Bone-afide Stress: Methodological Assessment of Cortisol and Osteocalcin Extraction from Archaeological Human Remains

Abstract Objectives This study presents a novel method for extracting cortisol from human archaeological cortical bone and evaluates its usefulness as a stress indicator by comparing it with established hair-cortisol extraction methods. We preliminarily investigate osteocalcin’s potential as a less destructive, more accessible biomarker for physiological stress assessment. Methods This study analyzed ten individuals from the 18th-century Fortress of Louisbourg. Fourier-transform infrared spectroscopy (FTIR) was employed to assess the effects of diagenesis. Cortisol was measured from hair and cortical bone samples, and osteocalcin was quantified from the bone samples using an enzyme-linked immunosorbent assay (ELISA). Results Cortisol was extracted and quantified from 19 hair and 10 bone samples. Bone cortisol significantly correlated with hair cortisol (ρ: 0.68; P: 0.029), whereas osteocalcin showed a significant inverse correlation with hair cortisol (ρ: -0.77; P: 0.009) and a similar, non-significant trend with bone cortisol (ρ: -0.59; P: 0.074). Diagenetic alteration significantly affected bone cortisol (ρ: -0.70; P: 0.007), with increased degradation linked to higher extracted cortisol. Normalizing bone cortisol by the carbonate-phosphate ratio strengthened correlations with osteocalcin (ρ: -0.65; P: 0.043). Discussion These findings help establish a reproducible protocol for extracting and properly normalizing cortisol for inter-individual comparisons in archaeological human bone, enabling cortisol-based research in regions where hair rarely preserves. The correlation between hair and bone cortisol confirms bone as a viable source of cortisol for the study of physiological stress archaeologically. The inverse relationship between osteocalcin and cortisol suggests osteocalcin's potential as a stress biomarker and merits further research.