title: A protocol for pressurized liquid extraction and processing methods to isolate modern and ancient bone cholesterol for compound-specific stable isotope analysis.
abstract: BACKGROUND
  Bone lipid compound-specific isotope analysis (CSIA) and bone collagen and apatite stable isotope ratio analysis are important sources of ecological and paleodietary information. Pressurized liquid extraction (PLE) is quicker and utilizes less solvent than traditional methods of lipid extraction such as soxhlet and ultrasonication. This study facilitates dietary analysis by optimizing and testing a standardized methodology for PLE of bone cholesterol.
  
  
  METHODS
  Modern and archaeological bones were extracted by PLE using varied temperatures, solvent solutions, and sample weights. The efficiency of PLE was assessed via quantification of cholesterol yields. Stable isotopic ratio integrity was evaluated by comparing isotopic signatures (δ13C and δ18O values) of cholesterol derived from whole bone, bone collagen and bone apatite. Gas chromatography/mass spectrometry (GC/MS) and gas chromatography isotope ratio mass spectrometry (GC/IRMS) were conducted on purified collagen and lipid extracts to assess isotopic responses to PLE.
  
  
  RESULTS
  Lipid yield was optimized at two PLE extraction cycles of 75 °C using dichloromethane/methanol (2:1 v/v) as a solvent with 0.25-0.75 g bone sample. Following lipid extraction, saponification combined with the derivatization of the neutral fraction using trimethylsilylation yielded nearly twice the cholesterol of non-saponified or non-derivatized samples. It was also found that lipids extracted from purified bone collagen and apatite could be used for cholesterol CSIA. There was no difference in the bulk δ13C values of collagen extracted from bone with or without lipid. However, there was a significant depletion in18O of bone apatite due to lipid presence or processing.
  
  
  CONCLUSIONS
  These results should assist sample selection and provide an effective, alternative extraction method for bone cholesterol that may be used for isotopic and paleodietary analysis. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Anthropology, University of Florida, 1112 Turlington Hall, Gainesville, FL, 32611, USA.
authors.forename: Ann O
authors.initials: AO
authors.lastname: Laffey
authors.affiliation: Department of Anthropology, University of Florida, 1112 Turlington Hall, Gainesville, FL, 32611, USA.
authors.forename: John
authors.initials: J
authors.lastname: Krigbaum
authors.affiliation: Department of Geological Sciences, University of Florida, 241 Williamson Hall, Gainesville, FL, 32611, USA.
authors.forename: Andrew R
authors.identifier: http://orcid.org/0000-0001-5137-4916
authors.initials: AR
authors.lastname: Zimmerman
mesh_terms: D000818:Animals; D001106:Archaeology; D001842:Bone and Bones / Q000737:chemistry*; D002247:Carbon Isotopes / Q000032:analysis*; D002417:Cattle; D002784:Cholesterol / Q000737:chemistry / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D059625:Liquid-Liquid Extraction / Q000379:methods*; D009587:Nitrogen Isotopes / Q000032:analysis*; D011312:Pressure; D013552:Swine
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D002784:Cholesterol