A protocol for pressurized liquid extraction and processing methods to isolate modern and ancient bone cholesterol...

Area of Science:

• Paleoecology
• Biogeochemistry
• Analytical Chemistry

Background:

• Bone lipid and stable isotope analyses are crucial for reconstructing past environments and diets.
• Pressurized liquid extraction (PLE) offers a faster, solvent-efficient alternative to traditional lipid extraction methods.
• Standardizing PLE for bone cholesterol extraction is essential for reliable paleodietary insights.

Purpose of the Study:

• To optimize and validate a standardized pressurized liquid extraction (PLE) methodology for bone cholesterol.
• To assess the efficiency and reliability of PLE for extracting cholesterol for compound-specific isotope analysis (CSIA).
• To evaluate the impact of lipid extraction on stable isotope ratios of bone collagen and apatite.

Main Methods:

• Bone samples (modern and archaeological) were subjected to PLE using varying temperatures, solvents, and sample weights.
• Cholesterol yields were quantified to assess PLE efficiency.
• Isotopic signatures (δ¹³C and δ¹⁸O) of extracted cholesterol, collagen, and apatite were analyzed using GC/MS and GC/IRMS.

Main Results:

• Optimal lipid yield was achieved with two PLE cycles at 75 °C using dichloromethane/methanol and 0.25-0.75 g bone samples.
• Saponification and trimethylsilylation significantly increased cholesterol recovery.
• Lipids from purified bone collagen and apatite are suitable for cholesterol CSIA; lipid extraction did not affect collagen δ¹³C but significantly altered apatite δ¹⁸O.

Conclusions:

• The optimized PLE method provides an effective and alternative approach for bone cholesterol extraction.
• This standardized methodology facilitates robust paleodietary and ecological analyses.
• Researchers should consider the impact of lipid extraction on bone apatite isotopic signatures.