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Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0000000000000280
Original Articles: Hepatology and Nutrition

Growth and Fatty Acid Profiles of VLBW Infants Receiving a Multicomponent Lipid Emulsion From Birth

Vlaardingerbroek, Hester*; Vermeulen, Marijn J.*; Carnielli, Virgilio P.; Vaz, Frédéric M.; van den Akker, Chris H.P.*; van Goudoever, Johannes B.§

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Author Information

*Department of Pediatrics, Division of Neonatology, Erasmus MC, Sophia Children's Hospital, Rotterdam, The Netherlands

Department of Mother and Child Health, Salesi Children's Hospital, Ancona, Italy

Department of Clinical Chemistry and Pediatrics

§Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, Amsterdam, The Netherlands.

Address correspondence and reprint requests to Johannes B. van Goudoever, Emma Children's Hospital, AMC, c/o Room H7-282, PO Box 22660, 1100 DD Amsterdam, The Netherlands (e-mail: h.vangoudoever@amc.uva.nl).

Received 11 December, 2013

Accepted 11 December, 2013

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.jpgn.org).

The study described in this article is part of a larger trial that has been registered at www.trialregister.nl trial registration no.: NTR1445.

The authors report no conflicts of interest.

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Abstract

Objectives: Very-low-birth-weight (VLBW) infants are dependent on parenteral nutrition after birth. A parenteral lipid emulsion with a multicomponent composition may improve growth and neurodevelopment and may prevent liver injury, which is often observed in association with long-term parenteral nutrition with pure soybean oil. Our aim was to evaluate the safety and efficacy of a multicomponent lipid emulsion containing 30% soybean oil, 30% medium-chain triacylglycerol, 25% olive oil, and 15% fish oil compared with a conventional pure soybean oil emulsion in VLBW infants.

Methods: We conducted a double-blind randomized controlled trial in VLBW infants randomized to parenteral nutrition with the multicomponent (study group) or pure soybean oil emulsion (control group) from birth at a dose of 2 to 3 g · kg−1 · day−1 until the infants were receiving full enteral nutrition. We assessed efficacy by growth rates and measuring plasma fatty acid profiles (representative subset). Safety was evaluated by assessing hematologic and biochemical parameters, potentially harmful phytosterol concentrations (same subset), and clinical neonatal outcome parameters.

Results: Ninety-six infants were included (subsets n = 21). The multicomponent emulsion was associated with higher weight and head circumference z scores during admission. Plasma eicosapentaenoic acid and docosahexaenoic acid concentrations were higher in the study group. The hematological, biochemical, and neonatal outcomes were not different between groups, whereas the plasma concentrations of phytosterols were higher in the control group.

Conclusions: The multicomponent lipid emulsion was well tolerated and associated with improved growth and higher plasma fatty acid profiles in VLBW infants in comparison with the pure soybean oil emulsion.

During intrauterine development, >80% of brain docosahexaenoic acid (DHA) accumulates between 26 and 40 weeks of gestation, mainly because of preferential placental uptake and metabolism. As a consequence, infants born before 32 weeks of gestation have low concentrations of brain DHA and low hepatic stores of DHA to function as a fatty acid reserve (1). Postnatally, this shortfall can only partially be replaced by (par)enteral intake. The amount of DHA in breast milk, however, varies considerably, depending on the maternal diet. In addition, the volume of enteral feeding is low during the first neonatal period and during disease. Moreover, they may have a limited capacity to form long-chain polyunsaturated fatty acids (LCPUFAs) from the precursor fatty acids. Indeed, randomized controlled trials in preterm infants fed either preterm infant formula or human milk have shown beneficial effects of supplemental LCPUFAs, particularly DHA, on visual and cognitive development (2,3). Presently, all preterm formulae are supplemented with LCPUFAs.

During the first days of life, very-low-birth-weight (VLBW) infants are largely dependent on parenteral nutrition. The most commonly used parenteral lipid emulsion was developed several decades ago and is purely soybean oil based, which we now know to have several disadvantages. First, pure soybean emulsions are high in n-6 polyunsaturated fatty acids (n-6 PUFAs), but do not contain LCPUFAs. Excess intake of n-6 PUFAs may increase the synthesis of proinflammatory eicosanoids (4) and increase oxidative stress in critically ill preterm infants (5). This increase occurs while the preterm infant is already susceptible to oxidative stress because of high levels of free radicals (eg, following oxygen administration and infections) and immature antioxidant systems (6), which can lead to tissue damage.

In newer lipid emulsions, soybean oil is combined with other lipid sources, such as coconut oil (providing medium-chain triacylglycerols [MCT]), olive oil, and/or fish oil. Each type of lipid has different characteristics and potential benefits or disadvantages. For example, MCTs are hydrolyzed more quickly than long-chain triacylglycerols (7), whereas olive oil is rich in monounsaturated fatty acids and naturally contains the antioxidant vitamin E, which may improve oxidative stress defense. Fish oil has a higher n-3:n-6 ratio compared with soybean oil, especially because of the high content of the n-3 LCPUFAs DHA (22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3). These n-3 LCPUFAs are not only crucial for neurodevelopment but may also reduce inflammatory and thrombotic responses while protecting tissue microperfusion and immunity (8). In addition, fish oil may play a role in the treatment and prevention of parenteral nutrition–associated liver disease/cholestasis (9), possibly because of the absence of phytosterols. Soybean oil contains high concentrations of phytosterols. Phytosterols are sterol alcohols that occur naturally in plants as a component of the cell membrane (similar to cholesterol in mammals). Phytosterols are associated with impaired biliary secretion (10) and may be the “hepatotoxic” or “cholestatic” component of soybean oil–based emulsions (11). Thus, lipid emulsions that contain fish oil may have several advantages over pure soybean emulsions, potentially leading to better clinical outcomes; however, because the requirements of the individual LCPUFAs for parenterally fed preterm infants are not known, and none of the available lipid emulsions are specially designed for (preterm) infants, it is not known whether the presently available multicomponent emulsions contain the most adequate dose for optimal function and development. Clinical data in preterm infants are scarce, and large-scale randomized controlled trials in preterm infants are needed to determine whether early initiation of lipids and lipid emulsions that are not based purely on soybean oil improves short- and long-term outcomes (12).

In this randomized controlled trial, VLBW infants received total parenteral nutrition from birth and were randomized to either a multicomponent (30% soybean oil, 30% MCTs, 25% olive oil, and 15% fish oil) lipid emulsion or a conventional pure soybean oil–based emulsion. This multicomponent emulsion contains 5 weight% of n-3 LCPUFA, whereas the conventional emulsion contained no n-3 LCPUFA. We hypothesized that the multicomponent emulsion would increase n-3 LCPUFA plasma concentrations (primary outcome) and would yield lower plasma concentrations of direct bilirubin and phytosterols. In addition, we hypothesized that the multicomponent emulsion would be safe and well tolerated, as measured by plasma concentrations of triacylglycerols (TGs), platelet count, other biochemical parameters, and neonatal outcomes. Finally, we hypothesized that growth in the group receiving the multicomponent emulsion would not be impaired compared with growth in the group receiving the pure soybean oil–based emulsion.

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METHODS

Study Design and patients

A randomized controlled trial was performed between December 2008 and January 2012 at the neonatal intensive care unit of the Erasmus MC–Sophia Children's Hospital, Rotterdam, the Netherlands. Inborn VLBW infants (birth weight <1500 g) with a central venous catheter for clinical purposes were eligible for the study. Written informed consent was obtained from the parents. Infants with congenital anomalies (including chromosome defects) and infants with metabolic diseases or endocrine, renal, or hepatic disorders were excluded. The study protocol was approved by the institutional medical ethical review board.

Within 6 hours after birth, the attending physician enrolled the infant in the study by opening a sealed opaque randomization envelope that was stratified by weight (<1000 g and 1000–1499 g) and sex. The envelopes were made by a research pharmacist who was not involved in clinical care, and they were based on a computer-generated block randomization list with variable block sizes provided by a statistician. Randomization to the lipid group remained double-blinded throughout the study and the analyses.

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Nutritional Intervention

The infants were randomly assigned to a multicomponent emulsion (composed of 30% soybean oil, 30% MCTs, 25% olive oil, and 15% fish oil [SMOFlipid 20%, Fresenius Kabi, Germany]) (study group) or to a pure soybean oil emulsion (Intralipid 20%, Fresenius Kabi, Germany) (control group). The emulsions contained similar amounts of phospholipids, but the study emulsion had higher EPA and DHA contents (3% and 2%, respectively) compared with the trace amounts in the soybean emulsion (Table 1). Lipids were first administered at a dose of 2 g · kg−1 · day−1 within 6 hours after birth. On the second day, the lipid dose was increased to 3 g · kg−1 · day−1. Glucose (at least 4.0 mg · kg−1 · min−1) and amino acids (Primene 10%, Baxter, Utrecht, the Netherlands; 2.4–3.6 g · kg−1 · day−1) were also administered from birth. This study was part of a larger randomized controlled trial in which infants were also randomized to 2 different amounts of amino acids administered in combination with lipids. In the larger study, infants were randomized to standard amino acids, early lipids plus standard amino acids, and early lipids plus high-dose amino acids. Within the 2 early lipid groups, infants were randomly assigned to the 2 lipid types that are compared in the present study. Outcome parameters were determined a priori as stated in the registered trial design. During the study design phase, we decided to publish the interventions in 2 separate articles because outcome parameters in the lipid study are mostly specific for lipid type and not for amino acid dosage. The specific effects of amino acid dosage and early lipid administration were presented previously (13). The effects on some general outcome parameters are presented in supplemental Tables 1 to 3 (http://links.lww.com/MPG/A296). In the present article, we present the data of the blinded part of the study on the type of lipid emulsion. Inherent in the design, amino acid dose was equally distributed between the intervention and control groups and amino acid dose was not associated with the outcome parameters described here. Therefore, the amount of amino acids was ignored in the final analysis.

Table 1
Table 1
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Minimal enteral feeding was initiated on the day of birth; if possible, the infants progressed to full enteral nutrition in the days following, according to the local protocol. After the third day of life, the nutritional regimen was left to the discretion of the attending physician. Throughout the study, the local protocol called for temporarily lowering the parenteral lipid intake by 25% to 50% when plasma TG concentrations were between 3 and 5 mmol/L (265–442 mg/dL) and lowering the parenteral intake of amino acids by 25% to 50% when plasma urea concentrations were between 10 and 14 mmol/L (28–39 mg/dL). Parenteral lipid administration and amino acid administration were temporarily stopped when plasma TG concentrations exceeded 5 mmol/L (442 mg/dL) and when urea concentrations exceeded 14 mmol/L (39 mg/dL), respectively. These guidelines were based on expert opinion.

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Data Collection and Analysis

Baseline data on sex, birth weight, birth weight z score (14), gestational age based on the best obstetric measurement (ultrasound in early pregnancy or the last menstrual period), the number of prenatal steroid doses (0, 1, or 2), and the severity of illness upon entry into the study (Apgar score at 5 minutes and the Critical Risk Index for Babies score (15)) were recorded. Nutritional intake was recorded daily until the infants successfully progressed to full enteral feeding (ie, no parenteral feeding occurred for 2 consecutive days).

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Primary Outcome

In the larger study, nitrogen balances were the primary outcome (13). In the present study on lipid type, the primary outcome was the fatty acid concentration in plasma TGs and phospholipids. The primary outcomes of this lipid study were determined a priori and power calculations were based on these outcomes to determine the number of infants in each study arm.

In all participating infants included between November 2010 and January 2012, blood was obtained on day 6 (±1 day) and on day 14 (±2 days) for the analysis of fatty acid concentrations in plasma TGs and phospholipids; see below for methods. During this period, 27 infants were included. Four infants died before day 6. In 2 infants, blood could not be drawn and analyzed because of logistic reasons (absence of researcher). Infants were randomly assigned to the different lipid emulsions and both attending physicians and analytical staff were blinded to the randomization. Therefore, the subset of 21 patients is representative of the total study population.

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Secondary Outcomes
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Safety Parameters

Safety was evaluated by measuring hematological and biochemical parameters, phytosterol concentrations, and clinical neonatal outcomes.

Blood gas and whole blood glucose concentrations were measured daily until postnatal day 7. Platelet count, electrolytes, plasma urea, TG, bilirubin (total and direct), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and cholesterol concentrations were measured on days 2, 4, and 7 (cholesterol on days 2 and 4 only). These analyses were performed in the hospital clinical chemistry laboratory using standard validated methods.

In all infants from whom blood was drawn for fatty acid concentrations (primary outcome), plasma phytosterol concentrations were analyzed in 60 μL of plasma collected on day 6 (±1 day) and on day 14 (±2 days); see below for methods.

The safety of the intervention was also monitored based on clinical outcome, that is, survival, duration of hospital stay, and neonatal morbidity. The definitions used for symptomatic patent ductus arteriosus, infant respiratory distress syndrome, bronchopulmonary dysplasia, necrotizing enterocolitis, late-onset sepsis, intraventricular hemorrhage, periventricular leukomalacia, and retinopathy of prematurity were described previously (13). Cholestasis was defined as a direct bilirubin concentration >20% of the total bilirubin concentration.

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Efficacy Parameters

In addition to plasma fatty acid profiles (primary outcome), efficacy was assessed using in-hospital growth rates. The time to regain birth weight, the growth rate during the first 28 days of life, the gain in lower leg length (knemometry) during the first month of life, and growth until discharge home (or until 40 weeks’ postmenstrual age, whichever event occurred first) were recorded. Growth until discharge home was adjusted for the number of days admitted to the hospital. Growth z scores were calculated (14). The gain in lower leg length was measured in triplicate as soon as possible after birth and weekly thereafter using a knemometer (absolute digimatic caliper, Mitutoyo, Best, the Netherlands), as described by Dixon et al (16). To determine the gain in lower leg length while taking into account the variations in the timing of the weekly measurements and some missing values, linear regression coefficients of the mean gain in length (millimeters per day) were calculated.

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Analytical Methods for Measuring Plasma Fatty Acid and Phytosterol Concentrations

Immediately after collecting the ethylenediaminetetraacetic acid blood, the samples were placed on melting ice and centrifuged (10 minutes, 3500g) to separate the plasma. The plasma used for the analysis of fatty acid composition was placed in tubes containing pyrogallol, closed under N2, and stored at −80°C until analysis. Plasma lipid classes and their fatty acid composition were analyzed as described previously (17). Briefly, lipids were extracted from the plasma following the method used by Folch et al (18) after adding appropriate internal standards for each lipid class. Lipid classes were isolated by thin-layer chromatography; plasma phospholipids and TG fatty acids were transesterified with HCl methanol. The individual fatty acid methyl esters were separated and identified using capillary gas chromatography (GC) (17). The data for plasma fatty acids with chain lengths from 8 to 24 carbon atoms were calculated as mole percentages.

Plasma sterols were analyzed after saponifying 60-μL plasma in 1 mL of 0.3-mol/L KOH in ethanol under incubation at 80°C for 2 hours in the presence of 65-nmol epicoprostanol as the internal standard. After cooling, 500 μL of distilled water was added, the solution was thoroughly mixed, and the aqueous layer was extracted with 2-mL hexane. This extract was evaporated until dry under nitrogen flow and resuspended in 120 μL of N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. This mixture was incubated at 80°C for 30 minutes. One microliter of this solution was injected into 2 separate systems: a gas chromatograph (Agilent HP6890N, Agilent Technologies, Amstelveen, the Netherlands) and a gas chromatograph-mass spectrometer (Agilent GC6890 + MSD5973) both equipped with a CPsil5, 25-m, 0.25-mm, and 0.25-μm GC column. Eluting trimethylsilylsterol ethers were detected with flame ionization detection and electron ionization mass spectrometry. Sterol concentrations were calculated using the known amount of internal standard and expressed as micromoles per liter using the GC data. The GC-mass spectrometry data were used to confirm the chemical identity of the different sterols.

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Statistical Analysis

The plasma fatty acid concentrations were the primary outcomes of the present study. A power calculation based on the n-3 fatty acid profile resulting from the administration of a multicomponent emulsion in a previous study (19,20) showed that 8 infants per group were required to find a statistically significant difference, with an α of 0.05 and a power of 0.80 (assuming an increase in the plasma EPA and DHA concentrations of 1.8% with a standard deviation of 1.4%). Because this study was part of a larger trial (13) and to allow for the comparison of safety outcomes, 48 infants were included in each group.

Depending on the distribution, the values are expressed as the mean ± standard deviation, as the median (interquartile range [IQR]), or as a number (percentage). The differences between groups were analyzed with χ2 tests, Student t tests, and Mann-Whitney U tests, as appropriate, on an intention-to-treat basis. The differences in time were analyzed with mixed models or Wilcoxon signed-rank tests. The significance level was set at P < 0.05. All statistical analyses were performed using SPSS version 20.0 (IBM SPSS, Armonk, NY).

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RESULTS

A total of 96 infants were randomly assigned to the study group (n = 48) or to the control group (n = 48) (Fig. 1). The demographics at baseline are displayed in Table 2. No differences were observed between group of infants included in the subset (primary outcome) and those not included in the subset. In the study group, 23 infants were randomized to the high amino acid group and 25 infants were randomized to the standard amino acid group. In the control group, an equal number of 24 infants were randomized to the high and standard amino acid groups.

Figure 1
Figure 1
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Table 2
Table 2
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Except for the allocated lipid emulsion, the average nutritional intake, including amino acid intake, was equal for both groups during the first week (Table 3) and until the second week (supplemental Table 4, http://links.lww.com/MPG/A296) of life. In approximately 40% of the infants in both groups, the amount of parenteral lipid administered was temporarily reduced because their TG concentrations exceeded 3 mmol/L during the first week of life (P = 0.818). Parenteral amino acid intake was temporarily adjusted in approximately 60% of infants in both groups because they had elevated urea concentrations (P = 0.935). The median duration of lipid administration was 11 days (IQR 9–14 days) in the study group and 12 days (IQR 9–16 days) in the control group (P = 0.825). In addition, there were no differences between the groups regarding the number of days required to reach full enteral feeding (median 13 [IQR 11–18] vs 16 [IQR 12–18] days, respectively; P = 0.233).

Table 3
Table 3
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Primary Outcome

The distribution of the total lipid classes in plasma (cholesterol esters, phospholipids, and TGs) did not differ between the groups (Fig. 2). On days 6 and 14, the concentrations of EPA and DHA in TGs and phospholipids were significantly higher in the study group compared with the control group (Figs. 3 and 4, supplemental Tables 5 and 6, http://links.lww.com/MPG/A296). With the expected ceasing of parenteral nutrition, the concentrations of these fatty acids were higher on day 6 compared with day 14 in the study group, but the concentrations did not change in the control group. The concentration of arachidonic acid (ARA) did not differ between the groups and remained constant over time. The TG and phospholipid ratios of n-3 to n-6 were significantly higher in the study group on days 6 and 14 than in the control group.

Figure 2
Figure 2
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Figure 3
Figure 3
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Figure 4
Figure 4
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Secondary Outcomes
Safety Parameters

On postnatal days 2, 4 (data not shown), and 7 (Table 4), there were no differences observed between the groups in acid-base status, platelet counts, and biochemical parameters, including TG, bilirubin (total and direct), and alanine aminotransferase; however, the potassium and ASAT concentrations were significantly higher in the study group on postnatal day 7, but they were still within the normal range for preterm infants. On day 6, the concentrations of individual phytosterols and total phytosterol were significantly lower in the study group compared with the control group (Fig. 5). On day 14, campesterol and β-sitostanol were still significantly lower in the study group.

Table 4
Table 4
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Figure 5
Figure 5
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Clinical outcomes, peak bilirubin concentrations, and mortality rates did not differ between the groups (Table 5). There was also no difference between the groups regarding the total length of hospital stay.

Table 5
Table 5
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Efficacy Parameters

The median time to regain birth weight was 8 days (IQR 3–11) in the study group and 8 days (IQR 6–12) in the control group (P = 0.359). At the time of discharge home, weight gain was significantly higher in the study group, resulting in an increase in the weight z scores from birth to the time of discharge in the study group, whereas the z scores for the control infants decreased (P = 0.012) (Table 6). There were no differences observed in the growth rates of head circumference and lower leg length, although the head circumference z scores for the infants in the study group increased significantly more from birth to discharge compared with the control subjects (P = 0.008).

Table 6
Table 6
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DISCUSSION

To the best of our knowledge, this study is the first randomized controlled trial comparing the administration of a multicomponent lipid emulsion to a pure soybean oil–based lipid emulsion in preterm infants from birth. In this study, we demonstrated that the administration of the multicomponent lipid emulsion prevented a decrease in the concentrations of DHA and EPA and resulted in a lower phytosterol concentration compared with the pure soybean oil emulsion, thus improving the plasma fatty acid profiles of VLBW infants. The general biochemical and neonatal in-hospital outcomes were comparable, demonstrating comparable tolerance of this multicomponent lipid emulsion. In addition, the multicomponent emulsion was associated with better gains in weight and head circumference for VLBW infants during their hospital stay compared with the pure soybean oil emulsion. Previous studies comparing the multicomponent emulsion with pure soybean oil in preterm infants with a birth weight of 500 to 2500 g did not demonstrate beneficial effects on growth (21–23). In these studies, however, only small amounts of parenteral lipids were supplied during the first week of life because lipids were generally not administered within the first few days and the target dose usually took several days to reach. Strikingly, the difference in growth was more significant from birth to discharge (around term age) than during the first 28 days of life, whereas the studied lipid emulsions were only administered during the first 2 weeks of life (until infants were receiving full enteral nutrition). No effect of amino acid intake on growth was found (supplemental Table 3, http://links.lww.com/MPG/A296) (13). We emphasize the possible explanations for the differences on growth outcome below.

In a recent meta-analysis, mixed lipid emulsions were associated with a 25% reduction in sepsis episodes compared with pure soybean oil emulsions (relative risk [RR] 0.75, 95% confidence interval [CI] 0.56–1.00, P = 0.05) (12). Our study was not powered to detect differences in the incidence of sepsis; however, when we added the data from our trial to the meta-analysis, we found a 28% reduction in sepsis episodes (RR 0.72, 95% CI 0.56–0.94, P = 0.02) based on a total of 293 VLBW infants. There is increasing evidence that n-3 LCPUFAs, particularly EPA and DHA found in fish oil, may prevent the development of inflammatory diseases in both adults (24) and infants (25,26) by affecting the immune response. Because the development of late-onset sepsis has been associated with lower in-hospital weight gain (27), a reduction in sepsis episodes with the use of a multicomponent lipid emulsion may have contributed to the improved growth rates observed in our trial. Whether this can also explain the delayed effect on growth is unknown. Because of the limitations in the amount of blood withdrawal in VLBW infants, we could not measure immune function. It would be interesting to focus on immune function and sepsis in a future study. In addition, ARA has been shown to positively affect growth and visual acuity (28), and a deficiency of ARA has been associated with decreased weight gain. Three trials have observed lower growth in preterm infants randomized to infant formula supplemented with n-3 LCPUFA without additional ARA versus control groups (29–31). Despite a significantly lower intake of the precursor of ARA (linoleic acid) and a fairly similar intake of ARA itself (Table 1), the plasma ARA concentrations in phospholipids and TGs were not significantly different between the 2 groups in our study. A recent Cochrane review demonstrated that the formula supplemented with both DHA and ARA did not hamper growth (32), and the present guidelines recommend supplementation of DHA combined with ARA in infant formula (33,34). This study was part of a larger trial on parenteral nutrition in VLBW infants. Infants in our lipid groups were also randomized to 2.4 or 3.6 g · kg−1 · day−1 of amino acids. Average amino acid dosage was not different between lipid groups in the study presented here (Table 3). Although protein accretion during the second day of life was significantly higher in the higher amino acid group (unpublished data), we could not demonstrate differences in growth between infants randomized to 2.4 of 3.6 g · kg−1 · day−1 amino acid during the first 28 days of life or until discharge home (supplemental Table 3, http://links.lww.com/MPG/A296) (13). Analyses on growth and type of lipid and amount of amino acid intake did not change results. Another explanation of improved growth in the study group may be because of different protein and energy intakes. In our trial, nutritional intake was recorded until infants were receiving full enteral nutrition, which was at a median age of 13 to 16 days. During this period, nutritional intakes were not different between groups, except for the allocated lipid emulsion. Because our study was a double-blind randomized controlled trial, differences in nutritional intake until discharge home, except for the allocated lipid emulsion, would be a matter of coincidence. Although small differences may occur, we believe that this cannot explain the differences in growth outcome. New trials should confirm whether lipid type is truly related to growth outcomes.

The safety parameters revealed no adverse effects of the multicomponent lipid emulsion. The platelet counts and biochemical parameters (including the incidence of hypertriacylglycerolemia) did not differ between the groups. These results are in agreement with previous studies, indicating that this multicomponent emulsion is safe and well tolerated (21–23). Unexpectedly, the mean ASAT concentrations were slightly higher in the group that received the multicomponent emulsion, but they were still within the normal range for preterm infants. We failed to demonstrate any beneficial effects of the multicomponent emulsion on biochemical markers of liver injury, but others have observed lower γ-glutamyltransferase (22) and bilirubin (total and direct) (21) concentrations. This difference may be explained by the relatively short duration (median 2 weeks) of parenteral nutrition in our study.

Emulsions containing fish oil are presently used as a rescue therapy in infants and children with parenteral nutrition–associated liver disease (PNALD) because fish oil has been demonstrated to reverse cholestasis and to reduce direct bilirubin concentrations in this condition (35). The preventive effects of fish oil on the development of PNALD have not yet been demonstrated in infants. Two infants in our study group and 2 in the control group developed cholestasis. All 4 of these infants received total parenteral nutrition longer than 14 days (160 and 22 days in the study group and 18 and 71 days in the control group), as opposed to an average of 15 days in the entire group. Several definitions of cholestasis are used in the literature. The standard definition is a cutoff value of direct bilirubin amounting >20% of total bilirubin. Others use a cutoff value of 1.5 to 2.0 mg/dL direct bilirubin in combination with the ratio between direct and total bilirubin (36,37), or a minimal time period during which direct bilirubin is elevated. When using the latter definitions, results did not change.

The mechanism of liver injury in PNALD is not yet completely understood. One suggested mechanism is that phytosterols in soybean oil may antagonize the bile acid–dependent activation of FXR target genes, leading to the persistent activation of bile acid synthesis and the accumulation of bile in hepatocytes (10,11), resulting in hepatic cholestasis (38,39). In our study, the phytosterol concentrations were indeed significantly lower in the infants receiving the multicomponent lipid emulsion compared with the group receiving the pure soybean oil emulsion. Another explanation for the development of PNALD is the high level of inflammatory n-6 fatty acids in pure soybean oil. Whether the effects of fish oil are caused by the presence of anti-inflammatory n-3 fatty acids or the low content of n-6 fatty acids and the absence of phytosterols is unclear (35).

In addition to the potential protective effects on liver function, n-3 fatty acids, especially DHA and EPA, play crucial roles in neurodevelopment (40,41) and immune function (4,42). To a certain degree, infants are capable of de novo synthesis of the LCPUFAs ARA and DHA from the precursors linoleic acid and α-linolenic acid, respectively (33,43); however, their synthesis rates are insufficient to maintain adequate plasma and erythrocyte membrane concentrations of these LCPUFAs, indicating that ARA and DHA may be conditionally essential fatty acids for preterm and term infants (33,44,45). Data from multiple studies in term infants have indicated that an exogenous supply of DHA that comprises at least 0.2% to 0.3% of total fatty acid intake enhances visual acuity and mental and psychomotor development (3,33,46). An even larger supply may be necessary for preterm infants because they have largely missed the physiologic intrauterine uptake of maternal DHA and ARA after the early termination of pregnancy (47) and because the endogenous synthesis rates of DHA and ARA from their precursors are insufficient (33,44,45). In utero, fetuses receive a constant supply of 5 to 10 weight% of DHA and 16 to 21 weight% of ARA between midterm and term gestation (48,49). In the present study, infants in the soybean oil group received no parenteral supply of DHA or ARA, whereas infants in the multicomponent lipid emulsion group received 3 weight% and 0.5 weight% of DHA and ARA, respectively. These amounts are significantly lower than the amounts the infants would have received while still in utero. Additionally, the multicomponent emulsion contains more EPA (the DHA precursor) compared with DHA, whereas in cord blood and human milk, EPA content is lower than the DHA content (49,50). Studies demonstrating the ARA, EPA, and DHA requirements of parenterally fed preterm infants are lacking and it is unknown whether the multicomponent emulsion used in this study provided the optimal composition of LCPUFAs. Supplemental studies in enterally fed (preterm) infants have resulted in LCPUFA supplementation of (preterm) infant formula. It would, therefore, be interesting to perform dose-response studies in parenterally fed infants to prevent DHA deficiency and to ensure an adequate plasma DHA content, including clinical measures on neurodevelopmental outcome.

In the present study, we demonstrated that parenteral nutrition with the multicomponent lipid emulsion prevented the decrease in DHA and EPA that was observed in association with the pure soybean oil infusion. These effects remained apparent on postnatal day 14, when a large proportion of the infants had already progressed to full enteral feeding. A previous study with a similar patient population and a study of infants and children receiving long-term parenteral nutrition also demonstrated higher plasma and red blood cell EPA and DHA concentrations after the infusion of emulsions partially composed of fish oil (19,21,51); however, another study did not observe a concomitant increase in red blood cell DHA after an infusion of the same type of emulsion (22), possibly because the researchers used a lower target dose of lipids (2.0 g · kg−1 · day−1). Whether the higher concentrations of plasma DHA will result in improved neurodevelopment will be investigated in the 2-year follow-up with the infants included in our study.

In conclusion, this study showed that the administration of an emulsion containing soybean oil, MCT, olive oil, and fish oil was associated with greater gains in weight and head circumference among VLBW infants during their hospital stay and prevented a decrease in weight and head circumference z scores from birth to the time of discharge home compared with a pure soybean oil emulsion. The biochemical and neonatal outcomes were comparable in the study group and in the control group, indicating that this multicomponent lipid emulsion was well tolerated in the direct postnatal phase; however, we did not observe a reduction in direct bilirubin concentrations. The plasma concentrations of phytosterols were lower in infants receiving the multicomponent emulsion compared with the control infants. The high LCPUFA content of the multicomponent emulsion prevented the decrease in plasma EPA and DHA concentrations that was noted in the pure soybean oil group. Long-term follow-up studies should be conducted to confirm the safety of the emulsion and the beneficial effects on neurodevelopment and visual function.

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Acknowledgments

The authors thank the children who participated and their parents for their consent. The nursing and medical staff was helpful in providing support for the study. The authors acknowledge Dr A. Niklasson for calculating the growth z scores and Dr P.E. Jira for advice on measuring phytosterols.

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Keywords:

cholestasis; growth; parenteral nutrition; phytosterol; preterm

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