Animals and management
Twelve dairy cows of the Holstein Frisian breed were included in this study. In order to reduce variability, all cows were selected from the same farm. The animals were housed in an open spaced barn comprising a total of 75 dairy cows. To be included in the study, the cows had to be multiparous, clinically healthy and not receiving any treatments at the beginning of the study. Also, they had to be confirmed pregnant with a known breeding date. Their health status was assessed by the herd veterinarian and clinicians of the Department of Farm Animals. All cows of the study were fed exactly the same diet and kept in the same barn. During the preceding dry period, the cows were fed grass silage, straw, hay and a mineral supplement. Two weeks before parturition, the cows were regrouped to the lactating animals receiving increasing amounts of a concentrate (0–1.5 kg). Post partum, the cows were fed grass and corn silage, beet pulp, soybean, grass, mineral supplement, and up to 4 kg concentrate. The body condition score was recorded concurrently with each blood sample collection.
The study was approved by the Veterinary Office of the Kanton Zurich and conducted in accordance with guidelines established by the Animal Welfare Act of Switzerland (permission No. 27/2013). Consent was obtained from the owner of the dairy farm to collect samples from his cows for the current study.
Collection of blood samples and liver tissue
Samples were obtained from the jugular vein at days −14, −7, 0, +7, +14, +21 and +28 relative to parturition. The samples were drawn always at the same time in the afternoon. Tubes supplemented with lithium heparin (Sarstedt AG and Co., Nümbrecht, Germany) were used to collect 10 ml of blood. The heparinized blood was centrifuged at 4000 g for 5 min. Separated plasma was stored in 2-ml tubes at −80 °C until analysis. Plasma samples were used for lipidome evaluation and measurement of -hydroxybutyrate and NEFA.
Liver biopsies were taken as described (Mølgaard et al. 2012) under ultrasonographic control and stained with hematoxylin-eosin (H & E). The histological lesions were staged into four categories: no abnormalities, mild fatty liver (only cells from one liver zone affected by lipid vacuoles), moderate fatty liver (fatty degeneration of the periportal to midzonal or midzonal to centrolobular areas), severe fatty liver (all three zones affected including the Kupffer cells).
Measurement of NEFA and β-hydroxybutyrate
Plasma concentrations of NEFA and β-hydroxybutyrate were determined by enzymatic analyses using the Wako NEFA-HR (2) (Wako Chemicals GmbH, Neuss, Germany) and the β-Hydroxybutyrate LiquiColor® kit (Stanbio Laboratory, Boerne, TX, USA), respectively. Spectrophotometric measurements were performed for both NEFA and β-hydroxybutyrate, using a Cobas Mira S Chemistry Analyzer (Roche, Basel, Switzerland).
Lipid nomenclature
For the designation of lipids, the common standard lipid language described by the Lipidomics Gateway (http://www.lipidmaps.org, National Institute of General Medical Sciences, National Institutes of Health) and by Schmelzer et al. (2007) was applied.
Chemicals and internal standards
All solvents were liquid chromatography-grade. Leucine-enkephalin was used as the lock mass at a concentration of 1 ng/μl in a solution of acetonitrile/water (50:50, v/v) supplemented with 0.1 % formic acid.
For the internal standards, an exogenous mixture of lipids was added to the organic solvent during the initial extraction step. These standards consisted of 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 17:0/0:0), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 19:0/0:0), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (PE 15:0/15:0), 1,2-diheptade-canoyl-sn-glycero-3-phosphoethanolamine (PE 17:0/17:0), 1,2-dipentadecanoyl-sn-glycero-3-phospho-(1’-rac-glycerol) sodium salt (PG 15:0/15:0), 1,2-diheptadecanoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG 17:0/17:0), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (PC 13:0/13:0), 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (PC 23:0/23:0), 1,2-dinona-decanoyl-sn-glycero-3-phosphocholine (PC 19:0/19:0) and 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphocholine [PC 16:0 (3me,7me,11me,15me)/16:0 (3me,7me,11me,15me)] purchased from Avanti Polar Lipids (Alabaster, AL, USA); 1,2,3-tripentadecanoylglycerol (TG 15:0/15:0/15:0) and 1,2,3-trihepta-decanoylglycerol (TG 17:0/17:0/17:0) were from Sigma-Aldrich (Buchs, Switzerland). The concentration of internal standards (350 nM) was calculated relative to the final amount of organic solvent in the extraction tube.
Sample extraction
Lipids were extracted from bovine plasma following a published protocol (Matyash et al. 2008). The extraction was validated by the addition of internal standards. For that purpose, 127 μl methanol was placed into 1.5-ml Eppendorf tubes and 23 μl of the 10 μM internal standard mixture was added (final concentration of 350 nM in the organic phase). A sample aliquot of 20 μl was added to each tube and the mixtures vortexed for 10 s. The mixtures were supplemented with 500 μl of methyl-tert-butyl ether and incubated for 60 min at room temperature in a thermo shaker (Vaudaux-Eppendorf AG, Schönenbuch, Basel) at 800 rpm. Then, a phase separation was induced by adding 125 μl of water and further vortexing. The samples were centrifuged for 10 min at 1000 g and 4 °C; 300 μl of the upper (organic) phase containing the lipids and non-polar components were collected and transferred into a new tube. The organic phases were dried in a vacuum centrifuge (Savant Speed Vac Plus SC 110A, Savant Instruments Inc., Holbrook, NY, USA), dissolved in 300 μl methanol and stored at −80 °C until measurements.
Liquid chromatography
An Acquity UPLC (Waters, Milford, MA, USA) system was used for ultra-pressure chromatographic sample separations. The plasma extracts were injected as triplicates and in a random order onto a HSS T3 column (Waters, Milford, MA, 1.8-μm particle, 100 × 2.1 mm id) heated to 55 °C. The average column pressure was 7000 psi. A binary gradient of two solvent mixtures was used for elution. Eluent A consisted of acetonitrile and water (50:40, v/v) with 10 mM ammonium acetate; eluent B consisted of acetonitrile and isopropanol (10:90, v/v) with 10 mM ammonium acetate. Eluent A was used for weak needle washes, whereas isopropanol was used for strong needle washes. The flow rate was 0.4 ml/min and the injection volume 10 μl. A linear gradient was performed for the sample analysis. The initial portion of the gradient was held at 60 % A and 40 % B. In the next 10 min, the composition was changed in a linearly ramped gradient (curve 6)–100 % B and held for 2 min. The system was switched back to the initial proportion (60 % A, 40 % B) and the column was equilibrated for 3 min. The total run time was 15 min.
Mass spectrometry
The UPLC inlet was coupled to a quadrupole time-of-flight mass spectrometer (SYNAPT G2 HDMS, Waters, MS Technologies Manchester, U.K.). Mass spectrometry was carried out following the protocol of Castro-Perez and Kamphorst (2010), whereby electrospray ionization (ESI) was employed in the positive and negative mode. A capillary voltage of 3 kV and cone voltage of 30 V were used for both polarities. The desolvation source conditions involved desolvation gas at 700 L/h and a temperature of 400 °C. The mass range during acquisition was 50–1200 Da and the signals were acquired in the centroid mode. Leucine-enkephalin was used as internal reference in each measurement.
During data acquisition, the first quadrupole was operated in a wide band RF mode, such that all ions were able to enter the T-wave collision cell. In this cell, two acquisition functions were applied. The first function with 5 eV resulted in non-fragmented ion molecules while the second function used a collision energy ramp of 20–30 eV to generate fragmented ions (MSE method). Argon gas was used for collision-induced dissociation. Using this interleaved acquisition, fragmented and non-fragmented ions could be used for quantification and initial ion identification. For final ion identification, a tandem mass spectrometry (MS/MS) method was applied by setting the energy ramp for collision-induced dissociation at 15–40 eV. The fragmentation pattern resulting from each parental mass was identified using the mass spectrometry database provided online the Lipidomics Gateway (http://www.lipidmaps.org, National Institute of General Medical Sciences, National Institutes of Health).
Data analysis
Descriptive statistics of the concentrations of NEFA and -hydroxybutyrate was performed using graphpad GraphPad Prism 6 (Graph Pad Software, La Jolla, CA, USA). The open source program MZmine (http://mzmine.sourceforge.net; Project GNU free software foundation, Boston, USA) was used for automatic alignment, denoising, deconvolution and extraction of peaks (Pluskal et al. 2010). All data were standardized using exogenous standards. The follow-up statistics were performed with the open source program R (R_Core_Team R Foundation for Statistical Computing 2014) and GraphPad Prism 6.