Assessment of Plasma Amino Acid Dynamics in Response to ACTH Stimulation by Liquid Chromatography Tandem-Mass Spectrometry.

BACKGROUND: The adrenocorticotropic hormone (ACTH) stimulation test is a widely used diagnostic tool to assess the adrenal gland function. Beyond that the ACTH test can be used in stress research to induce a biochemical stress response under standardized conditions. To study the impact of the stress response on protein metabolism, time-course plasma amino acid profiling in healthy individuals was performed with high performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS). METHODS: A set of 39 samples (pre/post 30´ and 60´ IV-ACTH) from 13 healthy individuals (age range 26 - 58, 3 female and 10 male) was investigated. Plasma amino acids were quantified by LC-MS/MS using the AbsoluteIDQ® p180 Kit (Biocrates Life Science, Innsbruck, Austria) including 19 biogenic amino acids, ornithine, and citrulline. RESULTS: Statistically significant decreases were observed for 11 proteinogenic amino acids (alanine, asparagine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, valine, methionine, aspartate, and threonine). The amino acids alanine, asparagine, and isoleucine showed markedly pronounced relative changes with short-term reduction of median inter-individual plasma concentrations of up to 25%. CONCLUSIONS: Amino acid profiling with LC-MS/MS revealed highly dynamic plasma alterations upon application of exogenous corticotropin as a stress model. Our findings provide novel insights into the biochemical stress response and improve our understanding of short-term metabolic consequences. Further studies should elucidate the impact of corticotropin mediated stress responses on amino acid catabolism.

Multicenter performance evaluation of a second generation cortisol assay.

BACKGROUND: Untreated disorders of the adrenocortical system, such as Cushing's or Addison's disease, can be fatal, and accurate quantification of a patient's cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva. METHODS: Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals. RESULTS: The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5-95th percentile) were 166.1-507 nmol/L and afternoon concentrations were 73.8-291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. CONCLUSIONS: The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.