Comparison of two lipid emulsions on interleukin-1β, interleukin-8 and fatty acid composition in infants post gastrointestinal surgery: a randomized trial [version 1; peer review: 1 approved with reservations, 1 not approved]

Background: Nutritional support plays an essential role for recovery in infants who undergo gastrointestinal surgery. The current standard type of intravenous lipid emulsion (IVLE) used as parenteral nutrition is the mixture of medium-chain triglyceride (MCT) and long chain triglyceride (LCT) rich in ω-6. Studies showed that ω-6 is associated with higher level of proinflammatory cytokines, leading to increased mortality rate, morbidity rate, and postoperative recovery time. The latest generation of  emulsion is a mixture of MCT, LCT, olive oil (OO), and fish oil (FO) which may optimize the ω6/ω3 ratio. This study aimed to compare the effect of MCT/LCT/OO/FO IVLE to standard IVLE on IL1β, IL-8 and plasma fatty acid composition in infants who had undergone gastrointestinal surgery. Methods: A single-blind, randomised controlled, pretest-posttest design study was done in twelve subjects that were classified into two groups. Group 1 received standard IVLE, group 2 received MCT/LCT/OO/FO IVLE. The type of standard and MCT/LCT/OO/FO IVLE used in this study were Lipofundin 20% and SMOFlipid 20%, respectively, both administered for three consecutive days in 1-4 gram/kilogram/day. IL-1β and IL-8 were examined using ELISA while fatty acid composition was analyzed using gas chromatography tandem mass spectrometry (GC-MS).  Statistical analyses were performed using SPSS for Mac 23. Results: No statistical difference was found in age, gender, birth weight and diagnosis, between both groups. Leukocyte level was Open Peer Review


Introduction
Surgical interventions may stimulate physiological inflammatory response as body's attempt towards general recovery 1 . The balance of inflammatory response brings about good recovery, while excessive level of proinflammatory cytokines such as interleukin (IL) )-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α, may cause organ damage and severe complications, leading to the rise of postoperative mortality and morbidity rate 2 . De Mooij stated that IL-1β plays an important role in infection control, homeostasis, and tissue repair, while IL-8 plays an important role in inflammation and wound healing [3][4][5] .
Nutritional support is essential in wound healing and plays an important role in growth and development of an infant after undergoing gastrointestinal surgery 6 . Patients who could not receive oral and enteral nutrition for two days should be considered for parenteral nutrition 7,8 . The current standard type of fat emulsion used as parenteral nutrition is a mixture of mediumchain triglyceride (MCT) and soy oil enriched with long-chain triglyceride (LCT) 9 . This emulsion is rich in ω-6 and contains linoleic acid (LA, C18:2 ω -6) and also α-linolenic acid (ALA, C18:3 ω-3). Several studies showed that ω-6 is associated with impaired cell-mediated immunity and higher potential risk of elevated proinflammatory biomarkers and severe inflammatory response. These mechanisms may bring about the rise in mortality rate, morbidity rate and may also prolong the duration of treatment and postoperative recovery time 10-12 .
The latest generation of fat emulsion, SMOFlipid, is a mixture of MCT, LCT, olive oil (OO), and fish oil (FO), optimizing the ω6/ ω3 ratio. Some studies showed that OO exerts indirect anti-inflammatory effect by replacing ω-6 with oleic acid, while the addition of ω-3 from FO in soy-oil based fat emulsion may inhibit inflammatory reactions, i.e. reducing cytokine secretion and adhesion molecule expression and balancing the immune system, since it contains eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) 6,[13][14][15][16] . ω-3 may also act as a regulator of the immune system and mitigator of inflammation since it acts as a precursor for lipid mediator 16 . Our previous study showed lower levels of proinflammatory cytokines IL-6 and TNF-α in subjects who received the mixture of MCT/LCT/OO/FO compared with subjects who received MCT/LCT IVLE 17 . To our knowledge, there been no study regarding the effects of MCT/ LCT/OO/FO IVLE on IL-1β and IL-8 levels and fatty acid composition in infants who undergo gastrointestinal surgery, compared to those who receive standard IVLE.

Study background and recruitment
This single-blind, randomized controlled, pretest-posttest design, parallel-group with 1:1 randomization study aimed to compare the effect of MCT/LCT/OO/FO IVLE to standard MCT/LCT IVLE on IL-1β, IL-8 levels and fatty acid composition in infants who had undergone gastrointestinal surgery. The primary outcomes of this study were IL-1β and IL-8 levels and fatty acid composition while the secondary outcomes were hemoglobin, leukocyte, C-reactive protein (CRP) and albumin levels. This study was conducted in April-July 2020. Our subjects were infants who had undergone gastrointestinal surgery at Soetomo General Hospital, Surabaya. Parents or legal guardian were recruited to the study through referrals from physicians who then contacted the research team. The number of subjects was determined based on the formula for calculating number of samples in non-comparative numerical analytical study, with type 1 error of 5% and type 2 error of 10%, and the minimum number samples for each group were 5 subjects 18 . Inclusion criteria for this study included subjects whose parents were willing for them to participate in this study, had undergone gastrointestinal surgery, and had received parenteral nutrition for at least 3 days. Exclusion criteria included subjects who had chronic diseases and subjects who were allergic to fish, egg, soy and/or nut proteins. Adverse effects were minimal or rare. However, if any harm was seen in the subjects, they would be recorded and reported at the end of the trial. Vital signs and allergic reaction signs were evaluated every 12 hours for all subjects.

Ethical considerations
This study was approved by the Ethical Committee of Dr. Soetomo General Hospital (No. 1922/KEPK/11/2020, March 27 th 2020). Written informed consent obtained from the subjects' parents or legal guardian.

Participant allocation and blinding
Subjects were randomly assigned to one of two IVLE groups following simple randomization procedures (computerized random numbers, https://www.random.org). Determination of whether a subject would get MCT/LCT standard IVLE or MCT/LCT/ OO/FO was made by reference to a statistical series based on random sampling numbers drawn up by the primary investigator. Except the primary investigator and the pharmacist in charge, all subjects and staff were kept blind to IVLE assignment of the subjects. Eight-folded numbered papers were placed into opaque sealed envelopes to be chosen by the subjects' parents or legal guardian. Investigators and pharmacy staff opened the envelope and used the lipid emulsion assigned to that patient. The trial is registered at ClinicalTrials.gov, number NCT04511299, registered on August 13 th 2020. The protocol of this study can be seen at https://doi.org/10.17504/protocols.io.bknmkvc6. The flow of this research is shown on Figure 1.

Interventions and measurement
The type of MCT/LCT IVLE used in this study was Lipofundin 20% which contained 50% coconut oil as the source of MCT and 50% soy oil as the source of LCT. Lipofundin 20% was given intravenously for three consecutive days after gastrointestinal surgery at 1-4 gram/kilogram/day dosing 8 . The type of MCT/LCT/OO/FO IVLE used in this study was Smoflipid 20%, which contained 30% soy oil as the source of LCT, 30% coconut oil as the source of MCT, 25% olive oil, and 15% fish oil. SMOFlipid® was given for three consecutive days in 1-4 gram/kilogram/day dosing 8 . The lipids used in this study were obtained from the manufacturers: Bbraun Indonesia (Lipofundin 20% I and Fresenius Kabi Indonesia (Smoflipid 20%). The comparison of the standard fat emulsion and ω-3-enriched fat emulsion is shown on Table 1.
Before surgery, blood samples of subjects were drawn (3-4 cm 3 ) in order to measure their IL-1β, IL-8 levels, fatty acid composition, also hemoglobin, leukocyte, CRP and albumin levels. After surgery, subjects were assigned to either MCT/LCT IVLE or MCT/LCT/OO/FO IVLE for three consecutive days. On 3 rd day (72 hours) after surgery, blood samples of subjects were drawn again in order to measure the same outcomes posttreatment. Once the samples arrive to the laboratory, samples are allowed to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. L-1β and IL-8 levels in the serum were examined using the Quantikine HS ELISA by R&D Systems (Catalog Number HSLB00D and HS800) at wavelengths of 540 nm and 650 nm, respectively. Fatty acid composition was analyzed using gas chromatography tandem mass spectrometry (GC-MS). This examination was measuring the levels of free fatty acid in human serum quantitatively, including the arachidonic acid (AA)/EPA ratio, EPA, DHA, and AA.This method consists of two techniques, namely gas chromatography, which is a separation technique based on the degree of polarity and vapor point and mass spectrophotometry, which is a quadupole scanning spectrometer that can measure masses of 7-250 atomic mass units. Reagents used in this study were FAME Standard Mix (Supelco), GLC Nonadecanoic ISTD (Supelco), N-Hexane MS grade (Merck), Chloroform (Merck), Methanol Hyper Grade (Merck), Capillary Column and Helium Gas for GCMS.

Statistical analysis
Statistical analyses performed in this study were Mann-Whitney U-test, Fishers' Exact test, independent sample t-test and chisquare test using SPSS for Mac 23.0. The analyses of the IL-1β, IL-8 levels, fatty acid composition, also hemoglobin, leukocyte, CRP and albumin levels will be done by Mann-Whitney U-test or independent sample T-test as appropriate to test the significance between the two groups. A p-value less than or equal to 0.05 will be considered statistically significant. The analysis of subjects' characteristic will be done by Mann-Whitney U-test, independent sample T-test, Fisher's exact test or chi-square test as appropriate to test the significance between the two groups.

Subject characteristics
This study enrolled 12 subjects at Soetomo General Hospital Surabaya who had undergone gastrointestinal surgery and met the inclusion and exclusion criteria. The recruitment flow of the subjects is shown on Figure 2. The subjects were classified into two groups: group 1 received intravenous MCT/LCT lipid emulsion, and group 2 received intravenous MCT/LCT/OO/FO lipid emulsion. Subject characteristics are shown in Table 2.
No statistical difference was found in age, gender, birth weight, and diagnosis between both groups. De-identified subject characteristics, alongside all parameters measured in this study, are available as Underlying data 19 .

Primary outcomes
Mean IL-1β levels among subjects are depicted in Table 3; no difference was found in IL-1β levels between both groups before surgery (p = 0.873) and on day three after surgery (p = 0.873).
We also did not find any difference in changes in IL-1β levels within 3 days (p = 0.906) in both groups.
Furthermore, there was no significant difference in mean IL-8 levels between both groups before surgery (p = 0.688) and on day three after surgery (p = 0.494), and no difference in IL-8 levels changes within 3 days (p = 0.837) in both groups.
The analysis of fatty acid composition is shown on Table 4. No significant differences were observed in ω6/ω3 ratio, AA/EPA ratio, and EPA, DHA, and AA levels between both groups. Nevertheless, ω-6 level was significantly lower in MCT/LCT/FO/ OO IVLE group on third day after surgery group (p=0,048) compared to the standard IVLE.

Secondary outcomes
The laboratory parameters are shown in Table 5. According to preoperative laboratory assessment, no statistical difference was found in hemoglobin, leukocyte, CRP and albumin levels between both groups. There was a statistically significant difference in leukocytes between both groups 3 days after surgery (p=0.025). CRP level was significantly lower in MCT/LCT/OO/ FO group 3 days after surgery (p=0.01) and in changes within 3 days (p=0.016) compared to the standard MCT/LCT IVLE.

Adverse effects
There were no adverse effects reported from all subjects in the study. The content of EPA and DHA in ω-3 lipids may inhibit inflammatory pathways in several ways, such as inhibiting chemotaxis of leukocytes, expression of adhesion molecules, and adhesive endothelial-leukocyte interaction 16 .

Discussion
CRP is an acute-phase protein synthesized by IL-6 induction from hepatocytes. The CRP level spikes in acute traumatic condition, e.g. after undergoing surgical intervention. CRP levels reflect rapid changes which occur in inflammatory conditions. A study showed that in the majority of patients, CRP levels rise for 3-12 hours after surgery, peaking at 24-72 hours, and return to baseline in 2 weeks after surgery 22 .
This study did not find any significant difference in IL-1β dan IL-8 levels between the two groups.     A proposed explanation to why our result is inconsistent with most of the previous studies is that in our study, intravenous lipid emulsion was only given for 3 days, while in other studies, which yielded significant reduction in mean proinflammatory cytokine levels, intravenous lipid emulsion was given for at least 6 days. In this study, levels of IL-1β and IL-8 were examined preoperatively and 72 hours after surgery. According to Lin and Lowry, systemic inflammation, which occurs after surgery, may trigger proinflammatory and anti-inflammatory cytokines. Among all types of cytokines, TNF-α is the earliest to emerge, followed by IL-6 as the cytokine with the highest level amongst all. TNF-α and IL-6 levels peak in 1-2 hours after surgery 25 . Our previous study showed a significant difference in mean IL-6 levels between subjects receiving MCT/LCT and subjects receiving MCT/LCT/OO/FO 17 . Lin and Lowry stated that the half-life of IL-1β in systemic circulation is less than 10 minutes, making it more difficult to detect during stressful periods than TNF-α. Proinflammatory cytokine mediators, such as IL-8, are released as part of the inflammatory cascade initiated by IL-1 25 .
This study also observed that ω-6 levels in the ω-3-enriched IVLE group was lower than the standard MCT/LCT IVLE group, on third day after surgery. This result is in accordance with several previous studies. Skouroliakou et al. found a significantly lower mean ω-6 level in preterm neonates who received ω-3 enriched IVLE for 15 and 30 days compared to soybean oil on the third day after abdominal surgery 26 . Grim et al. showed a significant decline in ω-6 levels in 33 adult patients after major abdominal surgery who received ω-3-enriched fat emulsion for 6 days 27 . The composition of fatty acids in cell membrane phospholipids has a significant role on cellular responses and cell function. Membrane order and lipid raft assembly are affected by the fatty acid makeup of membrane phospholipids. The fatty acid composition of the second messengers that are obtained from membrane phospholipid influences their biological activity and potency. Fatty acids that released from membrane phospholipids upon cellular activation are forming some lipid mediators 28 . Nevertheless, our results on profiling of phospholipid fatty acid composition, such as ω6/ω3 ratio, EPA level, and DHA level, contradict previous studies which found a significant decline in ω-3-enriched fat emulsion group 27,29-31 . This discrepancy might be due to dissimilarity of subjects' characteristics, and the duration of parenteral nutrition administration. In those studies, the standard IVLE used were 100% LCT/soybean oil-based lipid emulsion, not MCT/LCT IVLE like in our study.
To our knowledge, this is the first study in Indonesia to compare the effect of MCT/LCT/OO/FO IVLE with MCT/LCT IVLE on proinflammatory IL-1β and IL-8 levels and fatty acid composition in infants who had underwent gastrointestinal surgery. Further studies are needed to determine the difference in pathogenesis between adults and infants after undergoing gastrointestinal surgery, which may be associated with the difference in effects of MCT/LCT/OO/FO IVLE on their profiling of fatty acid compositions.
There were no adverse event or serious adverse event reported in this study. Limitation of this study include that it is a single-centre study with a small sample size. Our study did not have a long period of follow-up and is not a double-blind study.

Conclusion
In infants who underwent gastrointestinal surgery, MCT/LCT/ OO/FO IVLE can significantly lower leukocyte, CRP and ω-6 levels, and is comparable with standard IVLE on IL-1β & IL-8 levels. This project contains the underlying data for the study in SAV and CSV formats. endogenously synthesized from fatty acid precursors released from membranes. With this background: how should a 3 day-infusion influence these metabolite concentrations?

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes wound healing period occurs within 0-3 days.
4. It should be written as serum free fatty acid (revised already). Details on this were already included in the paper. The blood samples were harvested after stopping infusion for 3 hours.
5. It is already changed.
6. It is already added.
7. This study is the first study in Indonesia to examine the effects of intravenous fat emulsions on metabolites (such as cytokines) in postoperative infants. To be honest, we are making this study the baseline for our future studies. 3 days was chosen because this is the initial inflammatory period in the wound healing period. Looking at the results of this study, we probably will make further studies using lipid emulsion in infants after surgery over a longer time such as 7 days, 14 days and more than 14 days.

Philip C. Calder
School of Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK This manuscript describes findings from a clinical trial in neonates who underwent surgery and then were randomised to receive one of two lipid emulsions intravenously over the course of 3 days. One lipid emulsion was a mix of coconut and soybean oils (control) and the other was a mix of coconut, soybean, olive and fish oils (treatment). The main outcomes were two cytokines (IL-1b and IL-8) and serum fatty acids. In addition a number of secondary outcomes are reported including white cell count and CRP. This study is of interest. A major limitation is sample size: there were 6 infants per group. The cytokines were not different between groups, but white cells and CRP were lower and omega-6 fatty acids higher at 3 days in the treatment group. The article is well written, data are clearly presented and the discussion refers to relevant existing literature.
Comments: I believe the abstract should report findings for serum omega-3 fatty acids. 1.
For the sample size calculation what outcome(s) was used? This is not stated. Usually an 2. effect size in a specific outcome is needed to calculate sample size. More detail is required about the fatty acid analysis. In the methods section you refer to "free fatty acids in serum" but in the Discussion you refer to "profiling phospholipid fatty acids". It is necessary to a) clarify what you have actually measured and b) provide details of how the serum was processed prior to gas chromatography.

4.
Stat analysis section is written in future tense (will be) but should be in the past tese (were/was).

5.
Stat analysis. The key comparison is between groups either at day 3 controlling for baseline or the change to day 3. It is not clear whether this comparison has been done. 6.
Data display. What are the errors shown: SD or SEM? 7.
Data that does nth ae normal distribution should not be shown as mean and SD/SEM but as median and IQR.
8. Table 4. What are the units for total fatty acids? 9. Table 4. Huge variation is apparent here bringing the small sample size into focus. For example in the treatment group ALA changes from 0.39 pre to 18.35 post but this almost 50-fold increase is not significant.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
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