PARENTERAL NUTRITION ASSOCIATED LIVER DISEASE: THE EFFECTS OF ALUMINUM CONTAMINATION AND LIPID COMPOSITION ON BILE ACID TRANSPORTERS
Hall, Amanda 1986-
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Rationale: Infants on parenteral nutrition (PN) risk developing parenteral nutrition associated liver disease (PNALD). The pathophysiology is not fully understood, but it is a multi-factorial disease that likely stems from sepsis, hepatic immaturity, pro-inflammatory (omega-6) lipids, and PN contaminants such as aluminum (Al). Dysfunction of bile acid transporters may be an important mechanism for the development of cholestasis and PNALD. The objective of this project is to examine the effects of Al on bile acid transporter proteins, both with and without pro-inflammatory lipids, thereby determining whether further efforts to reduce Al are needed for the treatment of PNALD. Methods: We conducted four different experiments, including a clinical survey, two piglet PN models, and one rat hepatocyte model. In our first experiment, we collected thirty samples of PN from a neonatal intensive care unit and analyzed the Al content using inductively coupled plasma mass spectrometry. For the piglet work, we conducted two randomized control trials using a Yucatan miniature piglet PN model. Newborn piglets (aged three to six days) were placed into two groups of seven to eight animals each. All groups were maintained on a strict PN diet for two to three weeks. One group in each study received PN with 24µg/kg/day Al, while the other received 63µg/kg/day of Al in PN. The first trial used omega-6 lipids in the PN for all piglets, while the second study used a mixed lipid solution. We chose five bile acid transporters (Mrp2, Bsep, Mrp3, Ntcp, and Oatp8), a cytoskeletal protein (radixin), and a nuclear receptor (FXR) as targets important in bile flow. All transporters were examined by qPCR, but only Mrp2 and Bsep were studied by immunofluorescence confocal microscopy, while Western blot evaluated Mrp2 protein. The serum was analyzed for total bile acids and for C-reactive protein (a marker of inflammation). In the second study, we also conducted transmission electron micrography to evaluate hepatocyte ultrastructure. Our last study used sandwich-cultured primary rat hepatocytes. The hepatocytes were placed into six groups to compare Al, omega-6 lipids, and mixed lipids, both individually and combined. After 60 hours of exposure to the Al and/or lipids, the hepatocytes were collected and similar bile acid transporters were evaluated using qPCR, with additional Western blotting for Ntcp and Oatp2, as well as a cholyl-lysl fluorescein functional assay for Mrp2. Results: Our clinical work demonstrated that 90% of neonatal PN samples had potentially unsafe levels of Al (mean: 14.02 (SD: 6.51) µg/kg/day), as compared to the FDA recommendations of <5µg/kg/day. In the piglet trial using omega-6 lipids, qPCR demonstrated more mRNA for Mrp2, Bsep, Mrp3, and Ntcp in the lower Al group, as compared to the higher Al group. In the piglet study with mixed lipids, qPCR showed more mRNA for only Oatp8, Ntcp, and Mrp3, in the lower vs the higher Al group. This second piglet study also had a greater rise in C-reactive protein (p=0.03) and shorter canalicular microvilli (p=0.01) in the group with higher Al contamination, as compared to the lower Al group. Neither study showed any difference in the amount of Mrp2 protein between the groups and there were minimal changes in immunohistochemistry. There was also no difference in serum bile acids in either study. The hepatocyte experiment showed a wide array of mRNA changes following exposure to Al and/or lipids. For Western blot, there was no difference in Ntcp protein between any of the groups. However, there was significantly more Oatp2 protein in the isolated Al group as compared to the Al + omega-6 lipid group (p=0.04). Finally, the cholyl-lysl fluorescein assay demonstrated the least excretion in any group containing mixed lipids. Conclusions: Neonatal PN still has significant Al contamination. In piglet studies, high amounts of Al in PN have a negative effect on the mRNA of many of the bile acid transporters, in addition to inducing structural changes, and increasing inflammation. The less-inflammatory mixed lipid solution only partially mitigates the effects of Al. The hepatocyte work also suggests that there may be a synergistic negative effect for Al and omega-6 lipids, at least for Oatp2. Overall, Al does have a negative effect on bile acid transporters and could plausibly contribute to the pathogenesis of PNALD. Further efforts to reduce the Al contamination in infant PN are warranted.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorMiller, Grant; Zello, Gordon
CommitteeRadomski, Marek; Arnold, Chris; Alcorn, Jane; Keith, Roger
Copyright DateJune 2017