Free cortisol in serum assayed by temperature-controlled ultrafiltration before fluorescence polarization immunoassay

Area of Science:

• Endocrinology
• Analytical Chemistry
• Biochemistry

Background:

• Accurate measurement of free cortisol in serum is crucial for diagnosing adrenal disorders.
• Existing methods may be affected by variations in cortisol-binding protein levels.
• Temperature control is essential for reproducible ultrafiltration assays.

Purpose of the Study:

• To develop and validate a temperature-controlled ultrafiltration method for precise free cortisol measurement.
• To assess the impact of cortisol-binding protein variations on free cortisol levels.
• To establish reference intervals for serum free cortisol in a general population and oral contraceptive users.

Main Methods:

• Development of a specialized thermometer with an in-situ sensor for direct temperature measurement within the ultrafiltration device.
• Implementation of a temperature-controlled box to maintain sample temperature at 37°C ± 0.1°C during centrifugation.
• Validation of the assay's precision, with ultrafiltration contributing only 2.2-3.6% to the overall coefficient of variation (CV).

Main Results:

• The overall CV for the free cortisol assay ranged from 2.2% to 11.4%.
• Serum free cortisol concentrations in the reference population (n=114) were 12-43 nmol/L (4-9.5% of total cortisol).
• Oral contraceptive users (n=27) showed altered reference intervals (11-53 nmol/L, 1.5-4.5% of total cortisol), indicating minor but significant effects of increased cortisol-binding protein.

Conclusions:

• The developed temperature-controlled ultrafiltration method offers a precise and reliable approach for quantifying serum free cortisol.
• The assay demonstrates minimal contribution to overall variability, ensuring accuracy.
• Estrogen-containing oral contraceptives significantly impact serum free cortisol levels by affecting cortisol-binding protein, necessitating adjusted reference ranges.