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Frozen tibiae were homogenized in 40% methanol, and total lipids extracted from the bone using methanol-chloroform (1:2 ratio) with butylated hydroxytolulene (1 µM); 10-nonadecenoic acid 19:1 (Δ10 cis) (Nu-Chek Prep, Elysian, MN, USA) was added as a recovery standard at the time of extraction. Total lipids were saponified, and fatty acid methyl esters were prepared and analyzed by gas chromatography using an Agilent 7890 gas chromatograph equipped with a flame ionization detector (17). Methylated fatty acid standards were obtained from Nu-Chek Prep.
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