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Original Articles: Nutrition

Mixed Lipid, Fish Oil, and Soybean Oil Parenteral Lipids Impact Cholestasis, Hepatic Phytosterol, and Lipid Composition

Isaac, Daniela Migliarese; Alzaben, Abeer S.†,‡; Mazurak, Vera C.; Yap, Jason; Wizzard, Pamela R.; Nation, Patrick N.§; Zhao, Yuan-Yuan; Curtis, Jonathan M.; Sergi, Consolato§; Wales, Paul W.∗,¶,||,#; Mager, Diana R.∗,†; Turner, Justine M.∗,†

Author Information
Journal of Pediatric Gastroenterology and Nutrition: June 2019 - Volume 68 - Issue 6 - p 861-867
doi: 10.1097/MPG.0000000000002313

Abstract

What Is Known

  • Prolonged parenteral nutrition is a risk factor for developing intestinal failure-associated liver disease.
  • Fish oil containing parenteral lipids have hepatoprotective effects compared with standard soybean oil formulations, although it is not known if this is because of lower phytosterol or higher omega-3 fatty acid content.

What Is New

  • Parenteral lipids containing fish oil decreased cholestasis, hepatic lipid, and phytosterol accumulation.
  • Higher hepatic phytosterol content was the most important independent predictor of decreased bile flow.

Parenteral nutrition provides lifesaving nutritional support for infants and children with intestinal failure. Parenteral nutrition, however, can contribute to intestinal failure-associated liver disease (IFALD) (1). IFALD affects up to 60% of infants on parenteral nutrition, leading to increased morbidity and mortality (1,2). The clinical and histologic spectrum of IFALD includes cholestasis, steatosis, fibrosis, cirrhosis, and liver failure (2–5). Mechanisms underlying IFALD are multifactorial (6), and are divided into patient-related and parenteral nutrition-related risk factors (1,3). Patient factors include prematurity, lack of enteral feeds, and sepsis (1,3). Parenteral nutrition factors include prolonged use, excessive calories, higher intravenous (IV) lipid delivery (>2.5 · kg−1 · day−1), and the type of long-chain fatty acids (FA) in the lipid emulsion (1,3,6,7). Substantial research has focused on the impact of type and dose of parenteral lipid, given that lipids are a main source of calories and are critical for the biological function of the brain, immune system, and overall growth (8–11).

Traditional soybean oil (SO)-based lipid emulsions, such as Intralipid (Fresenius Kabi, Bad Homburg, Germany), are predominant in ω-6 FA and higher in phytosterol content (12). Increased ω-6 FA and excess phytosterols in peripheral blood have been implicated in liver toxicity (13–18). Alternatively, studies have shown the benefit of ω-3 predominant emulsions containing pure fish oil (FO), such as Omegaven (Fresenius Kabi), in reversing steatosis and cholestasis in infants (1,9,19–21); however, concerns exist regarding the risk of a specific ω-6 FA deficiency in arachidonic acid (ARA) with pure FO in animal studies (8,22,23). Mixed lipid (ML) emulsions provide an additional option for parenteral nutrition, including SMOFlipid (Fresenius Kabi; 30% SO, 30% medium-chain triglycerides, 25% olive oil, and 15% FO). SMOFlipid may be considered superior for infants with IF, given that it provides a more balanced ω-6:ω-3 FA ratio than pure FO and SO as outlined in Table 1, theoretically reducing oxidative stress and improving immune function regulation (24,25).

TABLE 1
TABLE 1:
Key fatty acid and phytosterol composition of parenteral lipids

Using a neonatal piglet model, this study aimed to identify the differential impact on the development of parenteral nutrition-induced IFALD by comparing the effect of ML, ω-3 predominant pure FO, and ω-6 predominant SO intravenous lipid emulsions on hepatic phytosterol composition, hepatic FA and neutral lipid composition, and the relationship with bile flow and biochemical cholestasis.

METHODS

Animals and Surgical Procedures

Ethics approval was obtained from the Faculty of Agricultural, Life and Environmental Sciences Animal Policy and Welfare Committee at the University of Alberta (AUP00000153). The study was conducted in a bio-secure swine research facility, according to the guidelines of the Canadian Council of Animal Care (8,26). Male Yorkshire-Landrace-Duroc piglets, 2 to 5 days old, weighing 1.5 to 2 kg were studied according to standardized protocols as published previously (8). Table 1 outlines the key features of the 3 lipid emulsions utilized in the study (12,16,27). The amino acid and dextrose content of the parenteral nutrition was previously published (8). Twenty exclusively parenteral nutrition-fed piglets received macronutrient intakes that varied only with respect to the lipid emulsion and hence total energy. Lipid doses reflected the rapid growth rate of piglets: 5 g · kg−1 · day−1 in a piglet is equivalent to a lipid sparing regimen in a human infant of 1 g · kg−1 · day−1; 10 g · kg−1 · day−1 is equivalent to a more standard human infant dose of 2 g · kg−1 · day−1. Piglets were randomized to receive either the mixed lipid SMOFlipid at 10 · kg−1 · day−1 (ML10 group, n = 5), the ω-3 predominant Omegaven at 5 g · kg−1 · day−1 (FO5 group, n = 5), or the ω-6 predominant Intralipid at 5 g · kg−1 · day−1 (SO5 group, n = 5) or 10 g · kg−1 · day−1 (SO10 group, n = 5) for 14 days. The lipid emulsion was added to the amino acid-dextrose solution immediately before infusion to create an all-in-one admixture. Sow-reared piglets (n = 4) observed for 14 days provided a reference range for health and growth of neonatal piglets, but were not statistically compared with the experimental groups.

Bile Flow and Liver Chemistry

Blood samples were collected on day 14, for measurement of the following liver biochemistry tests using an automated method at a veterinary laboratory (IDEXX, Edmonton, Canada): γ-glutamyl transpeptidase (GGT), alanine-transferase (ALT), total bilirubin, and bile acids (8). Bile flow was determined at terminal laparotomy by the method previously described by Van Aerde et al (28).

Phytosterol Determination

To assess the primary outcome of interest, the total phytosterol content of livers, including both the ester-bound and free forms, were determined by gas chromatography-mass spectrometry (6890N GC system coupled with 5975B inert XL MSD; Agilent Technology, Santa Clara, CA). Freeze-dried liver samples (approximately 50 mg) were spiked with the internal standard 5-α-cholestane. Then 2 mL freshly prepared 2 mol/L methanolic KOH was added for saponification at 70 °C for 30 min. Phytosterols were extracted into hexanes and the dried extracts were derivatized with Sylon BTZ (Millipore Sigma Canada Company, Oakville, Ontario, Canada).

Histology: Hematoxylin and Eosin Staining and Oil Red O Staining

A blinded veterinary hepatopathologist independently reviewed liver specimens that had been stained with hematoxylin and eosin staining as described previously (8). The total liver histology score was based on the following 9 histologic parameters: vacuolar degradation, spotty necrosis, cholestasis, apoptosis, Kupffer cell hyperplasia, sinusoidal dilatation, portal edema, siderosis, and leukocyte infiltration (8,26). Each parameter was scored as 0, 1, or 2, with 0 representing normal and 2 representing severe (8,26). The maximum total possible score for each sample was 18 (8).

Neutral lipid accumulation in the piglet liver was assessed in frozen liver sections stained using Oil Red O solution for 20 minutes (Sigma-Aldrich, St. Louis, MO), counterstained in Mayer's Hematoxylin (Sigma-Aldrich) and mounted with Geltol (Chem-Tel, Tampa, FL). The same blinded hepatopathologist quantitatively assessed neutral lipid load using a modified published index used for bronchiolar lipid-laden macrophages (29). This scoring approach was applied directly to lipid staining of hepatocytes in both peri-portal and peri-central regions. At least 100 hepatocytes per region were assessed, and the Oil Red O score ranged from 0 to 4, where 0 represented absence of staining in all hepatocytes and 4 represented diffuse staining of greater than 100 hepatocytes.

Lipid Extraction and Fatty Acid Analysis

Liver tissues (approximately 100 mg per sample) were frozen in liquid nitrogen and stored at −80 °C until analysis. Liver tissues were homogenized and lipids extracted using a modified Folch method as previously described (30). The triglyceride (TG) fraction was separated on G-plates as previously described (31). Bands were visualized and scraped. Individual phospholipids (PL) were directly methylated. An internal standard of known quantity was added to the TG band (15:0) and samples were saponified and methylated. A standard was added to the scraped liver PL (17:0) followed by direct methylation. FA methyl esters were prepared using 14% (weight/volume) BF3/methanol reagent and separated by automated gas-liquid chromatography (Varian 3900, Varian Instruments, Georgetown, ON). Peaks of FA methyl esters were identified by comparisons with known standards (GLC-461, GLC-087, and GLC-512, Nu-Chek Prep, Inc, Elysian, MN). FA content of liver lipid classes were calculated using the area peak of the internal standard. Total PL, TG, and FA contained therein were calculated on both an absolute and percentage basis (32,33).

Statistical Analysis

Statistical analyses were performed using IBM SPSS Version 24 statistical software. The data were assessed for normality using the Shapiro-Wilk Test. Data were expressed as mean ± standard deviation (SD). One-way ANOVA was used for continuous variables, and chi-square test was used for categorical variables. Tukey Honestly Significant Difference test was used for post hoc analysis. Comparisons that were statistically significant using the post hoc Tukey Honestly Significant Difference test are mentioned in the corresponding results section. An alpha value of P < 0.05 was considered significant.

RESULTS

Anthropometric Variables

As shown in Table 2, there were no differences in body weight at baseline between FO5, SO5, SO10, or ML10 Groups (P = 0.09). At day 14, there was a significant difference in total body weight between groups (P = 0.002), with ML10 being the highest, followed by FO5, SO5, and SO10. On day 14, only the ML10 group had a mean weight (5.33 ± 0.26 kg) that was within the sow-reared piglet weight range of 5.16 to 6.3 kg.

TABLE 2
TABLE 2:
Anthropometrics, cholestasis markers, liver histology scores and hepatic sterol composition in parenteral nutrition fed piglets on day 14

Bile Flow, Liver Chemistry, and Histology

Table 2 reports biochemistry, bile flow, and liver histology scores. Bile flow was adjusted to micrograms of bile per gram of liver as previously described (8,34). Bile flow was significantly different between groups (P = 0.001), with post hoc analysis showing FO5 and ML10 were the same, but were higher than SO5 and SO10. Total bilirubin was the highest in SO10, followed by SO5 > FO5 > ML10 (P = 0.02). The SO5 and SO10 groups had 3-fold higher total-serum bile acids than FO5 and ML10 groups (P = 0.04). GGT was elevated in all groups compared with the reference range. GGT and total liver histology scores were highest in SO10 and lowest in ML10, but the differences were not significant between groups (P = 0.19 and P = 0.16, respectively). Cholestasis was only found on histology in the livers of 3 piglets, 1 SO5 and 2 SO10. The lowest neutral lipid staining ORO score was for FO5, but there was no significant difference between groups (P = 0.3).

Hepatic Phytosterol Composition

Table 2 shows the hepatic phytosterol composition in parenteral nutrition-fed piglets on termination day. A wide variation in phytosterol content was observed (Fig. 1). The SO10 group contained the highest level of each phytosterol. Cholesterol showed a significant difference overall (P = 0.03); however, Tukey HSD did not show significant differences between parenteral nutrition groups. Campesterol was significantly different between all groups (P < 0.0001), from highest to lowest: SO10, SO5, ML10, and FO5. Stigmasterol was not detected in the FO5 group, but was significantly different overall among the remaining groups (P < 0.0001), with SO10 being significantly higher than SO5 (P < 0.0001) and ML10 (P < 0.0001). Stigmasterol was not significantly different between SO5 and ML10 (P = 0.13). β-sitosterol was not detected in the FO5 group, but was significantly different among the remaining groups (P < 0.0001) with SO10 being the highest and ML10 the lowest.

FIGURE 1
FIGURE 1:
Differences in hepatic phytosterol content in piglets given 4 different parenteral nutrition lipid emulsions. P < 0.0001; ∗∗ P < 0.0001; ∗∗∗ P < 0.0001; ML10 = SMOFlipid at 10 g · kg−1 · day−1; FO5 = Omegaven at 5 g/ · kg−1 · day−1; SO5 = Intralipid at 5 g · kg−1 · day−1; SO10 = Intralipid at 10 g · kg−1 · day−1.

Hepatic Fatty Acid Composition: Triglyceride Fraction

Table 3 represents FA composition of the hepatic TG fraction in parenteral nutrition-fed piglets on termination day. Total hepatic FA content in the TG fraction was 4-fold higher in the SO10 group compared with the FO5 group (P = 0.03). As a proportion of total FA in the TG fraction, the FO5 group had the highest proportion of ω-3 FA (EPA, DHA, total ω-3; 35.7 ± 14.3%), followed by ML10 (18.9 ± 4.2%), which were significantly higher than SO5 (5.5 ± 0.7%) and SO10 (6.1 ± 1.1%; P < 0.01). The FO5 and ML10 groups had significantly lower proportions of total ω-6 FA and ω-6:ω-3 ratios than SO5 and SO10 groups (P < 0.01).

TABLE 3
TABLE 3:
Hepatic fatty acid composition of triglyceride fraction in parenterally fed piglets on day 14

Predictors of Bile Flow

Univariate predictors of bile flow were: campesterol (r = −0.77, P = 0.002), β-sitosterol (r = −0.74, P = 0.002), stigmasterol (r = −0.74, P = 0.002), ω-6 FA (r = −0.72, P = 0.001), and ω-3 FA (r = 0.59, P = 0.02; Supplementary Table 1, Supplemental Digital Content, https://links.lww.com/MPG/B609). In stepwise multivariate linear regression analysis, only campesterol independently predicted bile flow (β = −0.769 [−1.23 to −0.412], P = 0.001).

DISCUSSION

This study confirms the presence of fish oil in lipid emulsions to be associated with a reduction in phytosterol accumulation, reduced hepatic lipid storage, and a shift in the proportion of ω-3 and ω-6 FA. The study also revealed that reduced bile flow was more strongly associated with a higher hepatic phytosterol content over the negative association with hepatic ω-6 FA and the positive association with hepatic ω-3 FA.

Increased phytosterol content in parenteral lipids appear to be associated with the development of IFALD. Excessive phytosterols in peripheral blood have been associated with liver toxicity and IFALD with parenteral nutrition use (13–17,35). Reducing phytosterol levels may assist in reducing the liver toxicity associated with lipid emulsions. The results of our study further support the findings from El Kasmi et al (18), which showed plant sterols in lipid emulsions are a major factor leading to IFALD in a mouse model. Although ML does contain phytosterols, the lower amount of phytosterols in ML10 compared with the SO groups seemed adequate to prevent cholestasis and provide similar benefits as FO5. Vlaardingerbroek et al (36) reported IFALD to be prevented in a piglet model with the use of pure FO and ML compared with SO. In a prospective cohort of preterm infants, Pupillo et al (35) also found accumulation of plasma phytosterols to contribute to IFALD development.

The phytosterol effect on cholestasis and liver injury is mediated by mechanisms including: suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Farsenoid X receptor (FXR), and failure to up-regulate hepatic sterol exporters (ABCG5/8) (15,17,18,35,37,38). Inhibition of FXR results in decreased hepatocyte expression of the bile salt export pump (BSEP), the principal determinant of bile secretion, which is encoded by Abcb11, and decreased expression of multidrug resistance protein 2 (MRP2), the canalicular multispecific organic anion transporter involved in conjugated bilirubin excretion, which is encoded by Abcc2. β-sitosterol, campesterol, and stigmasterol are the 3 major phytosterols in SO. In 2007, Carter et al reported that only stigmasterol had a significant effect on FXR antagonism in vitro, whereas campesterol had only a minimal effect on FXR antagonism and β-sitosterol had no effect (37). El Kasmi et al (18) further investigated stigmasterol, but not campesterol, and demonstrated that stigmasterol promoted cholestasis, liver injury, and liver macrophage activation in a mouse model. Overall the reduction or absence of plant sterols in IV lipid emulsions is one of the mechanisms for hepatic protection in infants requiring PN, and is thus a promising strategy to reduce IFALD incidence and severity.

Although not statistically significant, the lowest total liver histology score was observed in the ML10 group and the lowest hepatic fat deposition via ORO score was observed in the FO5 group, which is consistent with the prevailing literature (39). Although these differences were not significant, it is of interest that histologic cholestasis was only found in piglets provided SO. Further, we maintain that reduced bile flow is a key feature of early IFALD, although rarely measured in similar studies (8,34,36). Of note, hepatic fibrosis and cholestasis tend to be the clinically relevant histologic findings in human neonates over hepatic lipid accumualtion (5). Hepatic fibrosis is an end-stage finding in IFALD, and was likely not observed in this study, given it was structured as an early model of IFALD. The rapid growth rate and greater calorie requirements of neonatal piglets may also lead to more hepatic steatosis as compared with that typically seen in human infant liver histology.

The hepatic TG FA composition reflected that of the parenteral emulsion composition. As a reference, the hepatic TG fraction FA profile of the sow-reared piglet group was similar to sow milk (predominant in palmitic acid [16:0], oleic acid [18:1 ω-9], and LA) (40). Importantly ARA, a critical ω-6 FA in neonatal growth and development, did not appear to be compromised in the short-term by using the lower ω-6 containing FO5 and ML10 emulsions. In addition, DHA is a key FA for neonatal neurodevelopment and the higher levels found in pure FO and ML may be beneficial, especially in the preterm infant (34). These data further supports the existing literature that an increased supply of ω-3 FA (EPA and DHA) and decreased supply of ω-6 FA (LA) in fish oil-containing emulsions is hepatoprotective (1,6,9,19–21,28,34,41).

Importantly, hepatic ω-3 FA were higher when lipids containing fish oil were provided, without compromising the amount of ARA. In the TG fraction, the proportion of LA was higher in the SO groups than FO5 and ML10, with no differences in the proportion of ARA between groups. Another shorter term study in neonatal piglets similarly did not find differences in the proportion of ARA between the liver of piglets on ω-3 and ω-6 predominant lipid emulsions for 7 days in total phospholipid fractions (42). One potential explanation for these differences could be that a longer time frame is needed to induce changes in the expression of Δ5 and Δ6 desaturase enzymes in the liver (43). This implies that differences in conversion of LA to ARA may occur, given the biologic importance of ARA to be maintained regardless of diet.

Of clinical importance, the amount of parenteral lipid provided to the ML10 group was twice that provided to the FO5 group but still provided a similar benefit for reducing cholestasis. This suggests lipid restriction is not required when using ML, enabling provision of more calories for growth while providing similar protection from IFALD and the benefits of ω-3 FA. Compared with prolonged use of pure FO, there also should be less concern for a deficiency in ARA with the provision of ML, given that lipid restriction is not required and that it has a more balanced ω-6:ω-3 FA ratio (8,22,23,44). Another strength of this study was the comparison of the 3 most commonly used pediatric lipid emulsions in an animal model that mimics the neonatal infant. Neonatal piglets have been used as a model for human infants because of similarities in anatomy, physiology, and metabolism.

Limitations of this study include use of male piglets only, small sample size, and wide variance noted in some of the phytosterol data. The duration of the study was short, however, it is important to highlight that 14 days of parenteral nutrition in piglets likely equates to a longer duration of parenteral nutrition exposure, given the more rapid rate of piglet metabolism (approximately 5 times that of a human infant). An additional limitation was the difference in total calorie provision between the groups given 5 mg · kg−1 · day−1 versus 10 mg · kg−1 · day−1 of lipid, which confounds weight comparisons. In previous studies, however, we could not increase the carbohydrate calories via glucose without inducing unacceptable piglet mortality from diabetic nephropathy thereby making isocaloric diets unfeasible (8). We did not assess body composition of the piglets to assist in differentiating weight gain from fluid retention or edema versus weight gain from muscle or fat deposition, which is another significant limitation in interpreting weight outcomes. Finally, the addition of α-tocopherol to Intralipid may prevent IFALD in preterm piglets (45) but was not assessed in this study. In prior research, however, we did not observe a benefit of adding tocopherol to Intralipid in a piglet model (46).

In conclusion, the results of this study have important clinical implications for parenteral nutrition-dependent neonates at risk for IFALD. ML and pure FO lipid emulsions were associated with reduced cholestasis, improved bile flow, lower hepatic lipid accumulation, and more balanced ω-6:ω-3 FA ratios. Unique to this study was the documentation of elevated hepatic phytosterol content in SO-fed piglets, followed by ML and then pure FO. Importantly, hepatic campesterol was the sole independent predictor of decreased bile flow. Reducing or resolving cholestasis improves clinical outcomes in children with IFLAD, including survival, improved growth, and prevention or delay in the need for liver/intestinal transplantation (47). Overall, these data provide additional support for the use of fish oil-containing lipids in infants with intestinal failure before the development of features of IFALD. Although ML10 and FO5 did not differ significantly from one another in most parameters, the ability to provide an unrestricted lipid dose at a lower cost is an advantage to using ML over pure FO in pediatric intestinal failure. It is likely that a role for pure FO will continue to exist for pediatric patients with the most severe IFALD as a rescue therapy. The routine use of ML over SO as a first line lipid emulsion for the prevention of IFALD in at-risk pediatric intestinal failure patients remains to be clarified.

Acknowledgments

Personal funding for Abeer Alzaben was provided by the King Abdullah Program for Scholarship-Saudi Cultural Bureau. The authors gratefully acknowledge the assistance of Jessica Josephson in data collection, and Kelly Ann Leonard and Glen Shoemaker for their technical assistance in gas chromatography for FA analysis.

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Keywords:

intestinal failure-associated liver disease; parenteral nutrition; pediatric; phytosterol; SMOFlipid

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