ESPE Abstracts

Initially discovered in the 1970s, the C₂₁ steroidal acids α-cortolic acid, β-cortolic acid, α-cortolonic acid and β-cortolonic acid present the terminal oxidative products of cortisol metabolism. Undergoing renal elimination, these cortoic acids have been assumed to represent up to 25% of total urinary cortisol metabolites. However, their analysis has been difficult, only few data has been published in adults, and this class of steroids has become practically forgotten. Since data in children are lacking and nothing is known about their metabolism during human development, we aimed at establishing a more practical analytical method and determined their urinary concentrations in a high number of healthy subjects. 5 ml aliquots of 24-hour urine samples were used for sample work up consisting of solid phase extraction (C18 cartridges), strong anion exchange and derivatization to 2-propylester-trimethylsilylether derivatives. Subsequently, the cortoic acids were quantified by targeted GC-MS using a nonpolar GC column and selected ion monitoring. Baseline separation of all cortoic acids was achieved. Calibration graphs were linear in the chosen calibration range (R² > 0.98). Regarding precision and accuracy, coefficients of variation were smaller than 10%, the difference between measured and expected concentrations was less than 15%. The detection limit was 100 pg (injected) with a signal-to-noise ratio of 3. The method was used to analyze 24-hour urine collections from 240 healthy children (120 boys, 120 girls, aged 3-18 years, all participants in the DONALD study). The profile of cortoic acids was dominated by α-cortolonic acid with excretion rates up to 70 µg/d. When excretion rates of the four cortoic acids were summed, all subjects excreted less than 130 µg/d (range: 11.0 µg/d - 127,3 µg/d). In relation to neutral urinary cortisol metabolites, total cortoic acids only represented about 1% (range: 0,69% -1,51%) of overall daily glucocorticoid secretion. While higher age led to increased excretion rates, the children’s sex did not significantly affect the amount of daily excreted cortoic acids.

Conclusion: A new and less complicated method for quantification of urinary cortoic acids using GC-MS was developed, evaluated and successfully applied to 240 specimens from healthy children, adolescents and young adults and first reference values were established. Baseline separation of all four cortoic acids was achieved on a nonpolar GC column. Excretion of cortoic acids increased with age. However, cortoic acids’ proportion of total urinary cortisol metabolites only added up to about 1%, a percentage much lower as hitherto estimated.