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Lessons Learned. Cortisol. Why Measure Cortisol?. Hyper- cortisolism or hypo- cortisolism associated with several disease conditions Cushing’s Disease Addison’s Disease Congenital Adrenal Hyperplasia Depression Thyroid Disease Cortisol is an objective marker of the stress response.

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why measure cortisol
Why Measure Cortisol?
  • Hyper-cortisolism or hypo-cortisolism associated with several disease conditions
    • Cushing’s Disease
    • Addison’s Disease
    • Congenital Adrenal Hyperplasia
    • Depression
    • Thyroid Disease
  • Cortisol is an objective marker of the stress response
what medium to use
What Medium to Use?
  • Urine
  • Blood (serum or plasma)
  • Saliva
  • Hair
    • Pros and Cons for each
urine cortisol normal range 10 100 mcg 24hours
Urine Cortisol (Normal range 10-100 mcg/24hours)
  • Pros:
    • Non-invasive
    • 24-hr urine cortisol/creatinine ratio excellent diagnostic for disease states. Not influenced by diurnal rhythm.
  • Cons
    • Need a timed urine sample, so difficult to collect diurnal samples
    • Labor intensive for patients; can’t be used in patients with renal failure or dialysis
    • High rate of cross-reactivity (numerous excreted steroid metabolites in urine)
      • Most urine assays pick up cortisol metabolites (95%); only 2-3 % free cortisol
blood cortisol
Blood Cortisol
  • Pros
      • Can collect diurnal data or after stress exposure
      • Best measure for diagnosing certain hyper-cortisol states (e.g., Cushing’s via dex suppression test)
  • Cons
      • Blood draw expectation may increase cortisol release
      • Provide measurement of cortisol at a single point in time
      • Major fraction bound to corticosteroid binding globulin (CBG) and albumin (only 2-3% free).
        • CBG influenced by pregnancy, oral contraceptives, hyperthyroidism – so levels measured may be high, but not reflective of bioactive cortisol
      • Methods for assaying serum free cortisol are time consuming, expensive and require advanced techniques (ultrafiltration, gel filtration, gold standard: equilibrium dialysis)
salivary cortisol
Salivary Cortisol
  • Pros
    • Non-invasive – can collect from infants, children, patients with poor veins
    • Avoids stress-induced rise in secretion (like with a blood draw)
    • Can collect at home or in the wild: Do not need to freeze or keep refrigerated
    • Can collect one time or diurnal data
    • Provides measure of free cortisol – best indicator of active hormone
    • Correlates well with serum levels across a 24-hour period
      • r=.67 in healthy people; r= .73 in patients with Addison’s disease; and r= .61 in patients with Cushing’s)
  • Cons
    • Provides measurement of cortisol concentration at a single point in time
    • Periodontal disease, eating, tooth brushing can affect levels
    • Sample quantity may be low (e.g., dry mouth, impatient)
    • Messy
hair cortisol
Hair Cortisol
  • Pros
    • Biomarker of chronic stress
    • Relatively non-invasive
    • Only free cortisol represented
    • Easily transported and stored - put in an envelope or vial & store at room temp for months to years
    • Reflects cortisol levels over past months (Hair grows predictably about 1 cm/month) –
      • 1 cm closest to skull reflects last month; 2nd cm out reflects preceding month
        • So may not need to take multiple samples to evaluate chronic stress and perhaps compare months with and without a stressor
    • Not affected by hair color – but maybe by dying of hair
    • Significantly correlated with self-reports of stress (Kalra, 2007; Van Uum, 2008)
  • Cons
    • No clinically relevant reference range established to date
    • Exposure to exogenous steroids in lotions or creams may cause sample contamination
    • Mechanism of incorporation of cortisol into hair not known– is it from blood? Exocrine or sebaceous sources?
cortisol assays
Cortisol Assays
  • Radioimmunoassay
  • Immunoassay - most common but may be at risk of cross reactivity with steroids other than cortisol (problematic in critically ill patients and in urine samples)
  • High pressure liquid chromatography (HPLC)/mass spectrometry
measurement and analysis
Measurement and Analysis
  • A single sample
    • unreliable unless:
      • Collected accurately at a specific time of day for all subjects


      • Collected accurately at a specific time of day related to known time of awakening
  • Diurnal Rhythm
    • Unreliable unless collected accurately at correct times of day
measurement and analysis1
Measurement and Analysis
  • Diurnal Rhythm
    • Usually collect between 3-6 samples over the course of 1-3 days to evaluate:
      • Awakening level – considered a measure of allostatic load
      • Awakening response (15-45 mins after awakening) – considered a measure of acute stress response.
      • Then one or more collections across the day
        • High in morning, then falls over the day, lowest late in the evening (Kudielka& Kirschbaum, 2003)
      • If an individual or group deviate from that rhythm, indicates dysregulation
awakening response
Awakening Response
  • Awakening Response (Pruessner, 1997)

[30 min post awakening] – [awakening]


area under the curve
Area Under the Curve
  • Mathematically transforms multivariate data into a summary of the information (Fekedulegn et al., 2007).
  • Reflects the total cortisol released during the day
  • Provides information on the magnitude of the response
measurement and analysis2
Measurement and Analysis
  • Must validate correct collection times
    • Self-report
    • Phone calls
    • MEMs Caps (electronic monitoring)
special populations
Special Populations
  • Pregnant women
    • Levels very high but maintain a diurnal rhythm
    • Blood levels very high unless measure only free because CBG increases significantly

blood = bound + free

  • Infants and toddlers
thank you
Thank you!
  • Questions?
  • Fekedulegn, D.B., Andrew, M.E., Burchfiel, C.M, et al. (2007). Area under the curve and other summary indicators of repeated waking cortisol measurements. Psychosomatic Medicine, 69, 651-659.
  • Kalra, S., Einarson, A., Karaskov, T., Van Uums, S., & Koren, G., (2007). The relationship between stress and hair cortisol in healthy pregnant women. Clinical and Investigative Medicine, 30, E103-107.
  • Kudielka, B.M., Kirschbaum, C. (2003). Awakening cortisol response are influenced by health status and awakening time but not by menstrual cycle phase. Psychoneuroendocrinology, 28, 35-47.
  • Pruessner, J., C., Wolf, O.T., Hellhammer, D. H., et al. (1997). Free cortisol levels after awakening: a reliable biological marker for the assessment of adrenocortical activity. Life Science, 61, 2539-2549.
  • Russell, E., Koren, G., Rieder, M., & Van Uum. (2012). Hair cortisol as a biological marker of chronic stress: current status, future directions and unanswered questions. Psychoneuroendocrinology, 37, 589-601.
thyroid stimulating hormone and cortisol concentration
Thyroid Stimulating Hormone and Cortisol Concentration
  • Serum cortisol levels among individuals with TSH levels >2.0 uIU/L (N=31) were significantly higher than among those individuals with TSH levels ≤ 2.0 uIU/L (N=20) [13.83 ± 0.93 ug/dL vs. 8.66 ± 0.59 ug/dL, respectively, F(1,48)=14.24, p<0.0001].
  • Walter, Corwin, Ulbrecht, Demers, Bennett, Whetzel, & Klein (2012). Thyroid Research.
conversion factor
Conversion Factor
  • To change cortisol from
    • Microgram to nanomolar ;
      • Microgram x 2.76