Improved residual fat malabsorption and growth in children with cystic fibrosis treated with a novel oral structured lipid supplement: A randomized controlled trial

Virginia A Stallings; Alyssa M Tindall; Maria R Mascarenhas; Asim Maqbool; Joan I Schall

doi:10.1371/journal.pone.0232685

. 2020 May 8;15(5):e0232685. doi: 10.1371/journal.pone.0232685

Improved residual fat malabsorption and growth in children with cystic fibrosis treated with a novel oral structured lipid supplement: A randomized controlled trial

Virginia A Stallings ^1,^2,^*, Alyssa M Tindall ¹, Maria R Mascarenhas ^1,², Asim Maqbool ^1,², Joan I Schall ¹

Editor: Maret G Traber³

PMCID: PMC7209323 PMID: 32384122

Abstract

Background

In the primary analysis of a 12-month double-blind randomized active placebo-controlled trial, treatment of children with cystic fibrosis (CF) and pancreatic insufficiency (PI) with a readily absorbable structured lipid (Encala™, Envara Health, Wayne, PA) was safe, well-tolerated and improved dietary fat absorption (stool coefficient of fat absorption [CFA]), growth, and plasma fatty acids (FA).

Objective

To determine if the Encala™ treatment effect varied by severity of baseline fat malabsorption.

Methods

Subjects (n = 66, 10.5±3.0 yrs, 39% female) with baseline CFA who completed a three-month treatment with Encala™ or a calorie and macronutrient-matched placebo were included in this subgroup analysis. Subjects were categorized by median baseline CFA: low CFA (<88%) and high CFA (≥88%). At baseline and 3-month evaluations, CFA (72-hour stool, weighed food record) and height (HAZ), weight (WAZ) and BMI (BMIZ) Z-scores were calculated. Fasting plasma fatty acid (FA) concentrations were also measured.

Results

Subjects in the low CFA subgroup had significantly improved CFA (+7.5±7.2%, mean 86.3±6.7, p = 0.002), and reduced stool fat loss (-5.7±7.2 g/24 hours) following three months of Encala^TM treatment. These subjects also had increased plasma linoleic acid (+20%), α-linolenic acid (+56%), and total FA (+20%) (p≤0.005 for all) concentrations and improvements in HAZ (0.06±0.08), WAZ (0.17±0.16), and BMIZ (0.20±0.25) (p≤0.002 for all). CFA and FA were unchanged with placebo in the low CFA group, with some WAZ increases (0.14±0.24, p = 0.02). High CFA subjects (both placebo and Encala™ groups) had improvements in WAZ and some FA.

Conclusions

Subjects with CF, PI and more severe fat malabsorption experienced greater improvements in CFA, FA and growth after three months of Encala™ treatment. Encala™ was safe, well-tolerated and efficacious in patients with CF and PI with residual fat malabsorption and improved dietary energy absorption, weight gain and FA status in this at-risk group.

Introduction

The treatment of dietary fat malabsorption to optimize growth and nutritional status in patients with cystic fibrosis (CF) and pancreatic insufficiency (PI) is a key challenging component of standard clinical care[1]. Although CFTR modulators have shown great promise improving nutritional status[2], there is limited evidence on the effect of modulators on fat absorption. One study reported an improvement in fat absorption with Ivacaftor therapy[3], but there are no data in individuals with more severe (non-gating) mutations. Even with recommended doses of pancreatic enzyme medications, residual dietary fat malabsorption is common and contributes to weight and stature growth faltering in children and poor weight status in adults. These conditions are associated with a decline in lung function and reduced survival[4–8]. To address this clinical need, a readily absorbable structured lipid technology (Encala™ [previously LYM-X-SORB™], Envara Health, Wayne, PA) was developed and tested as an oral nutritional supplement to increase dietary fat absorption and to increase effective caloric intake. Encala™ was evaluated in a randomized placebo controlled trial[9] that reported the supplement was safe and well tolerated in children and adolescents with CF and PI, and treatment improved dietary fat absorption, growth, choline and essential fatty acid (FA) status in the participants over 12 months of treatment [10, 11]. The objective of this subgroup analysis was to determine if the effect of three months of Encala™ treatment varied by subject degree of dietary fat malabsorption at baseline.

Methods

Participants

Subjects ages 5.0 to 17.9 years with CF and PI and mild to moderate lung disease were recruited from ten CF Centers and evaluated between March 2007 and May 2011 (Fig 1). Informed, verbal assent was obtained from subjects 6.0 to <18.0 years and informed, written consent from parents/legal guardians of subjects <18 years.

Inclusion/exclusion criteria

The complete inclusion/exclusion criteria, study design and method details, Encala™ composition and the results for Encala™ impact on choline and fatty acid status on the full study cohort have been previously reported[10–12]. Liver disease and any other significant diagnosis that might impact dietary intake, growth or body composition were exclusion criteria for subjects to participate in this study. There were no subjects with cholestatic liver disease.

Design

In this double-blind placebo-controlled study, subjects were randomized in a 1:1 ratio to daily supplementation to be consumed as part of each meal and snack with either Encala™ or placebo. The random allocation sequence was generated by the CHOP research pharmacy who assigned participants to groups with stratification for age and sex; the rest of the research team was blinded to treatment allocation. Encala™ powder was mixed with a wide range of participant-selected foods and beverages and was comprised of lysophosphatidylcholine (LPC), monoglycerides and fatty acids. LPC is water-soluble, does not require lipase for digestion/absorption of associated fatty acids and generally fosters lipid absorption in the upper gastrointestinal tract [9, 13]. Encala™ was complexed to sugar and wheat flour, to provide a taste neutral, dissolvable powder. The placebo was a powder of similar appearance, taste and consistency, composed of trans-fat free vegetable shortening, flaxseed triglyceride, and sunflower triglyceride. The placebo and Encala™ had similar calories (152 kcal/32g packet), total fat (5.4g/32g packet), and macronutrient distribution (protein 6%, carbohydrate 58%, lipid 34% kcal). Subjects age 5.0–11.9 years received two packets/d (64 g powder) providing 304 kcal/d, and age subjects 12.0–17.9 years received three packets/day (96 g powder) providing 456 kcal/d. Both Encala™ and placebo were provided in sealed packages with identical appearances so participants and staff were blinded to group assignment. All subjects continued their pancreatic enzyme regime and other aspects of care (medications, physical therapy, and diet) as prescribed by their CF center.

All study visits were conducted at Children’s Hospital of Philadelphia (CHOP) at baseline, three, and 12 months and the protocol was approved by the CHOP Institutional Review Board (IRB) and each participating CF Center (Eastern Virginia Medical School IRB, Children’s National Medical Center IRB, Yale University School of Medicine IRB, University of Virginia IRB, Schneider Children’s Hospital IRB (now: Cohen Children’s Medical Center), St. Joseph’s Children’s Hospital IRB, and Monmouth Medical Center IRB). The data from the baseline and three-month visits were selected for this analysis. This protocol was registered as Study of Lym-X-Sorb (Encala™) to Improve Fatty Acid and Choline Status in Children with Cystic Fibrosis and Pancreatic Insufficiency, NCT00406536.

Pulmonary function was assessed and predicted percentage FEV₁ calculated[14, 15]. Body mass index (BMI) was calculated (kg/m²) from weight using a digital scale (Scaletronix, White Plains, NY) and standing height using a stadiometer (Holtain, Crymych, UK) measured by research staff. Weight, height and BMI were compared to Centers for Disease Control reference standards to generate age- and sex-specific Z scores[16]. Total body fat mass, lean body mass, and percentage fat were measured by whole-body dual-energy X-ray absorptiometry (DXA; Delphi A, Hologic, Inc., Bedford, MA).

Dietary intake was measured using 3-day weighed records and analyzed for energy and fat intake (Nutrition Data System, Minneapolis, MN)(17]. Subjects were trained for diet collection by research staff and provided food scales, measuring cups and spoons. Energy intake was reported as kcal/day and as percent Estimated Energy Requirement (%EER) for active children and adolescents[17, 18]. A 72-hour stool sample was collected and total fat content determined (Mayo Medical Laboratories, Rochester, MN), and CFA was calculated[19]. For the 3-month dietary assessment, the calorie and fat content of Encala™ and placebo supplements were included in the daily energy and fat intake data, adjusted for adherence[11].

Quantitation of morning fasting plasma FA was performed in two steps: 1) acid-base hydrolysis; 2) hexane extraction/derivatization with pentafluorobenzyl bromide (Mayo Medical Laboratories). Separation/detection was accomplished by capillary gas chromatography electron-capture negative ion-mass spectrometry, with quantitation based on analysis in selected ion-monitoring mode using stable isotope-labeled internal standards[20].

Statistics

Descriptive statistics are presented as frequency counts and percentages for categorical variables and mean ± standard deviation (SD) for continuous variables. In addition to the randomization groups (placebo vs. Encala™) for this analysis, subjects were assigned to one of two fat malabsorption subgroups based upon their CFA at baseline: 1) low baseline CFA subgroup with CFA below the cohort median of 88%; and 2) high baseline CFA subgroup with CFA equal to or above the median of 88%. Two-sample t-test (unpaired) or Mann-Whitney U test for continuous variables and chi-square tests of independence for categorical variables compared characteristics at baseline between randomization groups (placebo vs. Encala™), and also between low and high baseline CFA subgroups. Two-sample t-tests were also used to determine differences between randomization groups for outcomes at three months and for the 3-month change in outcomes. Paired t-tests were used to determine significance of change in outcomes within randomization groups for the low CFA and high CFA subgroups separately. Stata 12.0 (Stata Corporation, College Station, TX) was used with significance at 0.05.

Results

Eighty-six participants completed three months treatment on either Encala™ or placebo, and this subgroup analysis included the 66 children and adolescents (10.5±3.0 yrs, 39% female) who had both baseline CFA and 3-month visit assessments were the basis of this analysis. Table 1 provides the characteristics of the sample at baseline for all 66 subjects and for those in the high and low baseline CFA subgroups. At baseline, all subjects had mild to moderate lung disease (FEV₁ 99±22% predicted), and suboptimal growth status as indicated by Z scores for weight and height. Subjects in the low baseline CFA subgroup were significantly older, more likely to be homozygous for the F508del allele, and had lower BMI Z scores and %EER energy intake. Plasma fatty acid status, FEV₁ and daily dietary intake of fat and energy were similar between CFA subgroups at baseline.

Table 1. Characteristics at baseline for CFA subgroups and for randomization groups.

		By CFA Subgroup		By Randomization Group
	All	High CFA (≥88%)	Low CFA (<88%)	Placebo	Encala™
Characteristic	n = 66	n = 33	n = 33	n = 36	n = 30
Sex, % males	61	55	67	67	53
F508del homozygous, %	55	42	67^*	50	60
Age, yr	10.5 ± 3.0	9.6 ± 2.7	11.5 ± 2.9^*	10.4 ± 3.1	10.7 ± 2.9
FEV₁% ^a	99 ± 22	101 ± 24	98 ± 20	104 ± 22	94 ± 20
Growth and Body Composition
Height for age Z score	-0.52 ± 0.93	-0.54 ± 1.13	-0.49 ± 0.69	-0.57 ± 0.90	-0.46 ± 0.97
Weight for age Z score	-0.45 ± 0.72	-0.34 ± 0.88	-0.55 ± 0.52	-0.35 ± 0.77	-0.57 ± 0.66
BMI for age Z score	-0.17 ± 0.69	0.02 ± 0.73	-0.37 ± 0.60^*	0.00 ± 0.68	-0.39 ± 0.66^*
Whole Body DXA
FFM, kg	26.3 ± 9.7	23.9 ± 8.2	28.7 ± 10.6^*	26.7 ± 10.9	25.8 ± 8.2
FM, kg	7.1 ± 2.8	6.9 ± 2.8	7.2 ± 2.8	6.9 ± 2.5	7.3 ± 3.1
Fat, %	21.6 ± 5.8	22.7 ± 5.2	20.6 ± 6.3	21.4 ± 6.3	21.9 ± 5.3
Coefficient of Fat Absorption
Stool fat, g/day	16.4 ± 13.1	8.3 ± 4.8	24.4 ± 13.8^***	15.6 ± 14.6	17.3 ± 11.1
Dietary fat intake, g/day	103.3 ± 35.4	108.5 ± 41.4	98.0 ± 28.0	105.9 ± 38.7	100.2 ± 31.4
CFA, %	83.9 ± 11.2	92.4 ± 2.6	75.3 ± 9.9^***	85.1 ± 12.1	82.4 ± 10.2
CFA < Median (88%), %	50	0	100	39	63^*
Dietary energy intake, Kcal/day	2556 ± 692	2611 ± 792	2501 ± 582	2588 ± 740	2518 ± 639
EER %	124 ± 29	132 ± 37	116 ± 17^*	124 ± 31	123 ± 28
Plasma Fatty Acids, nmol/ L ^b
Linoleic acid	2292 ± 514	2348 ± 428	2239 ± 584	2308 ± 562	2271 ± 453
α-Linolenic acid	51.4 ± 24.4	52.0 ± 21.0	50.9 ± 27.6	52.6 ± 28.7	49.9 ± 18.0
γ-Linolenic acid	65.4 ± 28.7	61.4 ± 28.7	69.2 ± 28.6	63.7 ± 29.4	67.6 ± 28.1
Arachidonic acid	438 ± 153	445 ± 167	432 ± 141	441 ± 160	436 ± 145
Eicosapentaenoic acid	37.1 ± 23.4	37.3 ± 23.1	36.8 ± 24.1	36.7 ± 21.3	37.5 ±26.4
Docosahexaenoic acid	61.1 ± 23.6	62.3 ± 23.6	60.0 ± 28.8	62.1 ± 24.8	59.9 ± 28.4
Docosapentaenoic acid	36.2 ± 16.0	37.5 ±17.8	35.0 ± 14.3	37.5 ± 17.5	34.5 ± 14.1
Plasma Fatty Acid Group, mmol/L ^b
Saturated	3.2 ± 0.7	3.2 ± 0.7	3.2 ± 0.7	3.2 ± 0.7	3.1 ± 0.7
Monounsaturated	2.4 ± 0.5	2.3 ± 0.5	2.4 ± 0.7	2.4 ± 0.7	2.3 ± 0.5
Polyunsaturated	3.2 ± 0.7	3.1 ± 0.6	3.2 ± 0.8	3.2 ± 0.8	3.1 ± 0.6
Total Fatty Acids	8.8 ± 1.8	8.7 ± 1.7	8.8 ± 2.0	8.8 ± 2.0	8.7 ± 1.7

Open in a new tab

CFA: coefficient of fat absorption; DXA: dual x-ray absorptiometry; FFM: fat free mass; FM: fat mass; Fat: fat mass as percent total body mass; FEV₁%: Forced expiratory volume at 1 second percent predicted value.

Data are presented as mean ± SD and frequency (percentage) for categorical data.

Bolded values show statistical significance.

*Significantly different between randomization groups by unpaired t test for continuous variables and χ-squared test or Fisher’s exact test for categorical variables, p<0.05,

***p<0.001

^a All, n = 63; High CFA, n = 32; Low CFA, n = 31: Placebo, n = 36; Encala™, n = 27

^b All, n = 64; High CFA, n = 31; Low CFA, n = 33: Placebo, n = 36; Encala™, n = 28

Table 1 also provides characteristics of the sample at baseline by randomization groups (placebo vs. Encala™). Subjects randomized to Encala™ had significantly lower BMI Z scores than those randomized to placebo (-0.39±0.66 vs. 0.0±0.68, p<0.05), and were otherwise similar in growth, body composition, dietary intake and plasma fatty acid status. A greater proportion of the Encala™ group were in the low baseline CFA subgroup than the placebo group (63 vs. 39%, p<0.05), although there was no significant difference in mean CFA or stool fat loss at baseline.

Table 2 presents the change in outcomes from baseline to three-months for subjects receiving placebo or Encala™ within each of the baseline CFA subgroups. For those in the low CFA subgroup, three-month Encala™ treatment improved CFA significantly (7.5±7.2%, 78.9±7.5 to 86.3±6.7%, p = 0.002), with a significant drop in stool fat loss (-5.7±7.2 g/24 hours) and no change in dietary fat intake. At the same time, plasma linoleic acid increased 20%, α-linolenic acid 56%, γ-linolenic 51%, docosapentaenoic acid 45%, and arachidonic acid by 23% (p<0.05). Total plasma fatty acid concentration increased by 20% (p≤0.005), with similar increases in total saturated, monounsaturated and polyunsaturated fatty acids, and HAZ (0.06±0.08), WAZ (0.17±0.16), and BMIZ (0.20±0.25) all increased (p≤0.002). CFA status did not change with placebo treatment in the low baseline CFA subgroup. The low baseline CFA group receiving placebo treatment had significant reductions in dietary monounsaturated fatty acids (-0.3 ± 0.4 mmol/L) and plasma docosahexaenoic acid (-17%) with an increase in plasma eicosapentaenoic acid (31%) and WAZ (0.12±0.17, p’s<0.05). For subjects in the high baseline CFA subgroup, CFA% (-1.2±4.8% on placebo and -4.4±6.3% on Encala™), stool fat (0.3±4.5 g/d on placebo and 2.9±7.3 g/d on Encala™) and dietary fat intake did not change with either placebo or Encala™ treatment. WAZ improved in both groups (0.13±0.28 for placebo and 0.14±0.21 for Encala™, p<0.05).

Table 2. Outcomes by coefficient of fat absorption subgroups: Encala™ vs. placebo.

		CFA < Median (<88%)					CFA ≥ Median (≥88%)
Characteristic	n	Baseline	3 Months	3-Month Change	P	n	Baseline	3 Months	3-Month Change	P
CFA, %
Placebo	12	72.5 ± 11.8	71.1 ± 26.2	-1.4 ± 28.5	ns	18	92.8 ± 2.4	91.1 ± 5.9	-1.2 ± 4.8	ns
Encala™	14	78.9 ± 7.5	86.3 ± 6.7 ^Ɨ	7.5 ± 7.2	0.002	8	91.4 ± 2.9	87.1 ± 8.3	-4.4 ± 6.3	ns
Stool Fat, g/day
Placebo	12	30.4 ± 16.6	30.1 ± 25.3	-0.3 ± 32.1	ns	18	7.6 ± 4.2	7.9 ± 5.2	0.3 ± 4.5	ns
LXS	14	19.9 ± 10.7	14.3 ± 90.4 ^Ɨ	-5.7 ± 7.2	0.012	8	9.6 ± 4.7	12.6 ± 11.1	2.9 ± 7.3	ns
Dietary Total Fat Intake, g/day
Placebo	12	111.6 ± 28.1	109.2 ± 22.6	-2.4 ± 41.9	ns	18	108.4 ± 46.6	100.6 ± 33.6	-7.8 ± 35.5	ns
Encala™	14	92.5 ± 25.8	106.5 ± 33.8	14.0 ± 30.6	ns	8	109.3 ± 32.8	91.7 ± 38.3	-17.6 ± 36.1	ns
Dietary Saturated FAs, g/day
Placebo	12	41.0 ± 10.5	41.3 ± 11.5	0.3 ± 15.9	ns	18	36.9 ± 9.7	34.2 ± 11.3	-2.7 ± 10.0	ns
Encala™	14	35.6 ± 12.6	37.6 ± 13.3	2.1 ± 10.7	ns	8	40.7 ± 12.6	30.8 ± 16.5	-9.8 ± 13.0	ns
Dietary Monounsaturated FA, g/day
Placebo	12	40.2 ± 10.8	36.4 ± 8.0	-3.8 ± 16.0	ns	18	40.3 ± 22.7	34.3 ± 14.9	-6.0 ± 16.4	ns
Encala™	14	29.6 ± 6.9	34.1 ± 10.6	4.4 ± 8.9	ns	8	38.5 ± 12.0	32.1 ± 14.2	-6.4 ± 15.4	ns
Dietary Polyunsaturated FAs, g/day
Placebo	12	20.8 ± 7.8	22.8 ± 5.8	2.0 ± 8.9	ns	18	23.0 ± 18.1	25.2 ± 11.8	2.2 ± 12.7	ns
Encala™	14	19.5 ± 7.3	26.9 ± 12.3	7.4 ± 12.0	0.039	8	21.7 ± 9.4	22.3 ± 8.2	0.5 ± 7.1	ns
HAZ
Placebo	14	-0.51 ± 0.75	-0.50 ± 0.75	0.02 ± 0.08	ns	22	-0.60 ± 1.00	-0.50 ± 1.03	0.11 ± .20	0.026
Encala™	19	-0.48 ± 0.66	-0.41 ± 0.67	0.06 ± 0.08	0.002	11	-0.43 ± 1.40	-0.40 ± 0.41	0.03 ± .12	ns
WAZ
Placebo	14	-0.43 ± 0.62	-0.32 ± 0.59	0.12 ± 0.17	0.023	22	-0.30 ± 0.86	-0.17 ± 0.93	0.13 ± .028	0.038
Encala™	19	-0.64 ± 0.42	-0.47 ± 0.43	0.17 ± 0.16	<0.001	11	-0.43 ± 0.96	-0.29 ± 1.02	0.14 ± 0.21	0.046
BMIZ
Placebo	14	-0.17 ± 0.62	-0.04 ± 0.60	0.14 ± 0.24	ns	22	0.12 ± 0.71	0.19 ± 0.81	0.07 ± 0.38	ns
Encala™	19	-0.51 ± 0.56	-0.31 ± .51	0.20 ± 0.24	0.002	11	-0.18 ± 0.78	-0.00 ± 0.79	0.18 ± 0.38	ns
Linoleic Acid, nmol/L
Placebo	14	2309 ± 778	2159 ± 704	-150 ± 686	ns	22	2306 ± 390	2480 ± 513	173 ± 382	0.046
Encala™	19	2187 ± 404	2625 ± 695	438 ± 511 ^ƗƗ	0.002	9	2449 ± 522	2574 ± 653	126 ± 651	ns
α-Linolenic Acid, nmol/L
Placebo	14	57.6 ± 36.4	57.9 ± 41.8	-0.2 ± 44.7	ns	22	49.3 ± 22.9	62.8 ± 27.8	13.5 ± 24.4	0.017
Encala™	19	45.9 ± 18.5	71.5 ± 40.3	25.6 ± 31.1	0.002	9	58.4 ± 14.3	79.7 ± 33.2	21.2 ± 36.2	ns
γ-Linolenic Acid, nmol/L
Placebo	14	74.3 ± 29.8	77.4 ± 40.6	3.1 ± 32.8	ns	22	57.0 ± 27.7	67.1 ± 39.5	10.2 ± 38.2	ns
Encala™	19	65.4 ± 27.8	98.9 ± 69.2	33.5 ± 65.8	0.039	9	72.1 ± 29.7	92.8 ± 43.8	20.7 ± 39.4	ns
Arachidonic Acid, nmol/L
Placebo	14	457 ± 172	412 ± 157	-45 ± 133	ns	22	430 ± 156	417 ± 125	-13 ± 90	ns
Encala™	19	413 ± 114	509 ± 179	96 ± 128 ^ƗƗ	0.004	9	483 ± 196	470 ± 149	-13 ± 161	ns
Eicosapentaenoic acid, nmol/L
Placebo	14	35.6 ± 11.4	46.5 ± 22.6	10.9 ± 18.2	0.043	22	37.5 ± 25.9	46.1 ± 33.4	8.6 ± 27.0	ns
Encala™	19	37.8 ± 30.6	42.5 ± 18.8	4.7 ± 27.6	ns	9	36.9 ± 15.3	47.1 ± 14.1	10.2 ± 18.8	ns
Docosahexaenoic acid, nmol/L
Placebo	14	60.9 ±25.2	50.5 ± 15.0	-10.4 ± 17.1	0.04	22	62.8 ±25.1	64.8 ± 25.3	2.0 ± 15.9	ns
Encala™	19	59.3 ± 31.8	67.2 ± 46.9	7.9 ± 25.4 ^Ɨ	ns	9	61.1 ± 21.1	70.2 ± 30.4	9.1 ± 36.5	ns
Docosapentaenoic acid, nmol/L
Placebo	14	38.2 ± 16.0	38.5 ± 16.3	0.3 ± 16.4	ns	22	37.0 ± 18.7	42.4 ± 21.2	5.3 ± 13.9	ns
Encala™	19	32.7 ± 12.9	47.4 ± 22.1	14.6 ± 17.8 ^Ɨ	0.002	9	38.4 ± 16.4	43.9 ± 15.4	5.4 ± 18.0	ns
Saturated FAs, mmol/L
Placebo	14	3.4 ± 0.7	3.3 ± 1.1	-0.2 ± 0.6	ns	22	3.1 ± 0.7	3.4 ± 1.0	0.3 ± 0.9	ns
Encala™	19	3.1 ± 0.6	3.7 ± 1.1	0.6 ± 0.9 ^Ɨ	0.011	9	3.3 ± 0.7	3.8 ± 1.4	0.5 ± 1.1	ns
Monounsaturated FAs, mmol/L
Placebo	14	2.6 ± 0.6	2.3 ± 0.6	-0.3 ± 0.4	0.021	22	2.3 ± 0.7	2.4 ± 0.8	0.1 ± 0.9	ns
Encala™	19	2.3 ± 0.5	2.8 ± 0.9	0.5 ± 0.7 ^ƗƗ	0.012	9	2.4 ± 0.5	2.7 ± 0.9	0.3 ± 0.7	ns
Polyunsaturated FAs, mmol/L
Placebo	14	3.2 ± 1.1	3.1 ± 1.0	0.2 ± 1.0	ns	22	3.2 ± 0.7	3.4 ± 0.8	0.2 ± 0.6	ns
Encala™	19	3.0 ± 0.6	3.7 ± 1.1	0.7 ± 0.8 ^ƗƗ	0.001	9	3.4 ± 0.8	3.6 ± 0.9	0.2 ± 0.9	ns
Total FAs, mmol/L
Placebo	14	9.3 ± 8.1	8.6 ± 2.5	-0.6 ± 1.7	ns	22	8.6 ± 2.0	9.2 ± 2.4	0.6 ± 2.2	ns
Encala™	19	8.4 ± 1.6	10.2 ± 2.9	1.7 ± 2.4 ^ƗƗ	0.005	9	9.1 ± 1.8	10.2 ± 2.9	1.0 ± 2.5	ns

Open in a new tab

CFA: Coefficient of fat absorption; HAZ: height for age Z score; WAZ: weight for age Z score; BMIZ: BMI for age score; FA: fatty acid.

Data are presented as mean ± SD for normally distributed data.

P value is testing for Time effect within treatment group using student's paired t test.

Bolded values show statistical significance.

Ɨ Placebo and Encala™ groups different by student's unpaired t test p<0.05,

ƗƗ p<0.01.

Discussion

Encala™ was evaluated initially as a nutritional supplement in 2002 in children with CF and PI[9]. This novel compound was synthesized from known lipolysis products of dietary fats, including lysophosphatidycholine, monoglyceride and fatty acids. Lepage, et al.[9] published the results of two studies testing the safety and efficacy of Encala™. In the preliminary study, a liquid Encala™ test meal was consumed by healthy adults and by adolescents with CF and PI taking no pancreatic enzyme medication and plasma triglyceride absorption followed over 12 hours. Total triglycerides absorbed from Encala™ did not differ between the CF and healthy subjects and established that pancreatic enzymes were not required for Encala™ digestion and absorption in the context of CF and PI. In the 12-month double blind randomized, placebo-controlled CF clinical trial that followed, Encala™ and an active, isocaloric placebo supplements were tested in a cookie form in children and adolescents. The results showed that the Encala™ supplement was safe and was a well-absorbed source of dietary fat energy that Encala™ use led to better clinical outcomes in growth, fat-soluble vitamins and essential fatty acid status in this cohort of children with CF and PI. However, the cookie format and taste were not optimal (Dr. Yesair, BioMolecular Products, personal communication), and research and development continued to address the supplement taste and format characteristics. The improved Encala™, now a taste-neutral powder that was mixed with subject-selected, preferred foods and beverages, was used in the double blind, randomized, active placebo-controlled clinical trial reported here.

The main outcomes from our RCT study showed that in a large cohort of children and adolescents with CF and PI cared for at ten different centers and on recommended CF diet and pancreatic enzyme regimens, daily oral Encala™ powder was safe and more effective than the energy and macronutrient-matched placebo. Increased dietary fat absorption (CFA) and fasting plasma fatty acid status and growth were observed[11]. Previous reports also documented that Encala™ supplementation, compared with active placebo, improved choline (serum and muscle stores), amino acid, vitamin A and E, total body muscle stores and resting energy expenditure status in these school aged children with CF, PI and mild lung disease[10, 12, 21].

In this current subgroup analysis of subjects with baseline and three-month observations, Encala™ was particularly effective in subjects who experienced more severe dietary fat malabsorption prior to intervention. Low baseline CFA value indicated more dietary fat excreted in stool and a more severe malabsorptive clinical status. In this group, Encala™ treatment improved CFA by 7.5%, a magnitude that was both highly clinically and statistically significant and improved plasma fatty acid and growth status compared to placebo. Given the success of CFTR modulators in improving nutritional status, such as BMI, in patients with some genotypes more data are also needed to determine the efficacy of nutritional treatment in patients with CF taking CFTR modulator therapies. There are limited data on the impact on fat absorption with modulator therapy, particularly in patients with more severe mutations.

Encala™ was developed to improve dietary fat absorption, especially absorption long chain fatty acids including the two essential fatty acids, and to supply more calories per gram ingested than medium chain fatty acids, which are a common ingredient in therapeutic nutrition products. Investigators have documented persistent, residual dietary fat malabsorption in spite of optimized, modern pancreatic enzyme medication and treatment regimens for patients with CF and PI for the past two decades[4, 6]. Kalivianakis et al.[6] in CF and PI studies, suggested that this residual fat loss was due to reduced gut mucosal uptake of long chain fatty acids and/or to incomplete solubilization of long chain fatty acids in the gut lumen. As such, this residual malabsorption was not the result of impaired lipolysis (pancreatic lipase mechanism), and thus will not be reduced or eliminated by increased dose of pancreatic enzyme medication[6]. Their work in a fat malabsorption rat model showed that some of the long chain fatty acid absorption impairment was attributable to chronic bile acid deficiency. Chronic bile acid depletion is an established component of the spectrum of disease in patients with CF and PI[7].

Lysophosphatidylcholine (LPC) is the backbone of the Encala™ technology and has unique metabolic characteristics; most importantly LPC directly enhances dietary fatty acid absorption in the gut, enhances transfer of fatty acids to the lymphatic system and improves fatty acid retention in mucosa[22]. Intraluminal micellar LPC is extensively absorbed in the gut[23]. In contrast, phosphatidylcholine, the common phospholipid in the diet, directly suppresses dietary fatty acid absorption as described by Homan and Hamelehle[23] in the Caco-2 cell line. Peretti et al.[24] provided a full review of the mechanisms of lipid absorption in CF.

Few patients with CF and PI have a CFA ≥ 93%, the generally accepted range of absorption in health children and adults[3, 19]. Encala™ supplementation addresses this residual long chain fatty acid malabsorption, as the Encala™ -delivered fatty acids are not dependent upon the action of lipase or bile acids for effective digestion and absorption in the CF gut. Despite having similar daily dietary energy and total fat intake as subjects receiving placebo, those receiving Encala™ had significantly improved absorption of linoleic, α-linolenic, γ-linolenic, arachidonic, docosapentaenoic acid and total fatty acids including saturated, monounsaturated and polyunsaturated fat classes. The improvement in fatty acid status may have also affected growth. There was improvement in growth and fatty acid status with Encala™ treatment in subjects with high baseline CFA fat malabsorption, but this improvement was less than in the low baseline CFA subgroup. The placebo group showed some improvements, as this group also received additional energy from the active placebo. This analysis demonstrates Encala™ provided greater benefits to children with CF and PI with more severe baseline residual dietary fat malabsorption. This is particularly important as these were the patients with the greatest need for nutritional treatment as they had the poorer growth and more severe disease.

Malnutrition due to chronic malabsorption is an important intervention target in CF care as it affects growth, development, pulmonary function, immune system function, and survival[25]. Participants had dietary patterns that adhered to CF recommendations, with high calorie intake (124% EER) and high fat intake (42% energy; 103 g per day)[26–28]. Participants also followed the recommended pancreatic enzyme medication use regimen for meals and snacks[29] but had a mean baseline CFA of 84%, a value lower than the 93% reference for healthy people[6, 30]. Numerous studies have demonstrated pancreatic enzyme replacement therapy (PERT) decreases fat malabsorption[26], however, PERT does not fully remedy fat malabsorption in patients with CF and PI. Further, individuals at higher risk for fat malabsorption may not attain the same improvements with enzyme treatment as those with a lower risk[31]. Woestenenk and colleagues[31] completed a retrospective cross-sectional analysis of 224 children and adolescents with CF and PI prescribed PERT. The authors reported the median CFA varied (86–91%), and 24% had a CFA below 85% and CFA did improve following PERT. The lower CFA group also had significantly lower WAZ and BMIZ compared to the higher CFA group. Often, gastric acid-reducing agents are prescribed as adjunct therapy based on the theory that these medications reduce the gastric and duodenal acid environment and improve the efficacy of PERT. However, a recent Cochrane Database Systematic Review determined there was limited evidence that adjunct therapy to reduce gastric acid improved nutrition status[32]. New therapies, such as Encala™, are needed for patients that have residual fat malabsorption after optimizing PERT. Encala™ supplementation significantly improved CFA (+7.5%) when assessed after three months of treatment in the absence of change in dietary fat intake. The increase in CFA attained in the lower CFA group supplemented with Encala™ is clinically significant and warrants use as an adjunct therapy for individuals with CF on enzyme medications, with residual fat malabsorption.

Currently, enzyme medications are the only available treatment for dietary fat malabsorption and are a cornerstone of CF therapy. Enzyme products are a combination of all digestive enzymes and other components derived from porcine pancreas. There are non-capsule enzyme options to treat fat malabsorption, such as an immobilized lipase-only containing device available for use with enteral tube feeding (RELiZORB® Alcresta Therapeutics, Inc, Newton, Massachusetts, USA)[33], a bacterial-derived lipase-only product suspended in a liquid solution for use with meals (Burlulipase, Nordmark Arzneimittel, Uetersen, DE)[34] and a non-enteric coated enzyme powder (Viokase, Axcan Pharma US, Inc., Mont-saint-hilaire, Quebec, CA). Although lipase containing drugs improve triglyceride digestion in lipase-limited diseases, Encala™ is the only non-enzyme approach and has demonstrated improved fat absorption, growth, and nutritional status when used in conjunction with PERT. In the present study, we observed significant improvement in plasma fatty acid status following Encala™ treatment in individuals with greater fat malabsorption. Notably, supplementation increased essential fatty acid (linoleic and α-linolenic acid), arachidonic acid, docosapentaenoic acid and total fatty acid absorption. The increased dietary fat absorption and improved energy intake was reflected in the clinically significant growth in weight and height (HAZ, WAZ and BMIZ). Only WAZ improved with placebo supplementation in the lower CFA group, and this is likely due to the caloric density design of the placebo.

Given the exploratory nature of this analysis, there are some limitations. The lower and higher CFA groups in this study were defined for this study as the median CFA at baseline (88%) to provide a balanced distribution of participants. There is no generally accepted or proposed evidence-based definition for classification of individuals with CF and PI at high and low degree of fat malabsorption by CFA value. CFA is used mostly in the research setting and for FDA evaluation of new pancreatic enzyme products. For the cohort of subjects with a CFA both at baseline and the three month follow-up, there was a greater proportion of participants randomized to Encala™ in the lower CFA group. The unbalanced distribution was related to the original Encala™/placebo randomization procedure for all subjects with sex/age blocks that did not include CFA, as CFA was an outcome variable. There was no difference in mean CFA between subgroups at baseline and there were clinically significant improvements in CFA in the low CFA Encala™ group. There was variation in individual response as indicated by the standard deviation. The original study did not specify this subgroup analysis, however this additional evidence provided data and experience to inform future research and clinical care.

In summary, subjects with low baseline CFA and more severe fat malabsorption had a dramatic improvement in CFA with Encala™ treatment, accompanied by improved plasma fatty acid and growth status. Based on these results and clinical trial experience, Encala™ was well-accepted and reduced residual fat malabsorption in patients with CF and PI. These data indicate that Encala™ was efficacious in patients with CF and suggest efficacy in other exocrine pancreatic insufficiency diagnoses and malnutrition in need of improved energy absorption and weight gain. Further investigation is needed to evaluate the effectiveness of Encala™ supplementation to improve fat absorption and treat or prevent malnutrition in patients with other diagnoses.

Supporting information

S1 File

(PDF)

Click here for additional data file.^{(1.7MB, pdf)}

S1 Checklist

(DOC)

Click here for additional data file.^{(217.5KB, doc)}

Acknowledgments

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The study was registered as: Study of Lym-X-Sorb (Encala™) to Improve Fatty Acid and Choline Status in Children with Cystic Fibrosis and Pancreatic Insufficiency, NCT00406536. https://clinicaltrials.gov/ct2/show/NCT00406536.

The authors thank the subjects, parents, other care providers, and all of the CF Centers that participated in the study: Children’s National Medical Center, Washington, DC; Children’s Hospital of Philadelphia, Philadelphia, PA; Monmouth Medical Center, Long Branch, NJ; The Pediatric Lung Center, Fairfax, VA; Cystic Fibrosis Center of University of Virginia, Charlottesville, VA; Children’s Hospital of the King’s Daughters, Eastern Virginia Medical School, Norfolk, VA; Yale University School of Medicine, New Haven, CT; Cohen Children’s Medical Center, New Hyde Park, NY; St Joseph’s Children’s Hospital, Paterson, NJ, and the Pediatric Specialty Center at Lehigh Valley Hospital, Bethlehem, PA.. The authors also thank Norma Latham, MS, for study coordination, and Megan Johnson, Thananya Wooden, Elizabeth Matarrese, and Nimanee Harris, the staff of the CTRC at CHOP for their valuable contributions to the study.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The NIH provided all funding for this study through a Small Business Innovation Research (SBIR) program award. This grant funded the development and production of LXS (now known as Encala™) at Avanti Polar Lipids, the SBIR awardee and supplied the LXS and placebo for the clinical trial conducted at Children’s Hospital of Philadelphia. Envara Health was founded in 2018 to develop therapeutic nutrition products related to the LXS (now Encala™) technology. Envara Health provided no funding or products for this study.

References

1.Turck D, Braegger CP, Colombo C, Declercq D, Morton A, Pancheva R, et al. ESPEN-ESPGHAN-ECFS guidelines on nutrition care for infants, children, and adults with cystic fibrosis. Clin Nutr. 2016;35(3):557–77. 10.1016/j.clnu.2016.03.004 [DOI] [PubMed] [Google Scholar]
2.Borowitz D, Lubarsky B, Wilschanski M, Munck A, Gelfond D, Bodewes F, et al. Nutritional Status Improved in Cystic Fibrosis Patients with the G551D Mutation After Treatment with Ivacaftor. Dig Dis Sci. 2016;61(1):198–207. 10.1007/s10620-015-3834-2 [DOI] [PubMed] [Google Scholar]
3.Mennella JA, Inamdar L, Pressman N, Schall JI, Papas MA, Schoeller DA, et al. Type of infant formula increases early weight gain and impacts energy balance: an RCT. Am J Clin Nutr. 2018;(in press). [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Aldamiz-Echevarria L, Prieto JA, Andrade F, Elorz J, Sojo A, Lage S, et al. Persistence of essential fatty acid deficiency in cystic fibrosis despite nutritional therapy. PediatrRes. 2009;66(5):585–9. [DOI] [PubMed] [Google Scholar]
5.Bijvelds MJ, Bronsveld I, Havinga R, Sinaasappel M, de Jonge HR, Verkade HJ. Fat absorption in cystic fibrosis mice is impeded by defective lipolysis and post-lipolytic events. Am J Physiol Gastrointest Liver Physiol. 2005;288(4):G646–53. 10.1152/ajpgi.00295.2004 [DOI] [PubMed] [Google Scholar]
6.Kalivianakis M, Minich DM, Bijleveld CM, van Aalderen WM, Stellaard F, Laseur M, et al. Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids. Am J Clin Nutr. 1999;69(1):127–34. 10.1093/ajcn/69.1.127 [DOI] [PubMed] [Google Scholar]
7.Kalivianakis M, Minich DM, Havinga R, Kuipers F, Stellaard F, Vonk RJ, et al. Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption. Am J Clin Nutr. 2000;72(1):174–80. 10.1093/ajcn/72.1.174 [DOI] [PubMed] [Google Scholar]
8.Kalivianakis M, Verkade HJ. The mechanisms of fat malabsorption in cystic fibrosis patients. Nutrition. 1999;15(2):167–9. 10.1016/s0899-9007(98)00155-5 [DOI] [PubMed] [Google Scholar]
9.Lepage G, Yesair DW, Ronco N, Champagne J, Bureau N, Chemtob S, et al. Effect of an organized lipid matrix on lipid absorption and clinical outcomes in patients with cystic fibrosis. Journal of Pediatrics. 2002;141(2):178–85. 10.1067/mpd.2002.124305 [DOI] [PubMed] [Google Scholar]
10.Schall JI, Mascarenhas MR, Maqbool A, Dougherty KA, Elci O, Wang D-J, et al. Choline supplementation with a structured lipid in children with cystic fibrosis: a randomized placebo-controlled trial. J Pediatr Gastroenterol Nutr. 2016;62(4):618–26. 10.1097/MPG.0000000000001004 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Stallings VA, Schall JI, Maqbool A, Mascarenhas MR, Alshaikh BN, Dougherty KA, et al. Effect of oral lipid matrix supplementation on fat absorption in cystic fibrosis: a randomized placebo-controlled trial. J Pediatr Gastroenterol Nutr. 2016. 63(6):676–80. 10.1097/MPG.0000000000001213 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Bertolaso C, Groleau V, Schall JI, Maqbool A, Mascarenhas M, Latham NE, et al. Fat-soluble vitamins in cystic fibrosis and pancreatic insufficiency: efficacy of a nutrition intervention. J Pediatr Gastroenterol Nutr. 2014;58(4):443–8. 10.1097/MPG.0000000000000272 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Yesair DW. Phosphatidylcholine and lysophosphatidylcholine in mixed lipid micelles as novel drug delivery systems In: I H, G P, editors. Phospholipids. New York: Plenum Press; 1990. p. 83–106. [Google Scholar]
14.Wang X, Dockery DW, Wypij D, Fay ME, Ferris BG Jr. Pulmonary function between 6 and 18 years of age. Pediatr Pulmonol. 1993;15(2):75–88. 10.1002/ppul.1950150204 [DOI] [PubMed] [Google Scholar]
15.Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general U.S. population. Am J Respir Crit Care Med. 1999;159(1):179–87. 10.1164/ajrccm.159.1.9712108 [DOI] [PubMed] [Google Scholar]
16.Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, Flegal KM, Guo SS, Wei R, et al. CDC growth charts: United States. Adv Data. 2000(314):1–27. [PubMed] [Google Scholar]
17.Otten JJ, Hellwig JP, Meyers LD. Dietary DRI Reference Intakes: The Essential Guide to Nutrient Requirements. Washington, D.C.: The National Academies Press; 2006. [Google Scholar]
18.Trabulsi J, Ittenbach RF, Schall JI, Olsen IE, Yudkoff M, Daikhin Y, et al. Evaluation of formulas for calculating total energy requirements of preadolescent children with cystic fibrosis. Am J Clin Nutr. 2007;85(1):144–51. 10.1093/ajcn/85.1.144 [DOI] [PubMed] [Google Scholar]
19.Cohen J, Schall J, Ittenbach R, Zemel B, Scanlin T, Stallings V. Pancreatic status verification in children with cystic fibrosis: fecal elastase status predicts prospective changes in nutritional status. J Pediatr Gastroenterol Nutr. 2005;40:438–44. 10.1097/01.mpg.0000158222.23181.1c [DOI] [PubMed] [Google Scholar]
20.Lagerstedt SA, Hinrichs DR, Batt SM, Magera MJ, Rinaldo P, McConnell JP. Quantitative determination of plasma c8-c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol GenetMetab. 2001;73(1):38–45. [DOI] [PubMed] [Google Scholar]
21.Alshaikh B, Schall JI, Maqbool A, Mascarenhas M, Bennett MJ, Stalliings VA. Choline supplementation alters some amino acid concentrations with no change in homocysteine in children with cystic fiborosis and pancreatic insufficiency. Nutr Res. 2016;36(5):418–29. 10.1016/j.nutres.2015.12.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Viola G, Mietto L, Secchi FE, Ping L, Bruni A. Absorption and distribution of arachidonate in rats receiving lysophospholipids by oral route. J Lipid Res. 1993;34(11):1843–52. [PubMed] [Google Scholar]
23.Homan R, Hamelehle KL. Phospholipase A2 relieves phosphatidylcholine inhibition of micellar cholesterol absorption and transport by human intestinal cell line Caco-2. J Lipid Res. 1998;39(6):1197–209. [PubMed] [Google Scholar]
24.Peretti N, Marcil V, Drouin E, Levy E. Mechanisms of lipid malabsorption in Cystic Fibrosis: the impact of essential fatty acids deficiency. Nutr Metab (Lond). 2005;2(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Slae M, Wilschanski M. Prevention of malnutrition in cystic fibrosis. Curr Opin Pulm Med. 2019;25(6):674–9. 10.1097/MCP.0000000000000629 [DOI] [PubMed] [Google Scholar]
26.Borowitz D, Baker RD, Stallings V. Consensus report on nutrition for pediatric patients with cystic fibrosis. J Pediatr Gastroenterol Nutr. 2002;35(3):246–59. 10.1097/00005176-200209000-00004 [DOI] [PubMed] [Google Scholar]
27.Smyth R, Walters S. Oral calorie supplements for cystic fibrosis (review). Cochrane Database of Systematic Reviews. 2007(1):Art. No.:CD000406. [DOI] [PubMed] [Google Scholar]
28.Cystic Fibrosis Foundation Patient Registry: 2012 Annual Data Report. Bethesda, MD: Cystic Fibrosis Foundation; 2013 2013.
29.Taylor JR, Gardner TB, Waljee AK, Dimagno MJ, Schoenfeld PS. Systematic review: efficacy and safety of pancreatic enzyme supplements for exocrine pancreatic insufficiency. AlimentPharmacolTher. 2010;31(1):57–72. [DOI] [PubMed] [Google Scholar]
30.Borowitz D, Konstan MW, O'Rourke A, Cohen M, Hendeles L, Murray FT. Coefficients of fat and nitrogen absorption in healthy subjects and individuals with cystic fibrosis. J Pediatr PharmacolTher. 2007;12(1):47–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Woestenenk JW, van der Ent CK, Houwen RH. Pancreatic Enzyme Replacement Therapy and Coefficient of Fat Absorption in Children and Adolescents with Cystic Fibrosis. JPediatrGastroenterolNutr. 2015. [DOI] [PubMed] [Google Scholar]
32.Ng SM, Moore HS. Drug therapies for reducing gastric acidity in people with cystic fibrosis. Cochrane Database Syst Rev. 2016(8):CD003424 10.1002/14651858.CD003424.pub4 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Stevens J, Wyatt C, Brown P, Patel D, Grujic D, Freedman SD. Absorption and Safety With Sustained Use of RELiZORB Evaluation (ASSURE) Study in Patients With Cystic Fibrosis Receiving Enteral Feeding. J Pediatr Gastroenterol Nutr. 2018;67(4):527–32. 10.1097/MPG.0000000000002110 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Heubi JE, Schaeffer D, Ahrens RC, Sollo N, Strausbaugh S, Graff G, et al. Safety and Efficacy of a Novel Microbial Lipase in Patients with Exocrine Pancreatic Insufficiency due to Cystic Fibrosis: A Randomized Controlled Clinical Trial. J Pediatr. 2016;176:156–61 e1. [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0232685.r001

Decision Letter 0

Maret G Traber

26 Feb 2020

PONE-D-19-35347

Improved Residual Fat Malabsorption and Growth in Children with Cystic Fibrosis Treated with a Novel Oral Structured Lipid Supplement: A Randomized Controlled Trial

PLOS ONE

Dear Dr. Tindall,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Apr 11 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Maret G Traber, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Your manuscript describes an interesting approach to the continuing problem of fat malabsorption in patients with cystic fibrosis. The three reviewers were positive in their comments, but asked for some clarifications. One asked for a new statistical approach. Please address these comments and either justify your approach, or revise your statistical analysis.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for including your ethics statement: This study was approved by the Children's Hospital of Philadelphia Institutional Review Board and each CF center (#4611). Verbal assent was obtained from subjects 6 to <18 years and written consent from parents/legal guardians of subjects <18 years.

Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether written consent was informed (line 25).

4. Please add sub-sections to each part of your Methods section to aid in the reading of the manuscript.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"Supported by NIDDK (R44DK060302), and the Nutrition Center at the Children’s Hospital of Philadelphia. The project described was supported by the National Center for Research Resources, Grant UL1RR024134, and is now at the National Center for Advancing Translational Sciences, Grant UL1TR000003."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: none.

6. Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

8. Please include a separate caption for each figure in your manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: An team of nutrition investigators conducted a 2007-2011 double-blind placebo controlled dietary supplement study using LYM-X-SORBTM (LXS), a readily absorbable structured lipid technology developed to increase dietary fat absorption, in CF subjects with pancreatic insufficiency ages 5-18. The overall 12 month study demonstrated that the oral LXS supplementation was safe and more effective than the energy and micronutritent-matched placebo supplement and resulted in increase daily fat absorption and fasting plasma fatty acid status and growth as published in ref 9, related parts of the study being published in refs 8, 10 &11. In the present report the authors have harvested data gathered at baseline and the three month visit from 40 LXS supplemented and 46 placebo supplemented subjects. Findings in this secondary analysis showed LXS to be most effective in subjects with more severe baseline malabsorption of fat. The authors present solid data supporting their conclusions, the findings are of translational value to clinical medicine and the discussion is both informative and relevant. A story case is made for more widespread use of LXS in CF patients with continuing fat malabsorption after optimizations for dosing of PERT.

Comments

1. A major issue needing to be addressed by the authors concerns the modifying effects of CFTR modulator therapies on fat malabsorption. Unlike the subjects of a decade ago, most CF patients are now receiving CFTR corrector and potentiator therapies known to be modifiers of intestinal absorption... and this significantly impacts the translational value of this paper. There are now publications addressing this issue.

2. It would be useful if the authors could mention the effects of LXS to increase 20:5 and 22:6 absorption. To what extent are these values low in CF because of absorption issues vs. deficiencies in desaturases?

3. Line 127 and 180, what is CP?

4. Do the authors have any information on the effects of LXS on carotenoid absorption?

Reviewer #2: Stallings et al. performed a secondary analysis of a multicenter RCT focusing on the effects of 3-month supplementation with a readily absorbable structured lipid versus placebo in 66 patients with cystic fibrosis aged 10.3±3.0 years, which has been reported previously to be well tolerated and exert beneficial effects on fat absorption, growth and nutritional and essential fatty acid status.

In the present manuscript the effects in patients with coefficient of fat absorption (CFA) below versus above 88%, the median baseline CFA of the study group, were analyzed. Patients with lower than median CFA receiving the supplement showed improved CFA, anthropometry (weight, BMI z scores) and plasma essential fatty acids (linoleic, α-linolenic acid) and those receiving placebo only showed improved weight z scores (due to additional energy intake from the supplement), while patients with higher than median CFA (both treatment and placebo groups) had improvements in weight z scores and some plasma fatty acids.

The authors concluded that the supplement provided greater benefits to CF patients with more severe baseline residual dietary fat malabsorption and that it enhanced dietary energy absorption, weight gain and fatty acid status.

Overall, the RCT has been well designed and conducted using state-of-the-art methodologies and complying with human intervention study standards. However, this study has a few strong limitations:

(1) The main difficulty arises from the fact that the RCT was not designed and powered for detecting a difference in the treatment effects between patients with CFA below versus above the median CFA of the entire study group.

(2) The threshold of CFA 88% is not clinically or pathophysiology based, but determined by group characteristics and data distribution.

(3) There is an unbalanced distribution of 63% of the treatment group showing CFA <88%, while only 39% did so in the placebo group.

(4) Improvement of fat absorption at the level of the mean changes in CFA (7.5 � 7.2 %) and stool fat excretion (5.7 �7.2 g/d) reported for the group with CFA <88% receiving the supplement are clinically relevant, but the standard deviations are very high, suggesting that only a few patients may have responded that well.

(5) In the absence of dietary fatty acid intake data changes in plasma fatty acid concentrations are difficult to explain by improved intestinal fatty acid absorption.

Given that fat malabsorption-associated improvements are clinically relevant in CF patients, it is suggested that the authors address the following recommendations for further exploration of the results:

As a consequence of (1) some real differences might not have been detected while others could have been observed by chance. For instance, there were significant changes in plasma linoleic and alpha-linolenic acid in the CFA>88% group receiving placebo, which is difficult to explain. Student’s paired tests were applied for testing time effects within treatment groups. When using for instance 2-way repeated measures ANOVA across the treatment groups, the likelihood to observe significant changes by chance could be minimized.

To overcome (2) the limitation of an arbitrary threshold rather than one supported by clinical or pathophysiological criteria, it is suggested that differences in the treatment effects are studied across the entire range of baseline CFA instead of splitting the group by the median CFA and/or as an additional approach. It is suggested that the authors visualize and analyze the data in that way. This could possibly even help detect a real clinically relevant threshold (if such exists in this study).

Regarding (3), even though the differences in baseline CFA were not significant between treatment and placebo group, the impact of this imbalance on the results should be taken care of by additional statistical analysis.

Given (4) the high standard deviations of 7.5÷7.2 % for CFA and 5.7÷7.2 g/d for stool fat excretion in the CFA <88% and treatment group, a closer look at the data of this subgroup is suggested, including, for instance, the impact of absence versus presence of CF-associated cholestatic liver disease – which may have been more (or even exclusively) prevalent in CFA<88%.

(5) Even though the 3-day weighed dietary records performed before and after the intervention may be too short in duration to definitely exclude possible effects of changes in dietary intake, they could be helpful in this regard. It is therefore suggested to include fatty acid intake data in the present manuscript. Studying the improvements in plasma FA in relation to improvements in CFA by using regression analysis could help support a mechanistic explanation of improved intestinal absorption proposed in the manuscript.

Minor point:

λ-linolenic acid should read γ-linolenic acid.

Reviewer #3: This article is certainly very interesting and useful as it proposes to investigate whether the effect of 3 months of LXS treatment varied by the participants degree of dietary fat malabsorption at baseline. The paper is well written and highly readable, however they some minor comments worth addressing.

Minor

Perhaps not essential but it would it be good to include a rationale for looking at 3 month time-point. Clinical reason?, or more to do with immediate effects of the intervention?

It would perhaps be more clear and transparent that this was a subgroup analysis. Calling it a secondary analysis might imply that the analysis is based on one of the secondary objectives from the main RCT, but it isn’t.

In the methods section, it would be help if the sections were labelled clearly or made distinguishable, ‘outcomes’, ‘statistical analysis’

Result section, it’s worth mentioning that 86 participants completed 3 months (according to the Fig 1), however 66 children had data available for the ‘subgroup’ analysis.

Lines 118 – 121 (page 9), also give actual numbers as you have done for low CFA group.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 May 8;15(5):e0232685. doi: 10.1371/journal.pone.0232685.r002

Author response to Decision Letter 0

26 Mar 2020

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

We have added the full name of each of the other sites IRB:

We have added this information to the submission form.

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

We have amended the sentence to describe how consent was obtained: “Informed, verbal assent was obtained from subjects 6.0 to <18.0 years and informed, written consent from parents/legal guardians of subjects <18 years.”

4. Please add sub-sections to each part of your Methods section to aid in the reading of the manuscript.

We have added the following sub-sections: Participants, Inclusion/Exclusion Criteria, Design, Outcomes, and Statistics

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: none.

We have updated the funding statement in the online submission form and removed the funding information from the Acknowledgements section.

We have updated the Competing Interests by adding this information after the “Acknowledgements” section in the manuscript as there was not a “Competing Interests” section within the online submission form.

This has been added (https://orcid.org/0000-0002-2045-3002)

8. Please include a separate caption for each figure in your manuscript.

A caption has been added to the Figure 1 powerpoint file.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Review Comments to the Author

Comments

This is a great point. We conducted a literature search of modulator therapies and fat malabsorption and there is little information on the impact of fat absorption. A recent study from our group (Stallings VA et al., J Peds, 2018) that evaluated CFA and fecal calprotectin (marker of inflammation) in subjects with gating mutations and showed that CFA increased significantly (+1.5%) in participants who were pancreatic insufficient (PI) after ivacaftor treatment, and calprotectin was reduced in both PI and PS. Studies also report that Ivacaftor can alter microbial communities (Ooi CY et al., Sci Rep, 2018) and partially restored this disruption of bile acid homeostasis (van de Peppel IP et al., J Cyst Fibros, 2019), which could affect fat absorption. However, in individuals with more severe (non-gating) mutations, there are no data related to fat malabsorption. There is evidence of improved intestinal pH (Gelfond D et al., Clin Transl Gastroenterol, 2017) which could help pancreatic enzyme function, but this is speculative.

A comment about this this issue of impact of modulator therapy was added to the introduction and discussion.

Thank you for this suggestion. We added plasma eicosapentaenoic acid (EPA: C20:5w3), docosahexaenoic acid (DHA: C22:6w3), and docosapentaenoic acid (DPA: C22:5w3) concentrations at baseline comparing both CFA groups and treatment (randomization) groups in Table 1, and also the baseline, 3 month, and 3-month changes for each treatment/CFA group in Table 2.

There is evidence that fatty acid desaturase activity is actually increased in people with cystic fibrosis via stimulation of the AMP-activated protein kinase in the absence of a functional CFTR protein (Freedman SD et al., Proc Natl Acad Sci U S A. 1999; Mimoun M et al., J Nutr, 2009; Njoroge SW et al., Biochim Biophys Acta, 2011; Seegmiller AC, Int J Mol Sci, 2014). Low values in CF are a result of poor absorption and the impact of altered (increased and decreased pathways) metabolism may play a role.

3. Line 127 and 180, what is CP?

Thank you for this question. We have corrected this error to “CF”, not “CP”.

4. Do the authors have any information on the effects of LXS on carotenoid absorption?

Upon review, we do not have data on the effects of LXS on carotenoid absorption.

Overall, the RCT has been well designed and conducted using state-of-the-art methodologies and complying with human intervention study standards. However, this study has a few strong limitations:

We agree that the RCT a priori plan did not specify this analysis. However, the RCT results and interest in applications from our clinical colleagues led us to conduct this post hoc subgroup exploratory analysis. These facts are presented in the methods section, so the reader is aware that this was an exploratory analysis with associated limitations.

(2) The threshold of CFA 88% is not clinically or pathophysiology based, but determined by group characteristics and data distribution.

We agree there is no evidence-based, generally accepted cut-point for ‘typical’ or ‘best attainable’ CFA in people CF and PI. In addition, a cut-point is unlikely to be established since the CFA test is employed almost exclusively for research studies and for FDA evaluations of new pancreatic enzyme efficacy (all studies with small sample sizes). CFA reference value for healthy individuals is generally accepted as 93% or greater. A soon to be published paper from our group (Bashaw, et al 2020), confirms this value is still meaningful in a contemporary sample of healthy volunteers using the same study method as in this report.

After discussions with our biostatistician and consideration of feasibility (sample size, contrast between groups) with our data set, the approach to use the median for this subgroup analysis was employed.

The following comments were added to the limitations section:

The low and high CFA groups in this study were defined for this study as the median CFA at baseline (88%) to provide a balanced distribution of participants. There is no generally accepted or evidence based definition for classification of individuals with CF and PI at high and low risk of fat malabsorption by CFA value. CFA is used mostly in the research setting and for FDA evaluation of new pancreatic enzyme products.

(3) There is an unbalanced distribution of 63% of the treatment group showing CFA <88%, while only 39% did so in the placebo group.

Agree, and this is related to the original (Encala/placebo group) randomization procedure for all subjects with sex/age blocks. It did not include CFA, as CFA was an outcome variable. We have included this in the limitations section.

A higher standard deviation in the low CFA group was expected given that the range was wider (52-87%) than that of the high CFA where the range was lower (>88 and <100%) and has a ceiling effect since no one is above 100%. As part of preparing this response, we carefully reviewed the CFA for each individual; there were no outliers nor implausible data. In subjects who improved, all but 2 subjects improved between +1 and +16%, except 2 of 31 subjects, who improved >16%. In subjects who had decreased CFA, all subjects decreased between -1 and -16%, except 2 of 31 subjects who decreased >-16%. We confirmed that the results were not driven by ‘only a few patients may have responded that well’.

(5) In the absence of dietary fatty acid intake data changes in plasma fatty acid concentrations are difficult to explain by improved intestinal fatty acid absorption.

We appreciate the comment to reconsider the analysis. We are evaluating changes over time and this analysis is only a two measurement/data set-- from baseline to 3 months. A repeated measure ANOVA would be most suitable when there are comparisons among more than two groups or three or more time points (Park E et al., Korean J Lab Med, 2009; Bergh DD et al., Acad Manage J, 1995). We have chosen Student’s paired t-test for the time effects within each treatment groups (change from baseline to 3-months), and the Student’s unpaired t test for the difference between treatment groups in the 3-month change as the most suitable analysis since only two groups are compared. Therefore, we consider our analysis appropriate for this data set and subgroup analysis.

Baseline CFA Range Question: As a standard component of our analysis, we did visualize the baseline CFA data, looking for outliers, implausible data, and as you suggested, potentially obvious cut-points to consider. The results showed: n=4 subjects between 50-50.9% (lowest was 52%); n=5 between 60-60.9%; n=11 between 70-70.9%; and n=21 between 80-90%. There were n = 25 >90%, including n=14 between 90-<93%, and n=11 in the reference range for healthy people (≥93%). Our assessment was there was a good distribution across the %CFA results, there was no suggestion of specific clustering that was not expected in this outcome in CF/PI subjects, and all data were used in the analysis. The approach to use the median, with balanced sample size in both groups, was selected for the subgroup/exploratory analysis to determine if there was a different response to Encala vs Placebo intervention in subjects with more severe fat malabsorption at baseline.

We established a working definition of low and high baseline CFA for ‘use in this project’, and do not suggest it be used as a clinical endpoint. In the methods section, we have made it clear, that the cut point/definition is for the purpose of data analysis in the paper. CFA is not used in clinical care (burden of complete 3-day stool and accurate diet intake), so it is unlikely that clinicians will misinterpret/misuse this information for clinical care.

We recognize the unbalanced groups, however, this is an exploratory sub-group analysis of a study with balanced groups initially (110 subjects randomized to treatment [n=54] and placebo [n=56]). We suggest this analysis, with the reviewers-directed clarification and additions to the limitation section, is an acceptable approach for secondary, subgroup analysis. We agree and recommend future studies consider similar questions with appropriate design and adequate sample size.

Please see the response above (4) regarding the standard deviation values mentioned or we’ve also copied it here:

A higher standard deviation in the lower CFA group was expected given that the range was wider (52-87%) than that of the high CFA where the range was lower (>88 and <100%) and has a ceiling effect since no one is above 100%. As part of preparing this response, we carefully reviewed the CFA for each individual; there were no outliers nor implausible data. In subjects who improved, all but 2 subjects improved between +1 and +16%, except 2 of 31 subjects, who improved >16%. In subjects who had decreased CFA, all subjects decreased between -1 and -16%, except 2 of 31 subjects who decreased >-16%.

Low CFA Group: Upon review of individual, raw data, there were no outliers. Rather, there is a large range of CFAs in the low CFA group (52-87%).

Significant liver disease and any other significant diagnosis that might impact dietary intake, growth or body composition were exclusion criteria for subjects to participate in this study. There were no subjects with cholestatic liver disease.

(5) Even though the 3-day weighed dietary records performed before and after the intervention may be too short to definitely exclude possible effects of changes in dietary intake, they could be helpful in this regard. It is therefore suggested to include fatty acid intake data in the present manuscript. Studying the improvements in plasma FA in relation to improvements in CFA by using regression analysis could help support a mechanistic explanation of improved intestinal absorption proposed in the manuscript.

In nutrition studies, the prospective, three day, weighted food record is considered a high quality method to provide the food intake evidence/data (Johnson RK, Obesity, 2012;Yang JH et al., Nutr Res Pract. 2010; Crawford PB et al., JAND, 1994). Also note, our study specific research staff trains the subjects/families on food intake procedure, provides the digital food scales, measuring cups and spoons, and conducts the follow-up call when indicated, to clarify uncertain entry. The protocol did not call for modification to usual dietary intake, and the interventions (Encala and placebo products) were designed to be added to subjects self-selected, routine/preferred foods.

As suggested, we added the total, saturated, monounsaturated and polyunsaturated dietary fat intake data to Table 2 and dietary data from the full cohort of participants was reported in Stallings VA et al., J Pediatr Gastroenterol Nutr, 2016, supplemental Table 1. There were no significant differences in intake between groups for total, monounsaturated fat, polyunsaturated fat, or saturated fat classes. The subgroup subjects here are representative of the larger population and therefore, the lack of difference in dietary is not unexpected. Therefore, we respectfully disagree that changes in dietary intake were different between groups, and so unlikely to be the etiology of the plasma results.

Minor point:

λ-linolenic acid should read γ-linolenic acid.

Thank you. We have corrected this.

Minor

Perhaps not essential but it would it be good to include a rationale for looking at 3 month time-point. Clinical reason?, or more to do with immediate effects of the intervention?

The three-month time-point was selected because it was sufficient time for the intervention to have effect on the outcomes of interest, and the 3 month protocol visit provided the a sufficient number of subjects with all the data elements needed for the subgroup analysis.

We agree. We have changed the language from “secondary” to “subgroup”.

In the methods section, it would be help if the sections were labelled clearly or made distinguishable, ‘outcomes’, ‘statistical analysis’

As suggested, we have created the following labels: Participants, Inclusion/Exclusion Criteria, Design, Outcomes, and Statistics.

Result section, it’s worth mentioning that 86 participants completed 3 months (according to the Fig 1), however 66 children had data available for the ‘subgroup’ analysis.

We have included the following sentence to the results:

Eighty-six participants completed three months on either Encala or placebo, but this subgroup analysis included 66 children and adolescents (10.5±3.0 yrs, 39% female) with both baseline and 3-month visit CFA assessments (stool and diet records).

Lines 118 – 121 (page 9), also give actual numbers as you have done for low CFA group.

We added the actual values and amended the low CFA group language to:

For subjects in the higher baseline CFA subgroup, CFA% (-1.2±4.8% on placebo and -4.4±6.3% on Encala), stool fat (0.3±4.5 g/d on placebo and 2.9±7.3 g/d on Encala) and dietary fat intake did not change with either placebo or Encala treatment. WAZ improved in both groups (0.13±0.28 for placebo and 0.14±0.21 for Encala, p<0.05).

Attachment

Submitted filename: Response to reviewers PLOS ONE 032320.docx

Click here for additional data file.^{(35.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0232685.r003

Decision Letter 1

Maret G Traber

9 Apr 2020

PONE-D-19-35347R1

Improved Residual Fat Malabsorption and Growth in Children with Cystic Fibrosis Treated with a Novel Oral Structured Lipid Supplement: A Randomized Controlled Trial

PLOS ONE

Dear Dr. Tindall,

We would appreciate receiving your revised manuscript by May 24 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Maret G Traber, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please address the outstanding concerns from one of the reviewers, or provide a rationale why you think this action is unnecessary.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: The authors have addressed the concerns raised by this reviewer in their point-by-point reply and have added corresponding additions to their manuscript.

In contrast to the statement in the reply "As suggested, we added the total, saturated, monounsaturated and polyunsaturated dietary fat intake data to Table 2" and "There were no significant differences in intake between groups for total, monounsaturated fat, polyunsaturated fat, or saturated fat classes." these additional data do not appear in this table. These data should be added as described.

While it was proposed that "Regarding the unbalanced distribution of 63% of the treatment group showing CFA <88%, while only 39% did so in the placebo group ... this is related to the original (Encala/placebo group) randomization procedure for all subjects with sex/age blocks ... It did not include CFA, as CFA was an outcome variable ... We have included this in the limitations section", this explanation is still missing in the limitations section and should be appropriately addressed in a re-revised version of the manuscript.

Even though the inclusion/exclusion criteria have been previously reported (refs. 10-12), it would be helpful for the readers to include information regarding "Significant liver disease and any other significant diagnosis that might impact dietary intake, growth or body composition were exclusion criteria for subjects to participate in this study. There were no subjects with cholestatic liver disease" provided in the reply also in the manuscript as this is highly relevant for the present study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS One. 2020 May 8;15(5):e0232685. doi: 10.1371/journal.pone.0232685.r004

Author response to Decision Letter 1

15 Apr 2020

Reviewer #2: The authors have addressed the concerns raised by this reviewer in their point-by-point reply and have added corresponding additions to their manuscript.

We have amended the table to include these dietary fats and the statement "There were no significant differences in intake between groups for total, monounsaturated fat, polyunsaturated fat, or saturated fat classes” remains accurate.

We have added the following sentence to the discussion: “The unbalanced distribution was related to the original Encala™/placebo randomization procedure for all subjects with sex/age blocks that did not include CFA, as CFA was an outcome variable.” (line 227-229 in the marked up version)

We have added the following sentence under “inclusion/exclusion criteria”: “Liver disease and any other significant diagnosis that might impact dietary intake, growth or body composition were exclusion criteria for subjects to participate in this study. There were no subjects with cholestatic liver disease.”

Attachment

Submitted filename: PLOS LXS response to review 040920.docx

Click here for additional data file.^{(17.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0232685.r005

Decision Letter 2

Maret G Traber

21 Apr 2020

Improved Residual Fat Malabsorption and Growth in Children with Cystic Fibrosis Treated with a Novel Oral Structured Lipid Supplement: A Randomized Controlled Trial

PONE-D-19-35347R2

Dear Dr. Stallings,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Maret G Traber, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have responded acceptably.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0232685.r006

Acceptance letter

Maret G Traber

28 Apr 2020

PONE-D-19-35347R2

Improved Residual Fat Malabsorption and Growth in Children with Cystic Fibrosis Treated with a Novel Oral Structured Lipid Supplement: A Randomized Controlled Trial

Dear Dr. Stallings:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Maret G Traber

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File

(PDF)

Click here for additional data file.^{(1.7MB, pdf)}

S1 Checklist

(DOC)

Click here for additional data file.^{(217.5KB, doc)}

Attachment

Submitted filename: Response to reviewers PLOS ONE 032320.docx

Click here for additional data file.^{(35.5KB, docx)}

Attachment

Submitted filename: PLOS LXS response to review 040920.docx

Click here for additional data file.^{(17.6KB, docx)}

Data Availability Statement

All relevant data are within the manuscript.

[pone.0232685.ref001] 1.Turck D, Braegger CP, Colombo C, Declercq D, Morton A, Pancheva R, et al. ESPEN-ESPGHAN-ECFS guidelines on nutrition care for infants, children, and adults with cystic fibrosis. Clin Nutr. 2016;35(3):557–77. 10.1016/j.clnu.2016.03.004 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref002] 2.Borowitz D, Lubarsky B, Wilschanski M, Munck A, Gelfond D, Bodewes F, et al. Nutritional Status Improved in Cystic Fibrosis Patients with the G551D Mutation After Treatment with Ivacaftor. Dig Dis Sci. 2016;61(1):198–207. 10.1007/s10620-015-3834-2 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref003] 3.Mennella JA, Inamdar L, Pressman N, Schall JI, Papas MA, Schoeller DA, et al. Type of infant formula increases early weight gain and impacts energy balance: an RCT. Am J Clin Nutr. 2018;(in press). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref004] 4.Aldamiz-Echevarria L, Prieto JA, Andrade F, Elorz J, Sojo A, Lage S, et al. Persistence of essential fatty acid deficiency in cystic fibrosis despite nutritional therapy. PediatrRes. 2009;66(5):585–9. [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref005] 5.Bijvelds MJ, Bronsveld I, Havinga R, Sinaasappel M, de Jonge HR, Verkade HJ. Fat absorption in cystic fibrosis mice is impeded by defective lipolysis and post-lipolytic events. Am J Physiol Gastrointest Liver Physiol. 2005;288(4):G646–53. 10.1152/ajpgi.00295.2004 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref006] 6.Kalivianakis M, Minich DM, Bijleveld CM, van Aalderen WM, Stellaard F, Laseur M, et al. Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids. Am J Clin Nutr. 1999;69(1):127–34. 10.1093/ajcn/69.1.127 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref007] 7.Kalivianakis M, Minich DM, Havinga R, Kuipers F, Stellaard F, Vonk RJ, et al. Detection of impaired intestinal absorption of long-chain fatty acids: validation studies of a novel test in a rat model of fat malabsorption. Am J Clin Nutr. 2000;72(1):174–80. 10.1093/ajcn/72.1.174 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref008] 8.Kalivianakis M, Verkade HJ. The mechanisms of fat malabsorption in cystic fibrosis patients. Nutrition. 1999;15(2):167–9. 10.1016/s0899-9007(98)00155-5 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref009] 9.Lepage G, Yesair DW, Ronco N, Champagne J, Bureau N, Chemtob S, et al. Effect of an organized lipid matrix on lipid absorption and clinical outcomes in patients with cystic fibrosis. Journal of Pediatrics. 2002;141(2):178–85. 10.1067/mpd.2002.124305 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref010] 10.Schall JI, Mascarenhas MR, Maqbool A, Dougherty KA, Elci O, Wang D-J, et al. Choline supplementation with a structured lipid in children with cystic fibrosis: a randomized placebo-controlled trial. J Pediatr Gastroenterol Nutr. 2016;62(4):618–26. 10.1097/MPG.0000000000001004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref011] 11.Stallings VA, Schall JI, Maqbool A, Mascarenhas MR, Alshaikh BN, Dougherty KA, et al. Effect of oral lipid matrix supplementation on fat absorption in cystic fibrosis: a randomized placebo-controlled trial. J Pediatr Gastroenterol Nutr. 2016. 63(6):676–80. 10.1097/MPG.0000000000001213 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref012] 12.Bertolaso C, Groleau V, Schall JI, Maqbool A, Mascarenhas M, Latham NE, et al. Fat-soluble vitamins in cystic fibrosis and pancreatic insufficiency: efficacy of a nutrition intervention. J Pediatr Gastroenterol Nutr. 2014;58(4):443–8. 10.1097/MPG.0000000000000272 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref013] 13.Yesair DW. Phosphatidylcholine and lysophosphatidylcholine in mixed lipid micelles as novel drug delivery systems In: I H, G P, editors. Phospholipids. New York: Plenum Press; 1990. p. 83–106. [Google Scholar]

[pone.0232685.ref014] 14.Wang X, Dockery DW, Wypij D, Fay ME, Ferris BG Jr. Pulmonary function between 6 and 18 years of age. Pediatr Pulmonol. 1993;15(2):75–88. 10.1002/ppul.1950150204 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref015] 15.Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general U.S. population. Am J Respir Crit Care Med. 1999;159(1):179–87. 10.1164/ajrccm.159.1.9712108 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref016] 16.Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, Flegal KM, Guo SS, Wei R, et al. CDC growth charts: United States. Adv Data. 2000(314):1–27. [PubMed] [Google Scholar]

[pone.0232685.ref017] 17.Otten JJ, Hellwig JP, Meyers LD. Dietary DRI Reference Intakes: The Essential Guide to Nutrient Requirements. Washington, D.C.: The National Academies Press; 2006. [Google Scholar]

[pone.0232685.ref018] 18.Trabulsi J, Ittenbach RF, Schall JI, Olsen IE, Yudkoff M, Daikhin Y, et al. Evaluation of formulas for calculating total energy requirements of preadolescent children with cystic fibrosis. Am J Clin Nutr. 2007;85(1):144–51. 10.1093/ajcn/85.1.144 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref019] 19.Cohen J, Schall J, Ittenbach R, Zemel B, Scanlin T, Stallings V. Pancreatic status verification in children with cystic fibrosis: fecal elastase status predicts prospective changes in nutritional status. J Pediatr Gastroenterol Nutr. 2005;40:438–44. 10.1097/01.mpg.0000158222.23181.1c [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref020] 20.Lagerstedt SA, Hinrichs DR, Batt SM, Magera MJ, Rinaldo P, McConnell JP. Quantitative determination of plasma c8-c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol GenetMetab. 2001;73(1):38–45. [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref021] 21.Alshaikh B, Schall JI, Maqbool A, Mascarenhas M, Bennett MJ, Stalliings VA. Choline supplementation alters some amino acid concentrations with no change in homocysteine in children with cystic fiborosis and pancreatic insufficiency. Nutr Res. 2016;36(5):418–29. 10.1016/j.nutres.2015.12.014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref022] 22.Viola G, Mietto L, Secchi FE, Ping L, Bruni A. Absorption and distribution of arachidonate in rats receiving lysophospholipids by oral route. J Lipid Res. 1993;34(11):1843–52. [PubMed] [Google Scholar]

[pone.0232685.ref023] 23.Homan R, Hamelehle KL. Phospholipase A2 relieves phosphatidylcholine inhibition of micellar cholesterol absorption and transport by human intestinal cell line Caco-2. J Lipid Res. 1998;39(6):1197–209. [PubMed] [Google Scholar]

[pone.0232685.ref024] 24.Peretti N, Marcil V, Drouin E, Levy E. Mechanisms of lipid malabsorption in Cystic Fibrosis: the impact of essential fatty acids deficiency. Nutr Metab (Lond). 2005;2(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref025] 25.Slae M, Wilschanski M. Prevention of malnutrition in cystic fibrosis. Curr Opin Pulm Med. 2019;25(6):674–9. 10.1097/MCP.0000000000000629 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref026] 26.Borowitz D, Baker RD, Stallings V. Consensus report on nutrition for pediatric patients with cystic fibrosis. J Pediatr Gastroenterol Nutr. 2002;35(3):246–59. 10.1097/00005176-200209000-00004 [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref027] 27.Smyth R, Walters S. Oral calorie supplements for cystic fibrosis (review). Cochrane Database of Systematic Reviews. 2007(1):Art. No.:CD000406. [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref028] 28.Cystic Fibrosis Foundation Patient Registry: 2012 Annual Data Report. Bethesda, MD: Cystic Fibrosis Foundation; 2013 2013.

[pone.0232685.ref029] 29.Taylor JR, Gardner TB, Waljee AK, Dimagno MJ, Schoenfeld PS. Systematic review: efficacy and safety of pancreatic enzyme supplements for exocrine pancreatic insufficiency. AlimentPharmacolTher. 2010;31(1):57–72. [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref030] 30.Borowitz D, Konstan MW, O'Rourke A, Cohen M, Hendeles L, Murray FT. Coefficients of fat and nitrogen absorption in healthy subjects and individuals with cystic fibrosis. J Pediatr PharmacolTher. 2007;12(1):47–52. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref031] 31.Woestenenk JW, van der Ent CK, Houwen RH. Pancreatic Enzyme Replacement Therapy and Coefficient of Fat Absorption in Children and Adolescents with Cystic Fibrosis. JPediatrGastroenterolNutr. 2015. [DOI] [PubMed] [Google Scholar]

[pone.0232685.ref032] 32.Ng SM, Moore HS. Drug therapies for reducing gastric acidity in people with cystic fibrosis. Cochrane Database Syst Rev. 2016(8):CD003424 10.1002/14651858.CD003424.pub4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref033] 33.Stevens J, Wyatt C, Brown P, Patel D, Grujic D, Freedman SD. Absorption and Safety With Sustained Use of RELiZORB Evaluation (ASSURE) Study in Patients With Cystic Fibrosis Receiving Enteral Feeding. J Pediatr Gastroenterol Nutr. 2018;67(4):527–32. 10.1097/MPG.0000000000002110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0232685.ref034] 34.Heubi JE, Schaeffer D, Ahrens RC, Sollo N, Strausbaugh S, Graff G, et al. Safety and Efficacy of a Novel Microbial Lipase in Patients with Exocrine Pancreatic Insufficiency due to Cystic Fibrosis: A Randomized Controlled Clinical Trial. J Pediatr. 2016;176:156–61 e1. [DOI] [PubMed] [Google Scholar]

PERMALINK

Improved residual fat malabsorption and growth in children with cystic fibrosis treated with a novel oral structured lipid supplement: A randomized controlled trial

Virginia A Stallings

Alyssa M Tindall

Maria R Mascarenhas

Asim Maqbool

Joan I Schall

Roles

Abstract

Background

Objective

Methods

Results

Conclusions

Introduction

Methods

Participants

Fig 1. Outlines the enrollment of subjects, their allocation to treatment, disposition status and how they were analyzed in this trial.

Inclusion/exclusion criteria

Design

Statistics

Results

Table 1. Characteristics at baseline for CFA subgroups and for randomization groups.

Table 2. Outcomes by coefficient of fat absorption subgroups: Encala™ vs. placebo.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Maret G Traber

Roles

Author response to Decision Letter 0

Decision Letter 1

Maret G Traber

Roles

Author response to Decision Letter 1

Decision Letter 2

Maret G Traber

Roles

Acceptance letter

Maret G Traber

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases