Endocrinol Metab.  2022 Aug;37(4):599-607. 10.3803/EnM.2022.1549.

Clinical and Technical Aspects in Free Cortisol Measurement

• Choi MH

• KMID: 2532859
• DOI: http://doi.org/10.3803/EnM.2022.1549

Abstract

Accurate measurement of cortisol is critical in adrenal insufficiency as it reduces the risk associated with misdiagnosis and supports the optimization of stress dose. Comprehensive assays have been developed to determine the levels of bioactive free cortisol and their clinical and analytical efficacies have been extensively discussed because the level of total cortisol is affected by changes in the structure or circulating levels of corticoid-binding globulin and albumin, which are the main reservoirs of cortisol in the human body. Antibody-based immunoassays are routinely used in clinical laboratories; however, the lack of molecular specificity in cortisol assessment limits their applicability to characterize adrenocortical function. Improved specificity and sensitivity can be achieved by mass spectrometry coupled with chromatographic separation methods, which is a cutting-edge technology to measure individual as well as a panel of steroids in a single analytical run. The purpose of this review is to introduce recent advances in free cortisol measurement from the perspectives of clinical specimens and issues associated with prospective analytical technologies.

Keyword

Cortisol; Mass spectrometry; Immunoassay; Saliva; Adrenal insufficiency

Figure

• 

  Fig. 1. Distribution of cortisol in human blood. Corticoid-binding globulin (CBG) and albumin are main reservoirs of cortisol, binding 80%–90% and 10%–15% of total cortisol, respectively. Approximately 3%–5% of total cortisol is present as biologically active cortisol. States of altered CBG and albumin concentrations may lead to misrepresentation of cortisol action based on total cortisol levels.

• 

  Fig. 2. A schematic diagram of free cortisol analysis in serum. Zinc sulfate is used to precipitate large proteins in serum and an aqueous buffer exchange is then performed to further precipitate the remaining small proteins and zinc ions in sample solution. Samples are vortexed and centrifuged before analysis using mass spectrometry or immunoassay, and only the supernatant is used.

• 

Reference

1. Ross IL, Louw GJ. Embryological and molecular development of the adrenal glands. Clin Anat. 2015; 28:235–42.

2. Kim JH, Choi MH. Embryonic development and adult regeneration of the adrenal gland. Endocrinol Metab (Seoul). 2020; 35:765–73.

3. Lee DY, Kim E, Choi MH. Technical and clinical aspects of cortisol as a biochemical marker of chronic stress. BMB Rep. 2015; 48:209–16.

4. Staab CA, Maser E. 11beta-Hydroxysteroid dehydrogenase type 1 is an important regulator at the interface of obesity and inflammation. J Steroid Biochem Mol Biol. 2010; 119:56–72.

5. Juster RP, McEwen BS, Lupien SJ. Allostatic load biomarkers of chronic stress and impact on health and cognition. Neurosci Biobehav Rev. 2010; 35:2–16.

6. Lightman SL, Birnie MT, Conway-Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev. 2020; 41:bnaa002.

7. Seckl JR, Walker BR. Minireview: 11beta-hydroxysteroid dehydrogenase type 1-a tissue-specific amplifier of glucocorticoid action. Endocrinology. 2001; 142:1371–6.

8. Borresen SW, Klose M, Glintborg D, Watt T, Andersen MS, Feldt-Rasmussen U. Approach to the patient with glucocorticoid-induced adrenal insufficiency. J Clin Endocrinol Metab. 2022; 107:2065–76.

9. Teblick A, Peeters B, Langouche L, Van den Berghe G. Adrenal function and dysfunction in critically ill patients. Nat Rev Endocrinol. 2019; 15:417–27.

10. Gatti R, Antonelli G, Prearo M, Spinella P, Cappellin E, De Palo EF. Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clin Biochem. 2009; 42:1205–17.

11. El-Farhan N, Rees DA, Evans C. Measuring cortisol in serum, urine and saliva: are our assays good enough? Ann Clin Biochem. 2017; 54:308–22.

12. Cameron A, Henley D, Carrell R, Zhou A, Clarke A, Lightman S. Temperature-responsive release of cortisol from its binding globulin: a protein thermocouple. J Clin Endocrinol Metab. 2010; 95:4689–95.

13. Vogeser M, Briegel J. Effect of temperature on protein binding of cortisol. Clin Biochem. 2007; 40:724–7.

14. Diamandis EP. Immunoassay interference: a relatively rare but still important problem. Clin Biochem. 2004; 37:331–2.

15. Hammond GL, Smith CL, Paterson NA, Sibbald WJ. A role for corticosteroid-binding globulin in delivery of cortisol to activated neutrophils. J Clin Endocrinol Metab. 1990; 71:34–9.

16. Perogamvros I, Ray DW, Trainer PJ. Regulation of cortisol bioavailability: effects on hormone measurement and action. Nat Rev Endocrinol. 2012; 8:717–27.

17. Nader N, Raverot G, Emptoz-Bonneton A, Dechaud H, Bonnay M, Baudin E, et al. Mitotane has an estrogenic effect on sex hormone-binding globulin and corticosteroid-binding globulin in humans. J Clin Endocrinol Metab. 2006; 91:2165–70.

18. Qureshi AC, Bahri A, Breen LA, Barnes SC, Powrie JK, Thomas SM, et al. The influence of the route of oestrogen administration on serum levels of cortisol-binding globulin and total cortisol. Clin Endocrinol (Oxf). 2007; 66:632–5.

19. Hamrahian AH, Oseni TS, Arafah BM. Measurements of serum free cortisol in critically ill patients. N Engl J Med. 2004; 350:1629–38.

20. Ho JT, Al-Musalhi H, Chapman MJ, Quach T, Thomas PD, Bagley CJ, et al. Septic shock and sepsis: a comparison of total and free plasma cortisol levels. J Clin Endocrinol Metab. 2006; 91:105–14.

21. Annane D, Sebille V, Charpentier C, Bollaert PE, Francois B, Korach JM, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. JAMA. 2002; 288:862–71.

22. Boonen E, Vervenne H, Meersseman P, Andrew R, Mortier L, Declercq PE, et al. Reduced cortisol metabolism during critical illness. N Engl J Med. 2013; 368:1477–88.

23. Vogeser M, Mohnle P, Briegel J. Free serum cortisol: quantification applying equilibrium dialysis or ultrafiltration and an automated immunoassay system. Clin Chem Lab Med. 2007; 45:521–5.

24. le Roux CW, Sivakumaran S, Alaghband-Zadeh J, Dhillo W, Kong WM, Wheeler MJ. Free cortisol index as a surrogate marker for serum free cortisol. Ann Clin Biochem. 2002; 39(Pt 4):406–8.

25. le Roux CW, Chapman GA, Kong WM, Dhillo WS, Jones J, Alaghband-Zadeh J. Free cortisol index is better than serum total cortisol in determining hypothalamic-pituitary-adrenal status in patients undergoing surgery. J Clin Endocrinol Metab. 2003; 88:2045–8.

26. Pemberton PA, Stein PE, Pepys MB, Potter JM, Carrell RW. Hormone binding globulins undergo serpin conformational change in inflammation. Nature. 1988; 336:257–8.

27. Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R. Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 785:263–75.

28. Vogeser M, Briegel J, Jacob K. Determination of serum cortisol by isotope-dilution liquid-chromatography electrospray ionization tandem mass spectrometry with on-line extraction. Clin Chem Lab Med. 2001; 39:944–7.

29. Owen LJ, Adaway JE, Davies S, Neale S, El-Farhan N, Ducroq D, et al. Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory. Ann Clin Biochem. 2013; 50(Pt 4):345–52.

30. Vining RF, McGinley RA. The measurement of hormones in saliva: possibilities and pitfalls. J Steroid Biochem. 1987; 27:81–94.

31. Vining RF, McGinley RA, Maksvytis JJ, Ho KY. Salivary cortisol: a better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem. 1983; 20(Pt 6):329–35.

32. Thomson AH, Devers MC, Wallace AM, Grant D, Campbell K, Freel M, et al. Variability in hydrocortisone plasma and saliva pharmacokinetics following intravenous and oral administration to patients with adrenal insufficiency. Clin Endocrinol (Oxf). 2007; 66:789–96.

33. Kim YJ, Kim JH, Hong AR, Park KS, Kim SW, Shin CS, et al. Stimulated salivary cortisol as a noninvasive diagnostic tool for adrenal insufficiency. Endocrinol Metab (Seoul). 2020; 35:628–35.

34. Yaneva M, Mosnier-Pudar H, Dugue MA, Grabar S, Fulla Y, Bertagna X. Midnight salivary cortisol for the initial diagnosis of Cushing’s syndrome of various causes. J Clin Endocrinol Metab. 2004; 89:3345–51.

35. Viardot A, Huber P, Puder JJ, Zulewski H, Keller U, Muller B. Reproducibility of nighttime salivary cortisol and its use in the diagnosis of hypercortisolism compared with urinary free cortisol and overnight dexamethasone suppression test. J Clin Endocrinol Metab. 2005; 90:5730–6.

36. Papanicolaou DA, Mullen N, Kyrou I, Nieman LK. Nighttime salivary cortisol: a useful test for the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab. 2002; 87:4515–21.

37. Vieira-Correa M, Giorgi RB, Oliveira KC, Hayashi LF, Costa-Barbosa FA, Kater CE. Saliva versus serum cortisol to identify subclinical hypercortisolism in adrenal incidentalomas: simplicity versus accuracy. J Endocrinol Invest. 2019; 42:1435–42.

38. Ross IL, Lacerda M, Pillay TS, Blom DJ, Johannsson G, Dave JA, et al. Salivary cortisol and cortisone do not appear to be useful biomarkers for monitoring hydrocortisone replacement in Addison’s disease. Horm Metab Res. 2016; 48:814–21.

39. Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: a new laboratory tool for diagnostic and basic investigation. Clin Chim Acta. 2007; 383:30–40.

40. Whembolua GL, Granger DA, Singer S, Kivlighan KT, Marguin JA. Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva. Horm Behav. 2006; 49:478–83.

41. Garde AH, Hansen AM. Long-term stability of salivary cortisol. Scand J Clin Lab Invest. 2005; 65:433–6.

42. Lewis JG. Steroid analysis in saliva: an overview. Clin Biochem Rev. 2006; 27:139–46.

43. Wood P. Salivary steroid assays: research or routine? Ann Clin Biochem. 2009; 46(Pt 3):183–96.

44. Tomlinson JW, Stewart PM. Cortisol metabolism and the role of 11beta-hydroxysteroid dehydrogenase. Best Pract Res Clin Endocrinol Metab. 2001; 15:61–78.

45. Vogeser M, Groetzner J, Kupper C, Briegel J. The serum cortisol:cortisone ratio in the postoperative acute-phase response. Horm Res. 2003; 59:293–6.

46. Morita H, Isomura Y, Mune T, Daido H, Takami R, Yamakita N, et al. Plasma cortisol and cortisone concentrations in normal subjects and patients with adrenocortical disorders. Metabolism. 2004; 53:89–94.

47. Schiffer L, Barnard L, Baranowski ES, Gilligan LC, Taylor AE, Arlt W, et al. Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: a comprehensive review. J Steroid Biochem Mol Biol. 2019; 194:105439.

48. Mariniello B, Ronconi V, Sardu C, Pagliericcio A, Galletti F, Strazzullo P, et al. Analysis of the 11beta-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) in human essential hypertension. Am J Hypertens. 2005; 18:1091–8.

49. Duclos M, Marquez Pereira P, Barat P, Gatta B, Roger P. Increased cortisol bioavailability, abdominal obesity, and the metabolic syndrome in obese women. Obes Res. 2005; 13:1157–66.

50. Kim SH, Kim SE, Choi MH, Park MJ. Altered glucocorticoid metabolism in girls with central obesity. Mol Cell Endocrinol. 2021; 527:111225.

51. Wu JY, Hsu SC, Ku SC, Ho CC, Yu CJ, Yang PC. Adrenal insufficiency in prolonged critical illness. Crit Care. 2008; 12:R65.

52. Venkatesh B, Cohen J, Hickman I, Nisbet J, Thomas P, Ward G, et al. Evidence of altered cortisol metabolism in critically ill patients: a prospective study. Intensive Care Med. 2007; 33:1746–53.

53. Perogamvros I, Keevil BG, Ray DW, Trainer PJ. Salivary cortisone is a potential biomarker for serum free cortisol. J Clin Endocrinol Metab. 2010; 95:4951–8.

54. Debono M, Harrison RF, Whitaker MJ, Eckland D, Arlt W, Keevil BG, et al. Salivary cortisone reflects cortisol exposure under physiological conditions and after hydrocortisone. J Clin Endocrinol Metab. 2016; 101:1469–77.

55. Zhang Q, Chen Z, Chen S, Xu Y, Deng H. Intraindividual stability of cortisol and cortisone and the ratio of cortisol to cortisone in saliva, urine and hair. Steroids. 2017; 118:61–7.

56. Walker BR, Best R, Noon JP, Watt GC, Webb DJ. Seasonal variation in glucocorticoid activity in healthy men. J Clin Endocrinol Metab. 1997; 82:4015–9.

57. Choi S, Kim S, Yang JS, Lee JH, Joo C, Jung HI. Real-time measurement of human salivary cortisol for the assessment of psychological stress using a smartphone. Sens Bio-Sens Res. 2014; 2:8–11.

58. Sanghavi BJ, Moore JA, Chavez JL, Hagen JA, Kelley-Loughnane N, Chou CF, et al. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device. Biosens Bioelectron. 2016; 78:244–52.

59. Pinto V, Sousa P, Catarino SO, Correia-Neves M, Minas G. Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification. Biosens Bioelectron. 2017; 90:308–13.

60. Parlak O, Keene ST, Marais A, Curto VF, Salleo A. Molecularly selective nanoporous membrane-based wearable organic electrochemical device for noninvasive cortisol sensing. Sci Adv. 2018; 4:eaar2904.

61. Davison SL, Bell R, Montalto JG, Sikaris K, Donath S, Stanczyk FZ, et al. Measurement of total testosterone in women: comparison of a direct radioimmunoassay versus radioimmunoassay after organic solvent extraction and celite column partition chromatography. Fertil Steril. 2005; 84:1698–704.

62. Ghazal K, Brabant S, Prie D, Piketty ML. Hormone immunoassay interference: a 2021 update. Ann Lab Med. 2022; 42:3–23.

63. Roberts RF, Roberts WL. Performance characteristics of five automated serum cortisol immunoassays. Clin Biochem. 2004; 37:489–93.

64. El-Farhan N, Pickett A, Ducroq D, Bailey C, Mitchem K, Morgan N, et al. Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays. Clin Endocrinol (Oxf). 2013; 78:673–80.

65. Briegel J, Sprung CL, Annane D, Singer M, Keh D, Moreno R, et al. Multicenter comparison of cortisol as measured by different methods in samples of patients with septic shock. Intensive Care Med. 2009; 35:2151–6.

66. Cohen J, Ward G, Prins J, Jones M, Venkatesh B. Variability of cortisol assays can confound the diagnosis of adrenal insufficiency in the critically ill population. Intensive Care Med. 2006; 32:1901–5.

67. Jonsson BA, Malmberg B, Amilon A, Helene Garde A, Orbaek P. Determination of cortisol in human saliva using liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 784:63–8.

68. Wood L, Ducroq DH, Fraser HL, Gillingwater S, Evans C, Pickett AJ, et al. Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays. Ann Clin Biochem. 2008; 45(Pt 4):380–8.

69. Miller R, Plessow F, Rauh M, Groschl M, Kirschbaum C. Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry. Psychoneuroendocrinology. 2013; 38:50–7.

70. Miller WL. Congenital adrenal hyperplasia: time to replace 17OHP with 21-deoxycortisol. Horm Res Paediatr. 2019; 91:416–20.

71. Choi MH. Mass spectrometry-based steroid profiling: meeting unmet clinical needs. Tohoku J Exp Med. 2021; 253:171–80.

72. Choi MH, Chung BC. Bringing GC-MS profiling of steroids into clinical applications. Mass Spectrom Rev. 2015; 34:219–36.

73. Wudy SA, Choi MH. Steroid LC-MS has come of age. J Steroid Biochem Mol Biol. 2016; 162:1–3.

74. Storbeck KH, Schiffer L, Baranowski ES, Chortis V, Prete A, Barnard L, et al. Steroid metabolome analysis in disorders of adrenal steroid biosynthesis and metabolism. Endocr Rev. 2019; 40:1605–25.

75. Ahn CH, Lee C, Shim J, Kong SH, Kim SJ, Kim YH, et al. Metabolic changes in serum steroids for diagnosing and subtyping Cushing’s syndrome. J Steroid Biochem Mol Biol. 2021; 210:105856.

76. Lee C, Kim JH, Moon SJ, Shim J, Kim HI, Choi MH. Selective LC-MRM/SIM-MS based profiling of adrenal steroids reveals metabolic signatures of 17α-hydroxylase deficiency. J Steroid Biochem Mol Biol. 2020; 198:105615.

77. Casals G, Hanzu FA. Cortisol measurements in Cushing’s syndrome: immunoassay or mass spectrometry? Ann Lab Med. 2020; 40:285–96.

78. Kamrath C, Hochberg Z, Hartmann MF, Remer T, Wudy SA. Increased activation of the alternative “backdoor” pathway in patients with 21-hydroxylase deficiency: evidence from urinary steroid hormone analysis. J Clin Endocrinol Metab. 2012; 97:E367–75.