Eight-wk-old MCAD+/+ and MCAD−/− mice were fasted for 18 h (cold tolerance experiments) or 24 h (serum glucose, free fatty acid, organic acid, and carnitine experiments) prior to analysis. Glucose concentration was measured in 10 μl sera using an Ektachem DT II system (Johnson and Johnson Clinical Diagnostics, Rochester, New York, United States). Total non-esterified fatty acids (NEFA) were measured by an enzymatic, colorimetric method (“NEFA-C” reagents, Wako Diagnostics, Richmond, Virginia, United States). The assay was modified to accommodate a reduced sample size (10 μl) and use of a microplate reader for measurement of optical density at 550 nm. Urine organic acid analyses were performed using gas chromatography-mass spectroscopy as previously described [22,30], except tetracosane (C24; Sigma, St. Louis, Missouri, United States) was used as the internal standard, and quantitative determinations were made based on comparison with synthetic calibration standards. Acylcarnitine analyses in serum and bile were conducted using electrospray tandem mass spectrometry [31,32].