Effects of choline, lipopolysaccharide, and docosahexaenoic acid on the lipidome of bovine precision-cut liver slices.

Choline and fatty acids (FA) are involved in phospholipid synthesis and may influence hepatic triglyceride (TG) deposition and secretion in dairy cows. Lipopolysaccharide (LPS) can induce inflammation and may increase the propensity for fatty liver in nonruminants. Liver was biopsied from pregnant Holstein dry cows (parity = 2.4 ± 0.5) and precision-cut liver slices (~200 μm) were incubated in serum- free M199 medium on titanium inserts under 95% oxygen/5% carbon dioxide within a roller culture incubator. Slices were incubated for 8h with either unsupplemented medium (no choline, LPS, or FA), 2 mM choline chloride (CC), 100ng O55:B5 LPS/mL (LPS), CC with LPS (CC+LPS), or CC with LPS and 200 μM docosahexaenoic acid (22:6; CC+LPS+DHA). Untargeted lipidomics was performed using liquid chromatography and mass spectrometry. For each experiment, 3 cows with 3 slices per cow were utilized (9 total reps/experiment). Protein- normalized, generalized log-transformed and auto-scaled data were analyzed using ANOVA and Fisher’s LSD comparisons. Partial least squares discriminant analysis (PLS-DA) was performed. Analysis of variance identified differences for 32 metabolites including TG (n = 32), phosphatidylcholines (PC; n = 6), and Lysophosphatidylcholine (LPC; n = 2). The PLS-DA model distinguished slices treated with DHA, but no major lipid signatures distinguished CC from LPS. Changes in TG and PC were observed in response to treatment (variable importance of projection scores >3.0). Several TG and PC were distinctively enriched in slices treated with DHA (e.g., TG 18:0/22:4/22:6, TG 16:0/20:5/22:6, and PC 40:7, and PC 42:10, respectively; false discover rate [FDR] < 0.001). Treatment with DHA resulted in increased concentrations of TG enriched in 22:6, with no observable differences between all other treatments (e.g., TG 14:0/22:6/22:6, 14:0/22:6/22:6; P < 0.05). Similarly, highly unsaturated PC were increased only with DHA supplementation (e.g., PC 40:7, PC 40:8, and PC 42:10; P < 0.05). The robust response of the hepatic lipidome to the ex vivo supplementation with DHA appears to be independent of LPS or choline availability.