Salivary Cortisol and Cortisone After Low-Dose Corticotropin Stimulation in the Diagnosis of Adrenal Insufficiency

Ingrid Yin Fung Mak; Benjamin Yick Toa Au Yeung; Ying Wai Ng; Cheung Hei Choi; Heidi Yan Ping Iu; Chi Chung Shek; Sau Cheung Tiu

Disclosures

J Endo Soc. 2017;1(2):96-108. 

In This Article

Discussion

In our study, we established the cutoff value for post-LDSST peak serum cortisol, as assayed by chemiluminescent microparticle immunoassay using the Abbott Architect i2000SR system, from a group of healthy controls. Because the results had a normal distribution, we used mean − 2 SDs to establish this cutoff[3,6] and found it to be 376 nmol/L. This value appears to be lower than those commonly quoted in the literature. Previously used fluorimetric assays and older generation immunoassays had lower specificity for cortisol, measuring also other glucocorticoid metabolites.[10,11] A lower dose of Synacthen can also lead to a smaller rise in cortisol. A recent study in which the cortisol values after LDSST and 250 μg of SST [standard dose SST (SDSST)] were compared among 55 Turkish healthy volunteers showed that, for all who had cortisol responses >550 nmol/L during SDSST, the lowest cortisol response achieved during LDSST was only 345 nmol/L.[26]

To confirm the validity of this cutoff value, we explored the alternative approach of defining AI with salivary cortisol values of the healthy cohort. We reasoned that because CBG mutations can be completely silent,[27] the fact that our "healthy" controls were asymptomatic could not exclude the possibility of their having lower-than-usual CBG and thereby lower total serum cortisol levels. Although we measured their CBG levels, we were unable to find a convincing reference range on CBG either from the literature or from the manufacturer of the CBG assay. Defining AI by using salivary cortisol of the healthy subjects would presumably bypass the problem with the binding proteins. Although the post-LDSST peak salivary cortisol was not distributed normally because of 1 outlier who had a greater-than-usual response to LDSST, the distribution became normal after exclusion of this case, and we could again use the mean − 2 SDs to derive the cutoff, which was found to be 8.4 nmol/L ( Table 1 ). Defining AI as peak salivary cortisol of <8.4 nmol/L, the ROC curve analysis from the patient group showed that the optimal cutoff value for peak salivary cortisone was 33.5 nmol/L (AUC 0.982 ± 0.008, sensitivity 98.1%, specificity 91.5%), and that for peak serum cortisol was 361 nmol/L (AUC 0.960 ± 0.014, sensitivity 88.9%, specificity 92.3%). The value of 361 nmol/L was practically the same as 376 nmol/L when the assay variability was taken into account. This ROC curve also showed that peak serum cortisol levels of 291 and 438 nmol/L gave 100% specificity and sensitivity, respectively ( Table 4 ).

The convergence of these 2 approaches convinced us that the method-specific cutoff for peak serum cortisol of 376 nmol/L was a reasonable gold standard to define "AI" and "non-AI" among our patients. Table 2 showed that more subjects in the AI group had clinical features (40.7% in the AI group vs 27.7% in the non-AI group) and electrolyte disturbance (18.6% in the AI group vs 6.3% in the non-AI group) compatible with AI. Additionally, slightly more subjects in the AI group had impairment of other anterior pituitary hormones (44.0% in the AI group vs 36.6% in the non-AI group). We did not perform other objective "confirmatory" tests to obtain independent evidence of hypoadrenalism in the AI group, as our center had previously validated the LDSST against the insulin tolerance test (ITT) in a similar group of patients,[4] and the literature evidence on the performance of glucagon stimulation test in diagnosing AI was weak. Moreover, without performing the ITT in our normal controls (we did not think that it was justifiable to request them to undergo this risky test), we were uncertain about what cutoffs to use for this test with the current cortisol assay.

Among 233 patients with nonspecific symptoms, Corbould et al.[28] found that, at cutoff values of 550, 500 and 345 nmol/L, 45%, 26%, and 8% failed the LDSST respectively. The high rates of positive cases at the traditional cutoffs of 500 or 550 nmol/L were up to 50-fold above expected rates.[28] Applying this "reality testing" to our patient population, at the cutoff of 376 nmol/L, 34.5% had AI. Had we defined AI with the traditional cutoff of 500 nmol/L, 70.8% of our patient population would have been considered as having the condition. Many patients with serum cortisol levels between 400 and 500 nmol/L had been followed up by us for >3 years without the need to take regular or stress dose hydrocortisone, and they did not manifest any AI-related features such as hypotension or electrolyte disorder during the follow-up period even when they were exposed to various physical and mental stresses in life. Moreover, 22 (39.3%) of our healthy subjects would have been found to have AI at the cutoff of 500 nmol/L, making the proportion unreasonably high.

Our center participates in the external quality assurance program organized by the Royal College of Pathologists of Australia. We have a 2.4% positive bias from the Abbott analyzer median and a 4.3% positive bias from the medians of all analyzers of different brands. At the cutoff level of 376 nmol/L, the 2.4% bias is ~9 nmol/L, which is probably not significant given the analytical CV of most cortisol immunoassays of 4% to 8%. Centers contemplating adoption of our cutoff should review the difference in bias between their in-use immunoassay method and the Abbott method and adjust accordingly when the bias difference is considered significant.

Using serum cortisol of <376 nmol/L as the gold standard for classifying patients into AI and non-AI groups, we proceeded to derive the optimal cutoff values for peak salivary cortisol and cortisone. From our data, the cutoff value with the highest accuracy was 8.6 nmol/L for peak salivary cortisol. This value is very similar to the peak salivary cortisol reference value of 8.3 nmol/L (LC-MS/MS) established using mean − 2 SDs among 59 subjects with adequate SST or ITT response (serum cortisol > 500 nmol/L, measured by Siemens Centaur immunoassay) in the study by Perogamvros et al..[16] Cornes et al.[29] also attempted to establish the cutoff value for salivary cortisol (LC-MS/MS) during SST. They defined "normal response" as a peak serum cortisol [measured by electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany) shown by them to give results 20% higher than the Abbott assay] of ≥550 nmol/L after SDSST. Because only 4 of their 36 subjects had abnormal response during the SST, they could only extrapolate the salivary cortisol values from the serum cortisol value of 550 nmol/L, arriving at a cutoff of 15 nmol/L. Two older studies suggested higher cutoff values (20 and 24.3–27.6 nmol/L, respectively), but the number of subjects was small and radioimmunoassays were used to measure salivary cortisol.[17,30]

The ROC curve analysis for peak salivary cortisone showed that its accuracy in diagnosing AI was as high as peak salivary cortisol (Fig. 1; Table 3 ). The cutoff value associated with the highest accuracy was 33.5 nmol/L for salivary cortisone. The value derived by Cornes et al.[29] in the study mentioned previously was 45 nmol/L. We think that our methodology is a more robust one. To our knowledge, there is otherwise no similar study in the published literature.

We think that serum cortisol, salivary cortisol, and salivary cortisone have their individual roles in defining AI, and together they can give us a more comprehensive picture of the glucocorticoid status of a person, with little additional hardship for the patient and very little additional resources. Serum total cortisol has the advantage of being extensively studied, although the cutoff value is still controversial, making interpretation difficult; additionally, it is affected by variations in the binding proteins. Salivary cortisol theoretically better reflects the serum free cortisol, which is thought to be the active hormone. So far there is more information on salivary cortisol than salivary cortisone, but the latter is interesting in itself: an abnormally high cortisol to cortisone ratio would suggest recent intake of oral hydrocortisone or 11β-HSD type 2 inhibitors such as liquorice.[19,23,31] Similar to other investigators,[19,23,24] our study showed that salivary cortisone had a better and more linear correlation than salivary cortisol with serum total cortisol (on which a large body of literature on tests for AI was based) (Fig. 2), which may be an advantage for salivary cortisone. Salivary cortisone is partly contributed by serum cortisone.[23] Alhough an inert substrate in itself, circulating serum cortisone is converted back to cortisol via local 11β-HSD type 1 enzyme activities, enhancing glucocorticoid action at the tissue level and leading to various metabolic effects.[32] An altered serum cortisone to cortisol ratio may reflect differential activities of the 11β-HSD type 1 and 11β-HSD type 2; however, the physiological or pathological importance of cortisone levels, whether in the serum or in the saliva, still awaits further studies.

In addition to deriving the cutoff values, our data also showed that peak salivary cortisol and cortisone levels of 33.5 and 62.4 nmol/L yielded a sensitivity of 100%, and peak salivary cortisol and cortisone levels of 5.2 and 15 nmol/L yielded a specificity of 100% ( Table 4 ). These levels can be used to more definitively rule out or rule in AI, respectively.

In concordance with other studies,[33,34] we also showed that basal morning serum cortisol, salivary cortisol, or cortisone levels all performed less well in diagnosing AI than did the peak levels (Fig. 1). However, in situations when a stimulation test cannot be performed, our study suggested that cutoff values of 170, 1.7, and 12.5 nmol/L could be used for basal serum cortisol, salivary cortisol, and salivary cortisone, respectively ( Table 3 ). The reported reference values for basal salivary cortisol ranged from 3.2 to 13.3 nmol/L,[35–38] due largely to differences in the salivary cortisol assays and methodologies used in various studies.[39] The only study that had explored the basal salivary cortisone cutoff suggested that when AI was defined as post-LDSST serum cortisol of <350 nmol/L (measured by the Siements Immulite 2000 immunoassay), the morning basal salivary cortisone cutoff of 12.5 nmol/L (measured by immunoassay; Salimetrics, Carlsbad, CA) had a negative predictive value of 99.2% and positive predictive value of 30.1%.[38] This value is identical to the basal salivary cortisone cutoff established in our study.

One limitation of our study was that the mean age of the healthy subjects was significantly lower than that of the patient group. We included 4 older subjects with diabetes and hypertension because we were otherwise unable to recruit an adequate number of subjects in the older age range. We think that these conditions and their medications would not affect the hypothalamic-pituitary-adrenal axis and binding protein levels. A previous study,[12] however, showed no effect of age on cortisol response to SST. When reanalyzing the data after excluding these 4 subjects with diabetes and/or hypertension (n = 52), we found that the minor change in the reference cutoff values of interest among normal subjects (Supplemental Data 5) did not affect the results from the ROC curve analysis in the patient group (Supplemental Data 6). Some investigators recommended stricter rules in saliva collection, such as avoiding eating and brushing teeth for 2 hours, and mouth rinsing with water 10 to 15 minutes before sample collection,[20] but others suggested that 30 minutes of such avoidance should be adequate.[24]

Unlike immunoassays, LC-MS/MS enables accurate measurement of specific steroids, and results are less specific assay–dependent and are more comparable among laboratories. We think that LC-MS/MS will soon become the method of choice for measuring steroids; establishing cutoff values using this methodology would be important to facilitate clinical interpretation across different centers. The derivation of cutoff values for salivary cortisol and cortisone would also enable investigators to study the glucocorticoid axis in greater detail, and this would facilitate better understanding of the physiological and pathological roles of cortisone in future studies.

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