supply/media/11061/file/codex-and-rutf-specification-updates.pdf. [Accessed 4 March 2023].

[82] World Health Organization. Food and agriculture organization of the united nations. Joint FAO/WHO food standards programme codex alimentarius commission. Geneva: World Health Organization; 2021.