Long-term magnesium supplementation improves glucocorticoid metabolism 97 5 urine sample of the next morning after waking. Participants were requested to empty their bladder completely before going to sleep. On the day before blood sampling, participants were requested not to consume alcohol, nor perform strenuous physical activities. In addition, participants were requested not to consume any foods or drinks (except for water) after 08.00 PM on the day before blood sampling.Measurements were performed at the Metabolic Research Unit Maastricht (MRUM) research facilities. Fasting blood samples were taken from a forearm vein by venipuncture. Blood drawn in vacutainer serum tubes (Becton, Dickinson and Company) was first allowed to clot for at least 30 minutes at 21 °C. Serum separator tubes were then centrifuged at 1300×g for 15 minutes at 21 °C. Serum and urine magnesium were determined photometrically with a Magnesium and Calcium Gen.2 assay (COBAS, Roche Diagnostics GmbH) by the Central Diagnostic Laboratory Maastricht University Medical Center. Cortisol, cortisone, THF, allo-THF, and THE were measured in 24-h urine samples by isotope dilution liquid chromatography tandem mass spectrometryat theUniversityMedical Center Groningen (UMCG),as previouslydescribed 18. Total production of urinary cortisol, cortisone and their metabolites was calculated as the sum of the following metabolites: (urinary cortisol excretion (nmol/24-h) / 1000) + (urinary cortisone excretion (nmol/24-h)/ 1000) + urinary THF excretion (µmol/24-h) + urinary allo-THF excretion (µmol/24-h) + urinary THE excretion (µmol/24-h). Technicians at the MRUM and at the UMCG were not aware of the treatments of the participants. Figure 1. Schematic overview of glucocorticoid metabolism. 11β-HSD type 1 converts cortisone into cortisol causing increased intracellular cortisol levels, while 11β-HSD type 2 decreases intracellular cortisol levels by converting cortisol into cortisone. 5α- and 5β-reductase enzymes inactivates cortisol and cortisone to their inactive metabolites THF, allo-THF, and THE, respectively. Abbreviations: 11β-HSD, 11β-hydroxysteroid dehydrogenase; THF, tetrahydrocortisol; allo THF, allo-tetrahydrocortisol; THE, tetrahydrocortisone. Enzyme activities of 11β-HSDs and A-ring reductases Overall 11β-HSD enzymatic activitywas calculated using the ratio of urinarymetabolites of cortisol and cortisone (THF + allo-THF (THFs))/THE 13. The ratio of cortisol/cortisone
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