The addition of medium-chain triglycerides to a purified fish oil-based diet alters inflammatory profiles in mice
Introduction
Essential fatty acids (EFAs), necessary for growth, development, and a variety of biological functions, must be consumed in the diet. Historically, alpha linolenic acid (ALA, n-3 PUFA) and linoleic acid (LA, n-6 PUFA) have been considered the two essential fatty acids. However, recent studies have shown that the downstream metabolic products of ALA and LA, docosahexaenoic acid (DHA) and arachidonic acid (ARA), respectively, are sufficient to sustain growth, development and reproductive function [1], [2], [3].
For individuals dependent on parenteral nutrition (PN), EFAs must be provided intravenously as a lipid emulsion. Commercially available lipid formulations in the United States have been exclusively soybean oil-based and contain high levels of LA and lower levels of ALA. Soybean oil-based lipid emulsions are implicated in the development of parenteral nutrition-associated liver disease (PNALD), a progressive and often lethal complication affecting up to 74% of patients dependent on long-term PN [4], [5], [6], [7].
Alternative lipid sources have been investigated as a potential strategy to prevent liver disease in PN-dependent individuals. A fish oil-based lipid emulsion, provided at 1 g/kg/day, has been shown to reverse the progression toward liver failure in patients with PNALD [8], [9], [10]. However, PN formulations provided with low doses of lipids necessitate higher calories from carbohydrates (dextrose) in order to meet the daily caloric needs of the PN-dependent individual, a consideration that is especially important in developing infants and children. PN formulations high in dextrose predispose patients to hyperglycemia and increased central venous catheter infections, hepatic steatosis, and glycosuria, complications that can lead to significant morbidity and mortality in an already-fragile population [11], [12], [13].
Recently, attention has been given to non-essential fatty acids, so called “EFA-free” lipids, which may be utilized as additives to lipid emulsions in order to augment the total fat calories provided and decrease the requirement for additional dextrose in PN. Ling et al. examined the metabolic effects of combinations of EFAs with hydrogenated coconut oil (HCO), an EFA-free lipid source. Rats fed a diet with HCO as the sole source of calories and then given an endotoxin challenge demonstrated a lower inflammatory response, as measured by serum IL-6 and C-reactive protein, when compared to rats fed HCO supplemented with DHA and ARA [14]. These findings are in keeping with Cook et al., who also demonstrated a decreased systemic inflammatory response in states of essential fatty acid deficiency in rats [15].
Medium chain triglycerides (MCT) are an alternative source of EFA-free lipids. Studies have shown benefits of MCT oil in the prevention of alcohol-induced liver injury in animal models by a mechanism that is not fully understood but may be related to decreased inflammation [16], [17], [18]. Others have demonstrated that dietary supplementation of MCT reduces the degree of endotoxin-induced intestinal and hepatic injury in rats [19], [20]. In 2012, the American Society of Parenteral and Enteral Nutrition identified MCT oil as a potentially beneficial additive to lipid emulsions containing soybean and/or fish oil and recommended further research in identifying an optimal ratio of n-3 fatty acids, n-6 fatty acids, and MCT [21].
The creation of an optimized lipid formulation, which provides sufficient EFAs to sustain growth and development, while modifying the intensity of inflammation, is of high value to the PN-dependent population. Purified fish oil (PFO) is a rich source of DHA and ARA that meets essential fatty acid requirements in rodent models [2], [22] and in humans [23], [24]. Herein, we utilize a murine model to study several dietary lipids including soybean oil, PFO, HCO and MCT to determine the effects of these lipid sources on growth, serum EFA levels, liver histology, and inflammatory profiles with and without an endotoxin challenge.
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Animals and diets
Adult male C57/Bl6 mice (Jackson Laboratories, Bar Harbor, ME) were housed five to a cage and maintained in a climate-controlled facility with a 12:12-h light–dark cycle for a period of four days of acclimation prior to experimentation. During this period, animals had free access to water and a standard rodent chow diet. Experimental protocols were approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee. Animals were then assigned by cage to one of the following
Food intake and body weight
There were no differences in food intake among the six groups for the duration of the study period. Average food intake per group remained consistent at approximately 3–4 g/mouse/day for 12 weeks. Animals in all dietary groups gained weight throughout the study. Baseline weights ranged from 23 to 24 g, and at the end of 12 weeks final weights ranged from 29 to 31 g (Fig. 1). No significant differences in weight were noted among the six groups. No animal demonstrated physical signs of EFA deficiency
Discussion
The incorporation of fish oil into PN formulations has emerged as a viable approach to treat parenteral nutrition-associated liver injury. However, critics of the use of fish oil monotherapy have speculated that fish oil alone may be insufficient to support growth and prevent EFAD. Short-term animal studies have shown appropriate growth and prevention of EFAD after dietary treatment with native fish oil in mice when given as at least 20% of total calories, which supplies sufficient ARA to
Author contributions
SC, BB, KG, and MP designed the study. SC, AO, PN, VN, MC and EC performed the animal study and tissue preparation/analysis. PM performed statistical analyses. All authors interpreted the data. SC prepared the manuscript. All authors reviewed and approved the final manuscript.
Funding
Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number F532 HD 071715-02. Further research support was also given by the National Research Service Award Institutional Training Grant of the National Institutes of Health under award numbers 2T32DK007754-14, 5 T32 HD007466-16 and 5 T32 HD007466-17, the Joshua Ryan Rappaport Research Fellowship, and the Boston
Disclosures
The authors have no disclosures.
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