Wendel et al. BMC Pediatrics DY PROTOCOLOpen AccessEffects of nutrition therapy on growth,inflammation and metabolism in immatureinfants: a study protocol of a double-blindrandomized controlled trial (ImNuT)Kristina Wendel1* , Helle Cecilie Viekilde Pfeiffer1,2, Drude Merete Fugelseth1,3, Eirik Nestaas1,4, Magnus Domellöf5,Bjorn Steen Skålhegg6, Katja Benedikte Presto Elgstøen7, Helge Rootwelt7, Rolf Dagfinn Pettersen8,Are Hugo Pripp9, Tom Stiris1,3, Sissel J. Moltu1 and the ImNuT Collaboration GroupAbstractBackground: Current nutritional management of infants born very preterm results in significant deficiency of theessential fatty acids (FAs) arachidonic acid (ARA) and docosahexaenoic acid (DHA). The impact of this deficit onbrain maturation and inflammation mediated neonatal morbidities are unknown. The aim of this study is todetermine whether early supply of ARA and DHA improves brain maturation and neonatal outcomes in infantsborn before 29 weeks of gestation.Methods: Infants born at Oslo University Hospital are eligible to participate in this double-blind randomizedcontrolled trial. Study participants are randomized to receive an enteral FA supplement of either 0.4 ml/kg MCT-oil (medium chain triglycerides) or 0.4 ml/kg Formulaid (100 mg/kg of ARA and 50 mg/kg of DHA). The FAsupplement is given from the second day of life to 36 weeks’ postmenstrual age (PMA). The primary outcome isbrain maturation assessed by Magnetic Resonance Imaging (MRI) at term equivalent age. Secondary outcomesinclude quality of growth, incidence of neonatal morbidities, cardiovascular health and neuro-development. Targetsample size is 120 infants (60 per group), this will provide 80% power to detect a 0.04 difference in mean diffusivity(MD, mm2/sec) in major white matter tracts on MRI.Discussion: Supplementation of ARA and DHA has the potential to improve brain maturation and reduceinflammation related diseases. This study is expected to provide valuable information for future nutritionalguidelines for preterm infants.Trial registration: ID: NCT03555019. Registered 4 October - Retrospectively registered.Keywords: Arachidonic acid, Docosahexaenoic acid, Preterm, Nutrition of Neonatal Intensive Care, Oslo University Hospital, Oslo,NorwayFull list of author information is available at the end of the article The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence. The Creative Commons Public Domain Dedication waiver ) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Wendel et al. BMC Pediatrics 21:19BackgroundPreterm birth is the leading cause of child mortality inhigh and middle-income countries [1]. Very preterm infants need a combination of enteral and parenteral nutrition to meet their nutritional requirements duringhospitalization. Replacing the nutrition provided by theplacenta has proven difficult, resulting in postnatalgrowth restriction [2]. Growth and maturation of organsduring the last trimester rely on a steady supply of nutrients. Inadequate supply may lead to neurodevelopmentalimpairment, chronic lung disease, altered host defense,hypertension, and insulin resistance [3, 4]. The maintarget for feeding preterm infants is to achieve growthresembling normal fetal growth rates [5] as well as satisfactory functional development [6]. Despite establishedinternational recommendation, the nutritional management varies considerably between countries, hospitals,and even within institutions [7, 8]. Training in use ofparenteral nutrition (PN) and standardization of nutritional management is important to improve the implementation of nutritional guidelines [8]. Improving thequality and quantity of nutrition provided to extremepremature infants during their critical period of somaticgrowth and metabolic programming may be pivotal forshort-term clinical outcomes as well as long-term neurodevelopmental, cardiovascular and metabolic health. In a randomized, controlled trial conducted in our institution (the PRENU study) investigated the effect ofenhanced nutrient supply, including arachidonic acid(ARA) and docosahexaenoic acid (DHA), in very lowbirth weight (VLBW) infants compared to standard diet.The intervention group showed significant higher inhospital growth rates and catch-up growth in head circumference (HC) from birth to 36 weeks PMA [9] aswell as improved brain maturation on Magnetic Resonance Imaging (MRI) at term equivalent age (TEA) [10].Of note, this study was discontinued early due to a highoccurrence of a refeeding like syndrome among theintervention infants [11]. The risk of refeeding like syndrome has been confirmed by others [12–15] and theearly need for phosphate and potassium supplementation is highlighted in the revised European guidelines onPediatric Parenteral Nutrition [16, 17]. Moreover, thisunderlines the importance of conducting well-designedtrials on nutritional management in this patientpopulation.The long chain polyunsaturated fatty acids (LCPUFAs) linoleic acid and α-linolenic acid are essentialfatty acids (FAs) that must be supplied through the diet[18]. They provide energy and are used as precursors ofthe LC-PUFAs; ARA, DHA and eicosapentaenoic acid(EPA). Particularly ARA and DHA accumulate in thebrain during the last trimester and the first postnatalmonths, i.e. the period of rapid growth and brainPage 2 of 11development [19]. DHA is one of the main buildingblocks of the central nervous system including retinaand comprises 30–50% of neuronal plasma membranesby weight [20]. Extremely premature infants have lowendogenous capacity for conversion of linoleic acid andα-linolenic acid to ARA, DHA and EPA [21]. The lack ofadipose stores and limited provision of essential fattyacids through the parenteral solutions increase the riskof depletion. DHA deficiency may lead to reduced visualfunction and alterations in behavior or cognitive performance [22]. DHA and ARA supplementation in verypreterm infants have shown positive effects on growth,visual function and mental development [23].LC-PUFAs are not only essential cellular buildingblocks and important sources of energy, but they alsoact as signal molecules, important in sustaining and resolving inflammation [24]. Studies show that immatureinfants have elevated levels of inflammatory cytokinesduring the neonatal period, and that upregulated cytokine expression is associated with the development ofbronchopulmonary dysplasia (BPD), patent ductus arteriosus (PDA), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), white matter injury (WMI) ofthe brain and impaired neurodevelopmental outcomes[25–29]. A proposed mechanism behind this upregulated immune response is sustained activation andimpaired resolution of inflammation [27]. There is growing evidence that in addition to structural effects ongrowth and organ development, supplementation withARA and DHA, may reduce the incidence or severity ofBPD, ROP, NEC and WMI by modulating the immuneresponse [30–32]. Both omega-6 (ARA) and omega-3LC-PUFAs (DHA, EPA) serve as precursors for thesynthesis of bioactive mediators involved in immunemodulation. ARA is a precursor of pro-inflammatorymediators (such as leukotrienes of the n-4 series), and ofprostaglandins and thromboxanes of the n-2 series,which increase the vascular tone and promote plateletaggregation. However, ARA is also a precursor of lipoxins which are inflammation resolving mediators. Metabolites from DHA and EPA can modulate inflammationby decreasing the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) through the peroxisomeproliferator-activated receptor (PPAR) pathways. This inturn inhibits the nuclear transcription factor κB (NF-κB)and increases the production and secretion of antiinflammatory eicosanoids such as interleukin-10 [32].Resolvins, protectins, and maresins formed from bothDHA and EPA evoke anti-inflammatory and proresolving mechanisms, and they enhance microbialclearance [31].Perinatal infections or inflammation processes play animportant role in the pathogenesis of several comorbidities associated with preterm birth, such as BPD, PDA,

Wendel et al. BMC Pediatrics 21:19ROP, NEC and WMI [33]. Very preterm infants are susceptible to septicemia, possibly as a result of attenuatedinnate immune responses [27]. Interestingly, these infants also show signs of sustained systemic inflammationwith elevated pro-inflammatory cytokines [25–27, 34].Septicemia may be defined as “the host’s deleterious andnon-resolving systemic inflammatory response to microbial infection” [35]. The host response is similar to theactivation triggered by non-infectious tissue injuries likesurgery and ischemic reperfusion events [36]. The alarmin molecule, High Mobility Group Box 1 (HMGB1), isan activator of NF-κB and has been recognized as an important mediator of sepsis [36] and lung injury in preterm infants [37]. HMGB1 is released by necrotic cells,and sustains the inflammatory process after the resolution of the early stage of inflammation [37]. As mentioned, one of the anti-inflammatory potentials ofOmega 3-PUFAs is the ability to inhibit the activation ofNF-κB [32], and thereby possibly modulate an inappropriate inflammatory response.The pathogenesis of BPD is multifactorial, but intrauterine and postnatal growth restriction is an independent risk factor [38] disturbing pulmonary alveolar andvessel growth [39]. Along with sufficient early supply ofprotein and energy to promote growth, omega-3 PUFAsseem to protect against lung injury or reduce BPD severity by a DHA dependent activation of the PPAR pathways [37, 40], thereby accelerating lung maturation,pneumocyte growth and vasoproliferation [40]. Studiesshow conflicting results. Some studies suggest that lowDHA blood levels in premature infants are associatedwith increased incidence of BPD [41] and that fish oilsupplementation may improve lung function [42, 43].However, one study with enteral supplementation with60 mg/kg/d of DHA did not result in a lower risk ofBPD among preterm infants as compared to standardDHA intake and may have even resulted in a greater risk[44]. A controversy is the importance of balancing theamounts of ARA and DHA, since DHA supplementationalone may suppress ARA concentrations. Fetal plasmalevels of ARA are high, with an ARA:DHA ratio around3:1 at the beginning of the 3rd trimester compared toabout 2:1 in term infants. A low ARA:DHA ratio in extreme preterm infants (GA 28 weeks) has been associated with more severe BPD [45].ROP is a disorder of vascular development of theretina and the main reason for visual impairment inextreme premature infants. As for the lung, both nutritional and inflammatory factors seem to be importantmediators in disease progression. DHA is a majorstructural lipid in retina and accounts for approximately50–60% of the total fatty acid content within rod outersegments of photoreceptors [46]. Small RCTs haveshown that early lipid supply reduces the incidence ofPage 3 of 11ROP in VLBW infants [47, 48]. Two studies have demonstrated a significantly reduced incidence of ROP withfish-oil containing lipid emulsion as compared to standard soybean oil or a soybean and olive oil emulsion [49,50]. On the contrary, a trial that compared a multicomponent lipid emulsion (soybean oil, olive oil, fish oil andmiddle chain triglycerides) with a soybean and olive oilemulsion on the prevalence of ROP in extremely premature infants did not show any differences between thegroups [51]. Both decreased levels of DHA and ARAwere associated with the development of ROP. A recentRCT showed that enteral supplementation with DHAsignificantly reduced the incidence of stage 3 ROP inpremature infants [52].WMI of the brain accounts for the predominance ofneurological sequelae in surviving premature infants, including cerebral palsy and cognitive deficits [53]. The twomain mechanisms presumably responsible for the degeneration of immature oligodendrocytes are hypoxiaischemia and inflammation [54]. WMI of the prematurebrain include axonal damage, necrosis and periventricularleukomalacia (PVL) and is commonly categorized in diffuse WMI and focal WMI. MRI defined diffuse WMI ispoorly understood histopathological, but is thought tomainly result from damaged oligodendrocytes and lessfrom axonal damage [54]. In both forms of WMI an activation of microglia and astrocytes, as a diffuse inflammatory response is common [55]. Clinically, WMI isassociated with hemodynamic instability, poor postnatalgrowth, and inflammation [34, 54], suggesting that measures to optimize nutrition and reduce inflammationmight be beneficial in disease prevention. Other commonneurologic comorbidities in the preterm infant includesgerminal matrix hemorrhages, intraventricular hemorrhages (IVH) and diffuse atrophy, the cause of which aremultifactorial. Interestingly, inflammatory microglial andastrocytic activation following IVH has also been shownto be a de