Depletion and enrichment of phytosterols in soybean oil lipid emulsions directly associate with serum markers of cholestasis in preterm parenteral nutrition-fed pigs

Abstract

Background: Clinical reports show a positive correlation between phytosterol concentrations and severity of cholestatic liver disease markers in infants during long-term administration of parenteral lipid emulsions. Establishing a causal link between phytosterols and cholestasis has been complicated by confounding factors of lipid emulsion load, fatty acid composition, and vitamin E in many of these studies. The goal of this study is to determine whether altering the phytosterol concentration within a common soybean oil-based emulsion will alter the onset and severity of cholestasis in parenterally fed preterm piglets.

Methods: Preterm piglets were administered, for 21 days, either enteral nutrition (ENT) or parenteral nutrition (PN) prepared from a soybean oil-based emulsion containing either 24.0% (depleted [DEP]), 100% (Intralipid; normal phytosterol [NP] concentration), or 144% (enriched [ENR]) total phytosterol concentration.

Results: At the end of the study, plasma and liver phytosterol concentrations were highest in the ENR group, followed by NP and then DEP and ENT. Serum direct bilirubin, serum bile acids, and γ-glutamyltransferase were higher in the ENR and NP groups compared with either DEP or ENT groups. All PN lipid groups showed evidence of mild hepatic steatosis but no change in hepatic expression of proinflammatory cytokines or Farnesoid X receptor target genes.

Conclusion: The increase in serum direct bilirubin was lower in the DEP group vs the lipid emulsions with normal or ENR phytosterols. Our results provide additional evidence that phytosterols are linked to an increase in serum markers of cholestasis in preterm PN-fed pigs.

Keywords: bile acids; bile salt export pump; parenteral nutrition; parenteral nutrition-associated cholestasis; phytosterols; soybean oil.

Figure 1.

Plasma and liver tissue phytosterol content in piglet during the 21-day study. Plasma total phytosterol concentrations in each treatment group during the 21-day study (top panel). Liver tissue total phytosterol content at the end of the 21-day study period. Values are expressed as mean ± S.E.M and as individual piglet values. Statistical analysis performed was repeated measures two-way ANOVA with post-hoc pairwise comparisons using Tukey’s test. Main effects of treatment (P<0.001), day (P<0.001), and treatment x day (P<0.001) were significant. ^aP<0.05 ENT vs. DEP; ^bP<0.05 ENT vs. NP; ^cP<0.05 ENT vs. ENR; ^dP<0.05 DEP vs. NP; ^eP<0.05 DEP vs. ENR; ENT, enteral, DEP, depleted; NP, Intralipid; ENR, enriched.

Figure 2.

Serum markers of cholestasis and hepatic cholangiocyte injury from blood samples taken at end of the 21-day study. Direct bilirubin and GGT in serum shown in each treatment group (top panel) and correlated with plasma phytosterol concentrations (bottom panels). Values are expressed as mean ± S.E.M and as individual piglet values. Statistical analysis performed was one-way ANOVA with post-hoc pairwise comparisons using Tukey’s test. ^aP<0.05 ENT vs. DEP; ^bP<0.05 ENT vs. NP; ^cP<0.05 ENT vs. ENR; ^dP<0.05 DEP vs. NP; ^eP<0.05 DEP vs. ENR; ENT, enteral, DEP, depleted; NP, Intralipid; ENR, enriched; GGT, gamma glutamyl transferase.

Figure 3.

Plasma, liver, gall bladder, and small intestine bile acid pools after the 21-day study (panel A). Correlation between plasma and liver bile acids and plasma phytosterol concentrations (panel B). Values are expressed as mean ± S.E.M and as individual piglet values. Statistical analysis performed was one-way ANOVA with post-hoc pairwise comparisons using Tukey’s test. ^aP<0.05 ENT vs. DEP; ^bP<0.05 ENT vs. NP; ^cP<0.05 ENT vs. ENR; ^dP<0.05 DEP vs. NP; ^eP<0.05 DEP vs. ENR; ^fP<0.05 NP vs. ENR; ENT, enteral, DEP, depleted; NP, Intralipid; ENR, enriched;

Figure 4.

Hepatic lipid accumulation and histopathology scoring. (A) Oil red O staining of liver and quantification of lipid accumulation by percent coverage of oil red O stain. (B) Heat map of hepatic histopathology scores. ENT, enteral, DEP, depleted; NP, Intralipid; ENR, enriched;

Figure 5.

Hepatic gene expression and serum FGF-19 concentration. (A) bile acid synthesis regulatory genes: FXR, farnesoid x receptor; FGF19, fibroblast growth factor 19; SHP, small heterodimer partner; (B) bile acid transport genes: NTCP, sodium taurocholate co-transporting polypeptide; BSEP, bile salt export pump; OST-α, organic solute transporter; MDR1, multi-drug resistance 1; MRP2/3/4, multi-drug resistance-associated protein 2/3/4; (C) phytosterol transport genes: ABCG5/8, ATP-binding cassette transporter G5/8; (D) bile acid synthesis genes: CYP8B1/7A1, Cytochrome P450 8B1/7A1; (E) pro-inflammatory cytokine genes: IL-6/1β, interleukin-6/1β; (F) serum FGF-19. Values are expressed as mean ± S.E.M and as individual piglet values. Statistical analysis performed was one-way ANOVA with post-hoc pairwise comparisons using Tukey’s test. ^aP<0.05 ENT vs. DEP; ^bP<0.05 ENT vs. NP; ^cP<0.05 ENT vs. ENR; ^dP<0.05 DEP vs. NP; ^eP<0.05 DEP vs. ENR; ^fP<0.05 NP vs. ENR; ENT, enteral, DEP, depleted; NP, Intralipid; ENR, enriched.