Physiological response to fetal intravenous lipid emulsion

Abstract

In preterm neonates unable to obtain sufficient oral nutrition, intravenous lipid emulsion is life-saving. The contribution of post-conceptional level of maturation to pathology that some neonates experience is difficult to untangle from the global pathophysiology of premature birth. In the present study, we determined fetal physiological responses to intravenous lipid emulsion. Fetal sheep were given intravenous Intralipid 20® (n = 4 females, 7 males) or Lactated Ringer's Solution (n = 7 females, 4 males) between 125 ± 1 and 133 ± 1 d of gestation (term = 147 d). Manufacturer's recommendation for premature human infants was followed: 0.5-1 g/kg/d initial rate, increased by 0.5-1 to 3 g/kg/d. Hemodynamic parameters and arterial blood chemistry were measured, and organs were studied postmortem. Red blood cell lipidomics were analyzed by LC-MS. Intravenous Intralipid did not alter hemodynamic or most blood parameters. Compared with controls, Intralipid infusion increased final day plasma protein (P=0.004; 3.5 ± 0.3 vs. 3.9 ± 0.2 g/dL), albumin (P = 0.031; 2.2 ± 0.1 vs. 2.4 ± 0.2 g/dL), and bilirubin (P<0.001; conjugated: 0.2 ± 0.1 vs. 0.6 ± 0.2 mg/dL; unconjugated: 0.2 ± 0.1 vs. 1.1 ± 0.4 mg/dL). Circulating IGF-1 decreased following Intralipid infusion (P<0.001; 66 ± 24 vs. 46 ± 24 ng/mL). Compared with control Oil Red O liver stains (median score 0), Intralipid-infused fetuses scored 108 (P=0.0009). Lipidomic analysis revealed uptake and processing of infused lipids into red blood cells, increasing abundance of saturated fatty acids. The near-term fetal sheep tolerates intravenous lipid emulsion well, although lipid accumulates in the liver. Increased levels of unconjugated bilirubin may reflect increased red blood cell turnover or impaired placental clearance. Whether Intralipid is less well tolerated earlier in gestation remains to be determined.

Keywords: fetus; lipid metabolism; lipidomics; parenteral nutrition; preterm birth.

Figure 1. Increased plasma lipids during intravenous Intralipid

Fetal plasma cholesterol, phospholipid, and triglyceride levels in fetuses receiving intravenous Intralipid or Lactated Ringer’s Solution for 8 days. Number for Control female = 7, male = 4; Intralipid female = 4, male = 7. Individual data points shown with bar representing mean (sexes combined). Mixed measures three-way ANOVA (within factors by Day, and between factors by treatment and sex) with the Greenhouse-Geisser correction for sphericity. If indicated, multiple comparisons made with Bonferroni correction. For complete statistical analysis, see Table 1.

Figure 2. Fetal hemodynamic parameters following intravenous Intralipid

Fetal central arterial pressure, central venous pressure, and heart rate in fetuses receiving intravenous Intralipid or lactated Ringer’s solution. Number for control females = 7, males (M) = 4; Intralipid females = 4, males = 7. Individual data points shown with bar representing mean (sexes combined). Mixed measures three-way ANOVA (within factors by Day, and between factors by treatment and sex) with the Greenhouse-Geisser correction for sphericity. If indicated, multiple comparisons made with Bonferroni correction. For complete statistical analysis, see Table 1.

Figure 3. Arterial blood gas and chemistry parameters following intravenous Intralipid

Fetal arterial pH, plasma protein, hematocrit, total hemoglobin, partial pressure of CO₂ (PCO₂), partial pressure of O₂ (PO₂), O₂-hemoglobin (Hb) saturation, O₂ content, plasma protein, glucose, lactate, plasma insulin, plasma IGF-1, plasma IGF-2 and plasma norepinepherine in fetuses receiving intravenous Intralipid or lactated Ringer’s solution. Number for control females = 7, males = 4; Intralipid females = 4, males = 7; except for pH control females = 3, males = 3; Intralipid females = 2, males = 4. Individual data points shown with bar representing mean (sexes combined). Mixed measures three-way ANOVA (within factors by day, and between factors by treatment and sex) with the Greenhouse-Geisser correction for sphericity. If indicated, multiple comparisons made with Bonferroni correction. For complete statistical analysis, see Table 1.

Figure 4. Body and organ weights following Intralipid infusion

Fetal body, heart, and liver weights were measured following intravenous Intralipid or lactated Ringer’s solution for 8 days. Number for control females (F) = 7, males (M) = 4; Intralipid females = 4, males = 7. Individual data points shown with bar representing mean (sexes combined). Two-way ANOVA (between factors by treatment and sex). For complete statistical analysis, see Table 2.

Figure 5. Principal component analysis of fetal RBC lipids during Intralipid infusion

Relative concentrations of lipids within fetal RBCs were determined by LC-MS following intravenous Intralipid (n=11) or lactated Ringer’s solution (n=11) infusion for 8 days. Unsupervised principal component analysis clearly clusters control fetuses (days 0, 4, 8) and day 0 of Intralipid fetuses. Within the Intralipid group, day 4 is separate from day 8. There is no clear clustering by sex.

Figure 6. Lipid profiles of fetal RBCs following Intralipid infusion

Relative concentrations of lipids within fetal RBCs were determined by LC-MS. Following intravenous Intralipid (n=11), 37 of the 163 detected lipid species were elevated, and 15 were depressed compared with controls (n=11). Univariate analysis was performed in a linear mixed model 2 × 3 approach (experimental groups by time points) with individual fetus as a random effect (data from days 0 and 4 are in the supplement). All main effects and interaction terms are adjusted for multiple comparisons using the Benjamini and Hochberg false discovery rate correction.

Figure 7. Fetal liver function panel results following Intralipid infusion

Top: A panel of substances indicative of liver health were measured in fetal plasma on the terminal day. Liver damage may be indicated by elevated bilirubin, alkaline phosphatase, and aspartate aminotransferase, and by reduced albumin and globulin. Number for control females = 7, males = 4; Intralipid females = 4, males = 7. Individual data points shown with bar representing mean (sexes combined). Two-way ANOVA (between factors by treatment and sex). For complete statistical analysis, see Table 2. Bottom: Representative livers from a control (left) and an Intralipid-infused (right) fetus. Each square is 1 cm.

Figure 8. Oil Red O staining in fetal tissues

Accumulated neutral lipids and lipid droplets within parenchymal cells were stained with Oil Red O in fetal liver, lung, heart, and placental tissues. Raw and median values are shown. Number for control females = 7, males = 4; Intralipid females = 4, males = 7. Treatment effect was visually assessed by sex prior to analysis by Mann–Whitney test.