Edited by: Lucio Vilar, Federal University of Pernambuco, Brazil
Reviewed by: Zhanna Evgenjevna Belaya, The National Research Center for Endocrinology, Russia; Laurence Katznelson, Stanford University, United States
Specialty section: This article was submitted to Pituitary Endocrinology, a section of the journal Frontiers in Endocrinology
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Measuring salivary cortisol is both convenient and non-invasive for patients; however, its usefulness as a marker for monitoring medical therapy has not yet been established. The aim of this study was to assess the utility of multiple salivary cortisol measurements in patients with Cushing’s syndrome (CS) during medical therapy. Six patients with CS (three with cortisol-secreting adrenocortical adenoma and three with ACTH-secreting pituitary adenoma) were recruited. Samples for morning serum cortisol, urinary free cortisol (UFC), and multiple salivary cortisol levels were collected before and during metyrapone treatment. The area under the curve (AUC) and mean value (MV) of daily salivary cortisol levels were calculated. In five out of six patients, UFC were normalized; however, multiple salivary cortisol measurements revealed an impaired diurnal cortisol rhythm in these patients. To verify the usefulness of multiple salivary cortisol measurements, we performed a prospective case study of a patient in whom the excess secretion of cortisol was not controlled (UFC 211 μg/day) with 2,250 mg/day in four divided doses of metyrapone. Multiple measurements of salivary cortisol revealed that cortisol levels elevated before the next administration. Accordingly, we shortened the interval by increasing the number of administration from four to five times per day, with a slight increment of daily dose of 2,500 mg. These optimizations resulted in a drastic improvement of diurnal pattern as well as UFC level (101 μg/day). Changes in both the MV and AUC of salivary cortisol levels were more correlated with those in UFC levels (Correlation coefficient 0.75,
Cushing’s syndrome (CS) is associated with high mortality and serious morbidities such as infections, cardiovascular disease, stroke, and thromboembolism (
Collection of samples for salivary cortisol management is convenient for patients and is feasible in outpatient clinics (
This protocol was approved by the Kobe University Hospital ethical committee (IRB No. 1351). Written informed consent was obtained from all patients. Six patients with CS (three with cortisol-secreting adrenocortical adenoma (A);
Clinical characteristics of patients with Cushing’s syndrome.
Patient no. | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|
Cause of CS | A | A | A | P | P | P |
Age (years) | 48 | 43 | 64 | 35 | 55 | 22 |
M/F | F | F | F | F | M | F |
ACTH (pg/mL) | <5.0 | <5.0 | <5.0 | 32.6 | 179 | 79.9 |
Serum cortisol (μg/dL) | 21.2 | 21 | 19.1 | 19.2 | 38.6 | 29.4 |
UFC (μg/day) | 260 | 97.3 | 119.5 | 85.6 | 1,980 | 329.3 |
L-DST cortisol (μg/dL) | 18.1 | 18.7 | 15.7 | 14 | 38.6 | 25.3 |
Subjects rinsed mouth thoroughly with water 10 min before the sample is collected. They tilted the head forward, allowing the saliva to pool on the floor of the mouth, then passed the saliva into a polypropylene tube. Samples were centrifuged (1,500 ×
The salivary cortisol collection was performed five times in a day: at 6:00 (wake up time), 8:00 (before breakfast), 12:00 (before lunch), 18:00 (before dinner), and 22:00 (before sleep). A daily mean value (MV) and daily area under the curve (AUC) of salivary cortisol levels were calculated (MV: average of five measurements of salivary cortisol levels and AUC: integral of five measurements of salivary cortisol levels).
The samples were collected before metyrapone treatment, during metyrapone treatment, and when the dose of metyrapone was increased. The date of sampling was set for 2 days after the start or increase in the dose of metyrapone. The initial dose of metyrapone was 500–750 mg per day, and the maintenance dose was 500–2,000 mg per day. The administration timing was 14:00 and 22:00 for patients receiving the drug twice per day (500 mg per day); 6:00, 14:00, and 22:00 for patients receiving the drug three times per day (750–1,250 mg per day); and patients receiving the drug at 6:00, 12:00, 18:00, and 24:00 for four times per day (1,000–2,000 mg per day). The dosage and timing for each patient were determined based on the evaluation of the morning serum cortisol levels, the UFC levels, and the well-being of each patient. The target levels of morning serum cortisol and UFC were below the upper normal limit. Salivary cortisol measurements were performed, and its utility was analyzed retrospectively. In patient No. 6, salivary samples before treatment were not obtained. Therefore, only the changes in MV and AUC during treatment were analyzed in this patient.
Patient No. 4 was hospitalized again due to worsening of hypercortisolemia 1 year after the first hospitalization. To test whether the salivary cortisol is useful, we collected and measured multiple salivary samples from this patient during the treatment and adjusted the dose and timing of metyrapone administration as a prospective case study.
The serum cortisol levels were measured by an enzyme immunoassay (reference range: 6.4–21.0 µg/dL, EIA; TOSOH, Tokyo, Japan), and the daily UFC levels were measured by radioimmunoassay (reference range: 11.2–80.3 μg/day, RIA; TFB, Tokyo, Japan). Salivary cortisol levels were measured by enzyme immunoassay (Salimetrics Inc., State College, PA, USA).
All statistical analyses were performed using SAS 11.2 (Statistical Analysis Software release 11.2; SAS Institute Inc., Cary, NC, USA). Data are shown as mean ± SD. The paired
The clinical characteristics of the patients are described in Table
Both morning serum cortisol and UFC levels were decreased within the normal range after initiation of treatment in all patients except for Patient No. 5 (Figure
Salivary cortisol levels before and after initiation of metyrapone treatment.
In Patient No. 1, both the UFC and morning serum cortisol levels decreased after initiation of metyrapone treatment in a dose-dependent manner (Figure
In Patient No. 5, despite a marked reduction in UFC levels, the morning serum cortisol levels did not change with 1,250 mg of metyrapone. We then increased the dose to 2,000 mg, resulting in decrease in both morning serum cortisol and UFC levels, though still not up to target levels. This suggests that a further increase in the dose might be necessary. Salivary cortisol levels revealed that, even with the dose of metyrapone 2,000 mg, the CS remained uncontrolled, and obvious impairment of the diurnal cortisol rhythm remained (Figure
In Patient No. 6, UFC levels and morning serum cortisol levels were both normalized after metyrapone treatment. However, the diurnal rhythm was not fully reconstituted (Figure
Patient No. 4 was readmitted to the hospital 1 year after the first admission due to the worsening in hypercortisolemia despite a dose of 1,250 mg of metyrapone. During the second admission, although the dose of metyrapone was increased to 2,250 mg, the UFC level rather increased (Figure
Changes in the diurnal salivary cortisol pattern after the optimization of timing of metyrapone administration in Patient No. 4.
Both the MV and AUC of salivary cortisol levels decreased after initiation of metyrapone treatment (Table
Salivary cortisol levels before and after initiation of metyrapone treatment.
MV of salivary cortisol (μg/dL) | Area under the curve (AUC) of salivary cortisol (μg · h/dL) | |
---|---|---|
0 mg | 0.64 | 11.2 |
500 mg | 0.19 | 2.9 |
750 mg | 0.16 | 2.7 |
0 mg | 0.25 | 4.0 |
750 mg | 0.18 | 2.3 |
1,000 mg | 0.14 | 2.0 |
0 mg | 0.40 | 6.7 |
500 mg | 0.15 | 2.6 |
0 mg | 0.29 | 4.3 |
1,000 mg | 0.28 | 4.1 |
1,250 mg | 0.10 | 1.6 |
0 mg | 0.95 | 13.5 |
1,250 mg | 0.52 | 8.8 |
2,000 mg | 0.32 | 5.9 |
0 mg | ND | ND |
750 mg | 0.45 | 7.8 |
1,000 mg | 0.16 | 3.0 |
Correlation between changes in urinary free cortisol levels and other parameters.
Correlation coefficient | ||
---|---|---|
Changes in mean value of salivary cortisol levels | 0.75 | 0.007 |
Changes in area under the curve of salivary cortisol levels | 0.70 | 0.016 |
Changes in morning (6:00) serum cortisol levels | 0.42 | 0.200 |
Changes in morning (6:00) salivary cortisol levels | 0.70 | 0.005 |
Changes in midnight (22:00) salivary cortisol levels | −0.04 | 0.915 |
Correlation between changes in midnight salivary cortisol levels and changes in mean value (MV) or area under the curve (AUC) of salivary cortisol levels.
Correlation coefficient | ||
---|---|---|
Changes in AUC of salivary cortisol levels | 0.25 | 0.457 |
Changes in MV of salivary cortisol levels | 0.19 | 0.582 |
The present data suggested that multiple measurements of salivary cortisol levels may be useful for optimization of metyrapone treatment by visualization of diurnal cortisol levels. Although multiple collections are required, salivary collection is a less of a burden for patients. Furthermore, salivary samples are stable and can be easily stored (
We clearly demonstrated that measurements of multiple salivary cortisol levels revealed the impaired diurnal cortisol rhythm in patients even after normalization of UFC. The early morning serum cortisol levels and UFC levels remain within the normal range in most patients with subclinical CS (SCS). However, accumulating evidence suggests that the dysregulation of diurnal cortisol rhythms is associated with metabolic comorbidities such as hypertension and impaired glucose metabolism, even in patients with normal UFC levels. A recent systematic review suggested that normalization of the diurnal cortisol rhythms by surgical treatment resulted in an improvement in complications of SCS (
In our series, the salivary cortisol levels were constantly elevated throughout the day in patients with cortisol-secreting adrenocortical adenoma, and patients with ACTH-secreting pituitary adenoma had salivary cortisol levels that were elevated mainly in the morning and less in the evening or midnight. These results suggest that the diurnal secretion pattern was relatively preserved in Cushing’s disease as compared with adrenocortical adenomas, indicating that the significance of elevated morning cortisol levels is different depending on the disease etiology.
When determining the timing and dose of metyrapone treatment, it is important to not only normalize the total amount of daily cortisol secretion but also optimize the diurnal rhythm. In this aspect, multiple measurements of salivary cortisol are obviously advantageous for evaluation. Indeed, the prospective case study in Patient No. 4 showed that, although hypercortisolemia was not been controlled despite a high dose (2,250 mg) of metyrapone treatment, the evaluation for diurnal pattern by salivary cortisol levels enabled us to optimize not only the dose but also the timing.
In this study, the salivary cortisol collection was performed five times in a day: at 6:00 (wake up time), 8:00 (before breakfast), 12:00 (before lunch), 18:00 (before dinner), and 22:00 (before sleep). Several centers use the average values of serum cortisol levels from five measurements for evaluation (
The usefulness of salivary cortisol measurements for the diagnosis and prediction of the risk of the recurrence of CS has been well established (
This study has several limitations. The number of patients was small, especially the preliminary trial in the prospective study. In addition, the effect was evaluated over a short term. The protocol for the dosing and timing was not standardized. Although the symptoms were recorded, the changes in QoL were not evaluated. Thus, further investigation with a large-scale, long-term study is necessary.
In conclusion, although further study is necessary, the present study suggests that taking multiple salivary cortisol measurements could visualize the diurnal change of cortisol during medical treatment and may be a useful for optimizing metyrapone treatment in patients with active CS.
This study was carried out in accordance with the recommendations of the Kobe University Hospital ethical committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Kobe University Hospital ethical committee.
KY drafted the manuscript, and assembled and analyzed the data. HF and YT were responsible for the conception and design of the study. SN, MT, JI, YH, TY, HM, and NS contributed by collecting the data or caring for the patients. The other coauthors contributed to the acquisition, analysis, or interpretation of data for the work. HF was responsible for the critical revision of the manuscript for important intellectual content.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The salivary cortisol measurement kit was a kind gift by Dr. Yutaka Oki (Department of Family And Community Medicine, Hamamatsu University School of Medicine). We are grateful to Ms. M. Kitamura and Ms. C. Ogata for providing excellent assistance.