Bile Acid Profiling in Neonatal and Maternal Biological Fluids

Bile acid profiles for adult and neonatal mouse fecal samples, mouse breastmilk samples, and human breastmilk samples. Samples were collected to determine the bile acid composition in neonatal and adult feces and in murine and human breast milk.

Methods for Data

For mouse fecal bile acid profiling, stool was collected from naïve P4 and P5 neonatal mice and pooled, and from naïve 8-week-old adult mice. Each sample was mixed with 20 μL of acetonitrile per mg of raw material. The samples were then homogenized on a MM 400 mixer mill at 30 Hz for 3 min, followed by sonication for 3 min in an ice-water bath. The samples were centrifuged at 21,000 g for 10 min. 20 μL of the clear supernatant of each sample was mixed with 180 μL of the internal standard solution. For mouse breast milk bile acid profiling, lactating dams were milked daily on P8-P11 and pooled. On the day of milking, litters were separated from dams for 2 h and dams were given 2 international unit (IU)/kg of oxytocin diluted in saline (100 ml total volume) intraperitoneally (i.p.) and anesthetized using isoflurane. Eye lubricant was used for the duration of the procedure. Once anesthetized, milk was expressed from each mammary gland, collected using a micropipette, and frozen. Anesthesia time never exceeded 25 min total time. When the procedure was complete, the dams were monitored to ensure no side effects. An existing set of 24 human breast milk samples collected from women at various times 1-15 months following birth were used for bile acid profiling. The human study was approved by the Institutional Review Boards of the National Autonomous University of Nicaragua, León (UNAN-León, Acta Number 45, 2017) and the University of North Carolina at Chapel Hill (Study Number: 16-2079).  20 μL of each sample was mixed with 80 μL of acetonitrile. After vortexing, sonication for 5 min in an ice-water bath, and centrifugation at 21,000 g for 15 min, 80 μL of the clear supernatant was mixed with 40 μL of the internal standard solution and 880 μL of water. The mixture was loaded onto a reversed-phase solid-phase extraction cartridge (60mg/1mL). After sample loading under a positive pressure, the cartridge was washed with 2 mL of water. Bile acids were eluted with 1 mL of methanol under the positive pressure. The collected fraction was dried in a nitrogen gas evaporator. The residue was reconstituted in 40 μL of 50% acetonitrile. 10 μL aliquots of each fecal and milk sample solutions and each of the calibration solutions were then injected into an Agilent 1290 UHPLC system coupled to an Agilent 6495B QQQ mass spectrometer. The MS instrument was operated in the multiple-reaction monitoring (MRM) mode with negative ion detection. A Waters C18 column (2.1*150 mm, 1.7 μm) was used for LC separation and the mobile phase was 0.01% formic acid in water and in acetonitrile for binary-solvent gradient elution. A mixed solution of all the targeted bile acids at 10 μM of each compound was prepared in an internal standard solution of 14 deuterium-labeled bile acids in 50% acetonitrile. This solution was further diluted step by step to have 10 calibration solutions. Linear-regression calibration curves of individual bile acids were constructed with the data acquired from injection of the serially diluted calibration solutions. Concentrations of bile acids detected in the samples were calculated by interpolating the calibration curves of individual bile acids with the analyte-to-internal standard peak area ratios measured form injection of the sample solutions. Bile acid profiling was performed by Creative Proteomics.