Hair cortisol measures
HCC) were expressed as pg/mg with a method developed in-house using a competitive radioimmunoassay on methanol extracts of pulverised hair. A hair sample approximately 3 mm thick and 3 cm long was cut close to the scalp from the posterior vertex area of the head. The hair samples were further cut into 1.25 cm lengths to reflect the cortisol accumulations for each month, based on an assumption of an average hair growth rate of 1 cm per month (13). The hair samples analysed in this study weighed between 5 mg and 6 mg. In the laboratory, each sample was put into a 2 mL QiaGenRB sample tube along with a 0.5 mm QiaGen stainless steel bead and weighed on a Sartorius MC 210p microscale. The samples were put in specially made aluminium cylinders accommodating five 2 mL Eppendorf tubes, and frozen in liquid nitrogen for two minutes. This was followed by mincing in a Retch Tissue Lyser II at 23 Hz for two minutes to produce a fine hair powder. The cortisol was extracted by adding 1 mL of methanol (Chromasolv, Sigma-Aldrich) to each tube and placing the tubes in a metal holder on a plate with a 5-degree inclination on a horizontal shaker at room temperature, keeping the steel beads in constant gentle motion within the tubes for a minimum of 10 hours. Finally, the tubes were centrifuged for one minute at 13000 rpm at +4°C in a microcentrifuge, Thermo Scientific Heraus, Picotm & Frescotm 17/21, and 800 μL of the supernatant was moved to another plastic sample tube for lyophilisation in a SpeedVac Plus SC210A (Savant) using an Edwards XDS 5 vacuum pump for at least three hours. The samples were dissolved in radioimmunoassay buffer and analysed as described by Morelius et al. 2004 (24). A hair sample of 3–10 mg is needed to maintain a total inter-assay coefficient of variation below 8% for the combination of hair extraction and measurement of cortisol by the radioimmunoassay. The intra-assay coefficient of variation for the radioimmunoassay itself was 7% at 10 nmol/L. The antiserum cross-reacts 137% with 5α-dihydroxycortisol, 35.9% with 21-deoxycortisol, and 35.9% with prednisolone, but less than 1% with endogenous steroids (15).