Vitamin E in New-Generation Lipid Emulsions Protects Against Parenteral Nutrition-Associated Liver Disease in Parenteral Nutrition-Fed Preterm Pigs

Abstract

Introduction: Parenteral nutrition (PN) in preterm infants leads to PN-associated liver disease (PNALD). PNALD has been linked to serum accumulation of phytosterols that are abundant in plant oil but absent in fish oil emulsions.

Hypothesis: Whether modifying the phytosterol and vitamin E composition of soy and fish oil lipid emulsions affects development of PNALD in preterm pigs.

Methods: We measured markers of PNALD in preterm pigs that received 14 days of PN that included 1 of the following: (1) Intralipid (IL, 100% soybean oil), (2) Intralipid + vitamin E (ILE, d-α-tocopherol), (3) Omegaven (OV, 100% fish oil), or (4) Omegaven + phytosterols (PS, β-sitosterol, campesterol, and stigmasterol).

Results: Serum levels of direct bilirubin, gamma glutamyl transferase, serum triglyceride, low-density lipoprotein, and hepatic triglyceride content were significantly lower (P < .05) in the ILE, OV, and PS compared to IL. Hepatic cholesterol 7-hydroxylase and organic solute transporter-α expression was lower (P < .05) and portal plasma FGF19 higher in the ILE, OV, and PS vs IL. Hepatic expression of mitochondrial carnitine palmitoyltransferase 1A and microsomal cytochrome P450 2E1 fatty acid oxidation genes was higher in ILE, OV, and PS vs IL. In vivo (13)C-CDCA clearance and expression of pregnane X receptor target genes, cytochrome P450 3A29 and multidrug resistance-associated protein 2, were higher in ILE, OV, and PS vs IL.

Conclusions: α-tocopherol in Omegaven and added to Intralipid prevented serum and liver increases in biliary and lipidemic markers of PNALD in preterm piglets. The addition of phytosterols to Omegaven did not produce evidence of PNALD.

Keywords: cholestasis; life cycle; liver disease; neonates; nuclear receptors; phytosterols; research and diseases.

Figure 1

Plasma concentrations of total phytosterols and total alpha-tocopherol in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests. *P < .05 vs IL. ANOVA, analysis of variance; PN, parenteral nutrition; SEM, standard error of the mean.

Figure 2

Serum markers of hepatic injury and cholestasis including direct bilirubin, total bilirubin, bile acids, GGT, and ALT in preterm piglets after 14 days of treatment PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests. *P < .05 vs IL. ALT, alanine aminotransferase; ANOVA, analysis of variance; GGT, gamma glutamyl transferase; PN, parenteral nutrition; SEM, standard error of the mean.

Figure 3

Measurements of liver and serum triglycerides in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests. *P < .05 vs IL. ANOVA, analysis of variance; PN, parenteral nutrition; SEM, standard error of the mean.

Figure 4

Kinetics of plasma ¹³C-CDCA clearance measured between days 11–14 in preterm piglets given treatment of PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Values are means ± SEM; n = 8–10 pigs/group. *P < .05 vs IL. Panel A shows time-dependent changes in ¹³C-CDCA enrichment expressed as natural log of mole percentage excess (Ln mpe) during the 72-hour period; panels B and C show the parameters of whole-body half-life and mean retention time of ¹³C-CDCA calculated as described in the “Statistics” section. PN, parenteral nutrition; SEM, standard error of the mean.

Figure 5

Hepatic expression of genes involved in sensing (FXR, SHP), transport (BSEP, OSTα), and synthesis (CYP7A1) of bile acids in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Quantitative RT-PCR analysis showing relative transcript levels of BSEP, OSTα, CYP7A1, FXR, and SHP mRNA. Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests; *P < .05 vs IL. ANOVA, analysis of variance; BSEP, bile salt export pump; CYP7A1, cholesterol 7-hydroxylase; FXR, farnesoid X receptor; OST, organic solute transporter; PN, parenteral nutrition; RT-PCR, real-time polymerase chain reaction. SEM, standard error of the mean; SHP, small heterodimer partner.

Figure 6

Ileum expression of bile acid homeostatic genes (FXR, FGF19) and hormones (FGF19) in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Quantitative RT-PCR analysis shows relative transcript levels of FXR and FGF19 mRNA and plasma concentrations of FGF19 as described previously. Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests; *P < .05 vs IL. ANOVA, analysis of variance; FXR, farnesoid X receptor; PN, parenteral nutrition; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean.

Figure 7

Hepatic expression of genes involved in mitochondrial (CPT1A), peroxisomal (ACOX), and microsomal (CYP2E1) fatty acid metabolism in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Quantitative RT-PCR analysis shows relative transcript levels of mRNA. Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests; *P < .05 vs IL. ACOX, acyl-CoA oxidase; ANOVA, analysis of variance; CPT1A, carnitine palmitoyltransferase 1A; CYP2E1, Cytochrome P450 2E1; PN, parenteral nutrition; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean.

Figure 8

Hepatic expression of PXR target genes involved in microsomal bile acid metabolism (CYP3A29) and organic ion efflux (MRP2) in preterm piglets after 14 days of treatment with PN plus the following: Intralipid (IL), Intralipid plus vitamin E (ILE), Omegaven (OV), or Omegaven plus phytosterols (PS). Quantitative RT-PCR analysis shows relative transcript levels of mRNA. Values are means ± SEM; n = 8–10 pigs/group. Differences between treatment groups were analyzed using ANOVA, Kruskal-Wallis, and Mann-Whitney U tests; *P < .05 vs IL. ANOVA, analysis of variance; CYP3A29, cytochrome P450 3A29; MRP2, multidrug resistance-associated protein 2; PN, parenteral nutrition; PXR, pregnane X receptor; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean.

Figure 9

Fold-change in PXR luciferase assay in which HepG2 cells were transiently transfected with porcine PXR constructs, and luciferase activity was measured after 24 hours of treatment with PXR agonist rifampicin (0–50 µM) or natural d-α-tocopherol (0–125 µM) as described in detail in the “Material and Methods” section. All treated groups were compared statistically to control incubations with no treatment using ANOVA; *P < .05 vs control; regression analysis showed significant (P < .01) linear dose effects for both rifampicin and α-tocopherol. ANOVA, analysis of variance; PXR, pregnane X receptor.