Animals and Diet Conditions
Two-month-old male Sprague-Dawley rats were purchased from Charles River Laboratories and maintained on a 12 hour light-dark schedule (lights on at 6 am). Controls were fed standard NIH chow and water, and hyperlipidemia was induced by feeding a high-fat, high-sugar chow (Dyets #101842; Dyets Inc., Bethlehem, PA), with water containing 20% high-fructose corn syrup, as described (Stranahan et al., 2008
). Rats were weighed once per week. After three months on the diet, rats were euthanized by decapitation under light Isoflurane anesthesia. Hippocampi were dissected out, frozen, and stored at -80°C prior to lipid extraction and analysis. All procedures were approved by the Animal Care and Use Committee at the National Institute on Aging and followed the NIH Guide for the Care and Use of Laboratory Animals
Serum Chemistry Analyses
Total postprandial serum cholesterol (high- and low-density lipoprotein; HDL, LDL) and triglycerides were measured using a Roche Cobas Fara II robotic chemical analyzer according to the manufacturer's specifications. Total cholesterol levels were determined using a kit, as were triglyceride levels, HDL levels, and LDL levels. All reagents for these analyses were purchased from Wako Diagnostics USA (Richmond, VA).
Lipid Extraction from Liver and Hippocampus
Rats on the high-fat diet with serum cholesterol values falling in the top third of the distribution (n=6 high-fat diet, n=6 control diet) were selected for measurement of lipids and oxidative stress markers. A modified Bligh and Dyer procedure was used for extraction of total lipids from brain and liver samples, as described (Cutler et al., 2004
). Briefly, each sample was homogenized at room temperature in 10 volumes of deionized water, then in 3 volumes of 100% methanol containing 30 mM ammonium acetate, and vortexed. Four volumes of chloroform then were added, and the mixture was vortexed and then centrifuged at 1,000 g for 10 minutes. The bottom (chloroform) layer was removed and analyzed by direct injection into a tandem mass spectrometer. Lipid extractions were performed using borosilicate-coated glass tubes, pipettes, and injectors.
Extracted lipids were analyzed using an electro-spray ionization API 3000 tandem mass spectrometer. The ion spray voltage (V) was 5,500 at a temperature of 80°C with a nebulizer gas of 8 psi, curtain gas of 8 psi, and the collision gas set at 4 psi. The declustering potential was 80 V, the focusing potential 400 V, the entrance potential −10 V, the collision energy 30 V, and the collision cell exit potential was 18 V. The MS/MS scanned from 300 to 2,000 atomic mass units (amu) per second at a step of 0.1 amu. Each species of sphingolipids, phospholipids, cholesterol esters, and lipid peroxides initially was identified by a Q1 mass scan, then by precursor ion scanning or neutral loss scanning of a purified standard. Samples were injected into the ES/MS/MS for 3 minutes, where the mass counts accumulated and the sum of the total counts under each peak was used to quantitate each species.
Sphingomyelins, ceramides, cholesterol, and cholesterol ester standards (C16:0, C18:0, C18:1, and cholesteryl-arachidonate C20:0) were purchased from Sigma. Ceramides dC18:1/C16:0 - C24:0, C24:1, phosphatidylcholine C16:0/C18:1, C18:0/C18:1, phosphatidylethanolamine C16:0/C18:1, phosphatidylglycerol C16:0/C18:1, phosphatidylserine C16:0/C18:1, phosphatidylinositol C16:0/C18:1, and phosphatidic acid C16:0/C18:1 were purchased from Avanti Polar Lipids (Alabaster, AL). Palmitoyl-lactosyl ceramide dC18:0/C16:0, stearoyl-lactosyl-ceramide dC18:1/C18:0, lignoceryl-glucosyl-ceramide dC18:1/C24:0, lignoceryl-galactosyl-ceramide dC18:1/C24:0, and sulfatide (stearoyl-galactosyl-ceramide-sulfate dC18:1/C24:0) were purchased from Matreya Inc. (Pleasant Gap, PA). 4-hydroxynonenol and adducts were purchased from Cayman Chemicals.
For the serum measures, raw values for cholesterol, HDL, LDL, and triglycerides were first converted to percent of control values, then compared across diet groups using bidirectional student's t-tests. For the mass spectrometry measures, peak values were first normalized to total lipids, then compared between the diet conditions using bidirectional student's t-tests. All statistical analyses were conducted using Graphpad Prism 5.0 (La Jolla, CA) with significance set at p < 0.05.