outcome.
Aken, M.O. van
Citation
Aken, M. O. van. (2005, March 17). Cushing's Syndrome : hormonal secretion patterns,
treatment and outcome. Retrieved from https://hdl.handle.net/1887/3748
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Salivary cortisol measurement in the diagnosis of Cushing’s
syndrome
M.O. van Aken, J.A. Romijn, J.A. Miltenburg, E.G. Lentjes
D ep artment of End oc rinology and Metabolic D iseases, Leid en U niversity Med ic al C enter, Leid en, th e N eth erland s
Published in part in
ABSTRACT
INTRODUCTION
T he diagnostic tests req uired for the biochemical confi rmation of the underlying cause of Cushing’s syndrome (CS ) have been among the most puz z ling problems in clinical endocrinology and remain controversial (1 ;2 ). Distinguishing betw een CS and situations referred to as pseudo-Cushing states, such as the metabolic syndrome, depression, alcoholism can be diffi cult, w ith confusing results of biochemical tests. Moreover, w ith increasing aw areness among docters and the w idespread availability of biochemical testing, patients are screened in an earlier phase of their disease, in w hich only minor abnormalities of cortisol secretion are present, making biochemical discrimination of the different causes of hypercortisolism even more diffi ccult.
T he diagnosis of CS should begin w ith a careful case history and a thorough physical ex amination, looking for the characteristic features w hile ex cluding ex ogenous intake of oral, parenteral, inhaled or topical glucocorticoids. In the case of clinical suspicion of CS , biochemical screening for hypercortisolism can be performed. T w enty-four hour urine collection for the measurement of urinary free cortisol and the low -dose dex amethasone suppression test (1 mg) have been used ex tensively as fi rst line screening tests for CS , but neither test has proven fully capable of distinguishing all cases of CS from other individuals.
Twenty-four-hour urinary free cortisol (UFC)
T w enty-four hour urine collection for the measurement of urinary free cortisol (UF C) ex cretion has been considered a gold standard for the diagnosis of CS , w ith sensitivity of 1 0 0 % and specifi city of 9 8% (3 ). H ow ever, this method has several limitations. Collecting urine for 2 4 hr is cumbersome, hindering repetitive urinary collection in case of suspected intermittent hypercortisolism. Incorrect or incomplete collection can give false, negative results. In addition, impaired renal function (glomerular fi ltration rate below 3 0 ml/min.) decreases cortisol ex cretion, resulting in normal UF C despite the presence of hypercortisolism (4). Conversely, some medications, such as carbamaz epin and digox in, can give false elevations of UF C (5). Mild elevations of urinary cortisol can also be found in pseudo-Cushing’s states and pregnant w omen (6 ). F inally, urinary cortisol ex cretion may be normal, and therefore not identify patients w ith subclinical CS in w hich hypercortisolism is still mild. F or these reasons, UF C cannot be used as a single screening test for the detection of CS (2 ).
L ow-d ose d ex am ethasone sup p ression test (D S T)
original described 138 nmol/l (6), to the recently advised 50 nmol/l (2;5). The lower cutoff value increases sensitivity, obviously at the cost of lower specifi city (7 ). Specifi city is further reduced by possible insuffi cient suppression in cases of increased concentrations of cortisol binding globulin, acute and chronic illness and pseudo-Cushing states.Other interfering conditions causing apparent insuffi cient suppression are decreased dexamethasone absorption and drugs enhancing hepatic dexamethasone metabolism (phenytoin, carbamazepin, rifampicine) (8;9). Finally, in a recent series of 103 patients with Cushing’s syndrome, six (8%) patients had suppressed serum cortisol-concentrations after 1 mg dexamethasone below 54 nmol/l, showing that this test should not be used as the sole criterion to exclude the diagnosis of Cushing’s syndrome (7 ).
Late-night salivary cortisol
The measurement of cortisol-concentrations in saliva has been described since the 7 0’s (10-12). The study of Laudat et al. (13)was among the fi rst to document the effectiviness of diurnal salivary cortisol sampling to diagnose CS, with an elevated salivary cortisol level in all patients with CS.
Since that study, new assay technologies in measuring cortisol concentration in saliva have emerged and several clinical studies have been performed using salivary cortisol as a fi rst line test in screening for CS. The purpose of the present report is to describe the technical aspects of measuring salivary cortisol concentration, especially the validation of an automated assay on the Roche immunoanalyzer. Furthermore, published data on the clinical use of late-night salivary cortisol measurement in the diagnosis of CS are discussed.
R elationship b etween plasma and salivary cortisol concentrations
blood to saliva can occur by passage through the tight junctions and pores in the cell-membrane and by passive diffusion through the acinar membranes.
Consequently, free cortisol in plasma is in equilibrium with cortisol in saliva and is not affected by the rate of saliva production (15). An increase in plasma cortisol is refl ected by a change in salivary cortisol concentration within a few minutes (10). The circadian rhythm of plasma cortisol is similarly refl ected in the diurnal variation of salivary cortisol concentration, with a peak in the early morning and nadir around midnight (13;15;16).
Collection of saliva
The need for expectorating saliva into a test tube has been obviated by the development of different saliva collection devices, from which the Salivette (Sarstedt) has been used extensively in measuring salivary cortisol concentrations. This device consists of a plastic centrifugation tube containing a cotton roll. This roll is placed in the mouth and the subject chews gently on it for 2 – 3 minutes. The roll is then placed back in the plastic tube and transferred to a laboratory. Saliva obtained in this way can be stored at room temperature for at least a week and the salivettes can be transferred to the laboratory by regular post without infl uencing the salivary cortisol concentration (17-19). In the laboratory, the salivette is centrifuged to obtain saliva from the cotton role, and the saliva can be frozen for later measurement of cortisol concentration.
MEASURING CORTISOL CONCENTRATION IN SALIVA
Validation of an automated salivary cortisol assay
For this study, saliva samples were collected with a Salivette® (Sarstedt), with an insert containing a sterile polyester swab for collection of the saliva, yielding a clear and particle-free sample. The salivettes were used according to the instructions provided by the manufacturer. Samples collected this way are stable at room temperature for at least a week and therefore offer the opportunity to collect samples at home in a patient friendly way (16;19). Salivettes containing saliva were centrifuged at 2000 g for 10 min and the fi ltrates were stored frozen (-20 ° C). Prior to analysis the samples were thawed, mixed and placed on the Elecsys analyzer without pretreatment.
The Roche cortisol assay is a competitive electrochemiluminescence immunoassay (ECLIA) using a sheep polyclonal antibody. Endogenous cortisol contained in the sample is liberated from the binding proteins by danazol, and subsequently competes with a cortisol derivative (a cortisol - peptide - Tris bipyridyl ruthenium complex) for the binding sites on the biotinylated antibody. After the addition of streptavidine-coated paramagnetic particles the biotin on the antibody can bind to the streptavidin of the microparticle and form a complex. This complex is then captured on the surface of the magnetic electrode. Electrical stimulation of the ruthenium complex induces chemiluminescent emission which is measured by a photomultiplier. The assay was calibrated against Enzymun-Test-Cortisol which in turn was calibrated via isotope dilution-massspectrometry. The cortisol assay was used as instructed by the manufacturer without modifi cations. The sample volume for the assay was 20 µL. Processing time is 18 minutes.
The linearity of the cortisol determinations in saliva was studied according to the NCCLS EP-6 protocol, using a saliva sample with a low cortisol concentration with added cortisol (Sigma Chemical Co) at a concentration of 240 nmol/L (28). This sample was diluted with the untreated sample. The correlation between added and measured cortisol was linear with slope = 0.99, intercept = -2.9 nmol/L, r2 = 0.998.
A precision profi le was established using saliva samples with or without cortisol additions. Each sample was aliquoted ten times and stored frozen. Analysis of the samples was performed on 10 different days over a time span of 4 months (fi gure 1). The functional sensitivity (20% interassay coeffi cient of variation (CV )) as determined from these measurements is 2.0 nmol/L. The performance of the Elecsys cortisol assay in the low concentration range renders it suitable for the measurement of cortisol concentrations in saliva.
Inter-assay precision evaluated according to the NCCLS EP-5 protocol (29). Two pools of saliva with different cortisol concentration were aliquoted and cortisol was measured on 20 separate days, in each sample twice. Interassay CV was 11.5% at 11 nmol/L and 5% at 50 nmol/L. The somewhat higher CV ’s, compared with those found in the former experiment, refl ected by a shift in the concentrations after a calibration.
5 nmol/L. Apparent cortisol concentration at 10, 100 and 1000 nmol/L of the cross reactant cortisone and 11-deoxycortisol never exceeded respectively 0.6% and 1.8% of the added amount. However, for 6 -hydroxycortisol there was an increasing interference, i.e. apparent cortisol at 10 nmol/L 30 %, at 100 nmol/L 40 % and at 1000 nmol/L 50 % of the added amount. For 21-deoxycortisol at a level of 10, 100, 1000 nmol/L, apparent cortisol concentrations were 44, 24 and 14 % respectively. Importantly, interference by cortisone, an abundant steroid metabolite in saliva, is negligible. There are by contrast no reports on the presence of 6 ß -hydroxycortisol or 21-deoxycortisol in saliva. These steroids are easily excreted in urine (6 ß -hydroxycortisol) or present only at low concentrations in serum, which makes it unlikely that the concentrations in saliva will interfere with the cortisol assay.
We compared the Elecsys cortisol assay with an in-house radioimmunoassay (30)using salivary samples. Correlation of salivary cortisol measured by ECLIA (Elecsys) vs RIA was: Elecsys = 0.92 x RIA – 1.6 (n=34, r = 0.84, SD of slope = 0.1, SD intercept 1.9, Sy.x = 4.3) (Deming regression analysis).
Reference intervals were estimated from an unselected group of healthy individuals, 26 male and 32 female (20-80 yr old). Saliva samples were obtained between 07:00 and 08:00 hr and between 23:00 and 24:00 hr (late night). The mean (SD) for morning salivary cortisol was 13.4 (3.2) and for late night cortisol was 3.55 (0.94). No sex differences were observed. Although these values similar to previously reported reference intervals, they should be established in each laboratory as different assays show different results (31).
This is the fi rst report of a fully automated nonisotopic assay for the measurement of cortisol in saliva. Sensitivity and reproducibility in the low nanomolar concentration range suggest it will be a useful tool in the assessment of the activity of the hypothalamic-pituitary-adrenal axis. This method offers several advantages over isotopic assays and commercially available EIA’s (26;32). It is automated, samples need no pretreatment, results can be obtained within 20 min and there is no need for collecting a certain number of samples for effi cient use. This makes it
precision profile 0 5 10 15 20 25 1 10 100 1000
cortisol concentration (nm ol/L)
C
V
(
%
)
Figure 1. Precision profi le of t h e E lecsy s sa liv a ry cort isol a ssa y .
a suitable test for daily laboratory and clinical use, as recently advocated for the diagnosis of Cushing’s syndrome (33).
Confounding factors
Salivary cortisol measurement has several possible pitfalls. Above all, the contamination of the saliva sample with traces of blood is a risk for falsely elevated salivary cortisol concentration, because of the 20-fold higher cortisol concentrations in plasma compared to in saliva (20). To minimize this risk, patients are advised not to eat, drink or brush their teeth at least 30 minutes before collecting saliva. The possible impact of these factors is uncertain, as one study found no effect of eating or dental care on salivary cortisol levels (19). Visual inspection of the saliva sample for the detection of blood contamination and discarding samples with a slight reddish color is usually suffi cient. For more sensitive assessment, dipsticks designed for the detection of blood in urine can be used.
The concentration of cortisol in saliva is, in theory, independent of changes in cortisol binding globulin (CB G) levels, because salivary cortisol is a refl ection of free plasma cortisol. However, several studies showed that salivary cortisol is elevated in patients with estrogen therapy or in pregnant women, suggesting that estrogen stimulates the activity of the HPA axis in addition to increasing CB G concentration (34;35).
In salivary glands, 11`-Hydroxysteroid dehydrogenase type II (11`-HSD II) is expressed, which converts cortisol into inactive cortisone (36). Substances which interfere with 11`-HSD-II activity, like licorice or chewing tobacco, could infl uence salivary cortisol levels. However, the administration of glycyrrhetinic acid to normotensive or primary hypertensive subjects had no effect on the salivary cortisol/cortisone ratio (37). Two patients were reported having extremely elevated late-night salivary cortisol levels (> 500 nmol/l), without other biochemical fi nding’s of Cushing’s syndrome (38). B oth patients were on statin therapy. Salivary cortisol/ cortisone ratio was elevated in both patients and normalized after discontinuation of statin therapy. Urinary cortisol/cortisone ratio was normal during statin therapy. These fi ndings suggest a mechanism for statin therapy to inhibit 11`-HSD-II activity in salivary glands but not in kidney epithelium. However, there have been no further reports of patients o statins with falsely elevated salivary cortisol levels. In young infants, breast milk might induce false elevated salivary cortisol levels, but for cow's milk this effect has not been demonstrated (31;39). Finally, the use of exogenous steroids, including inhalers and topic agents (contamination of salivette roll) should be avoided before collection of a saliva sample.
Reference ranges
and should be established in each laboratory. In healthy elderly subjects, late-night salivary levels have been shown to be slightly increased compared to young subjects, possibly by reduced responsiveness to glucocorticoid feedback inhibition in human aging (40-43).
DIAGNOSIS OF CUSHING’S SYNDROME USING SALIVARY CORTISOL MEASUREMENT Late-night Salivary Cortisol Concentration
The biochemical diagnosis of Cushing’s syndrome requires the documentation of hypercortisolism. Measurement of elevated UFC and/or insuffi cient suppression of morning cortisol after overnight low-dose dexamathasone testing have been used as the primary markers of hypercortisolism. However, these tests have their limitations, as discussed previously.
Disruption of the normal circadian rhythm of cortisol secretion is considered as one of the characteristics of endogenous CS, with absence of the late night nadir in serum cortisol concentration(44-48). However, more recent studies have shown that the diurnal pattern of cortisol secretion is preserved in certain patients but with levels that are set abnormally high (49-53). (49-53). At midnight, the overlap of serum cortisol levels between patients with Cushing’s syndrome and the normal range was shown to be minimal (54). Assessment of the late night serum or salivary cortisol concentration may therefore be a useful instrument in the biochemical confi rmation of CS.
In two studies, the measurement of midnight serum cortisol concentration in the documentation of hypercortisolism was studied (55;56). In the fi rst study, patients were hospitalized and had to be asleep before taking a midnight blood sample (55). Blood was sampled within 2 minutes of waking the patient. An elevated midnight serum cortisol higher than 50 nmol/l was found in all 150 patients with CS, in contrast to midnight serum cortisol values below 50 nmol/l in 29 healthy subjects. However, as no patients with pseudo-Cushing’s states were included, the specifi city of a midnight serum cortisol concentration greater than 50 nmol/l in the screening for hypercortisolism cannot be calculated from this study. In the second study, patients were also hospitalized, but were not asleep when blood samples were taken via an indwelling venous catheter from 2300 to 0100 h at 30 minutes intervals (56). A midnight serum cortisol value greater than 207 nmol/l correctly identifi ed 225 of 234 patients with CS, in contrast to midnight serum cortisol values below 207 nmol/l in 23 patients with pseudo-Cushing’s syndrome (96% sensitivity at 100% specifi city). However, the need for hospitalization to obtain an unstressed midnight blood sample for the measurement of cortisol makes this a very impracticle test for the primary assessment of hypercortisolism, unsuitable for daily clinical practice.
A u to m a te d m ea su re m en t o f s a liv a ry c
A uthor (R ef) Published (yr)
Setting Collection
method
salivary cortisol assay L ate-night Salivary cortisol concentration (nmol/ l,mean ± SE) (N umber of subjects) Cut-off value for diagosis CS (nmol/ l) Sensitiv. (% ) Specifi c. (% )
H ealthy O bese Cushing’s R uled
out
Cushing’s Syndrome
L audat (13 ) 19 8 8 In-patient spitting R IA 3 .9 ± 0.2 (101) n.d. n.d. 3 5 .8 ± 5 .0 (14 ) 4 .2 100 100
R aff (6 4 ) 19 9 8 O ut-patient? Salivette R IA , Coat-a-Count, D iagnostic Products, L os A ngeles 1.2 ± 0.1 (7 3 ) n.d. 1.6 ± 02 (3 9 ) 2 4 .0 ± 4 .5 (3 9 ) 3 .6 9 2 9 2 M artinelli (6 3 ) 19 9 9 In-patient, children spitting R IA n.d. 3 .3 ± 2 .2 (2 1) n.d. 2 8 .3 ± 13 .4 (11) 7 .7 100 9 5 .2
Castro (6 2 ) 19 9 9 O ut-patient spitting R IA ref 2 2 2 .6 ± 0.2 (3 0) 3 .7 ± 0.7 (18 ) n.d. 2 5 .2 ± 2 .6 (3 3 ) 7 .7 9 3 .3 9 3 .3
G afni (6 5 ) 2 000 O ut-patient, children
spitting R IA , Covance L aboratories, inc., V ienna, V a)
3 .6 ± 0.1 (6 0) n.d. n.d. 3 5 .6 ± 6 .1 (15 ) 7 .5 9 3 100
Papanicolaou (3 3 )
2 002 In-patient spitting R IA , Covance L aboratories, inc., V ienna, V a)
7 .2 ± 0.6 (3 4 ) n.d. D ata not stated (2 2 )
D ata not stated (12 2 )
15 .2 9 3 100
Putignano (6 6 )
2 003 In-patient Salivette R IA , B yk -Sangtek D iagnostik a, D ietz enbach, G ermany
5 .0 ± 0.6 (2 7 ) 5 .5 ± 0.3 (19 9 ) 6 .3 ± 0.6 (3 3 ) 2 6 .7 ± 3 .6 (4 1) 9 .7 9 2 .7 9 3 .1
Y aneva (6 7 ) 2 004 In-patient Salivette R IA , CIS B iointernational, G if-sur-Y vette, F rance
Low-dose dexamethasone testing using salivary cortisol measurement
Failure to suppress morning serum cortisol after taking 1 mg dexamethasone the night before is considered as a second hallmark of CS. The limitations of the low-dose dexamethasone test have been summarized above. Apart from these problems, the stress of drawing blood in the morning can give false positive results. The measurement of morning salivary cortisol concentration after 1 mg dexamethasone the night before might therefore be a rational alternative, making the test an unstressed at-home procedure.
In four studies this approach has been evaluated, all showing a clear separation between unsuppressed salivary cortisol in patients with CS, compared to undetectable or very low levels in controls (table 2). Again, limitations of these studies are the relatively small number of patients, the lack of patients referred for suspected CS but in whom CS was ruled out, and the inpatient setting. Moreover, performing a dexamethasone-test on two separate occasions, using a dose of 0.25 dexamethasone, morning salivary cortisol concentration was shown to be more variable compared to morning serum cortisol (61). This experiment has not yet been performed using the 1 mg dexamethasone test. Finally, a limited interassay variation at very low (1 nmol/l) cortisol concentrations is a prerequisite for reliable measurement of dexamethasone-suppressed salivary cortisol levels.
Combination of late night salivary cortisol and low-dose dexamethasone suppression test using salivary cortisol measurement
A u to m a te d m ea su re m en t o f s a liv a ry c
Author (Ref) Published (yr) Setting Collection
method
salivary cortisol assay
Salivary cortisol concentration after 1 mg dex (nmol/l,mean ± SE) (Number of subjects)
Sensitiv. (%)
Specifi c. (%)
Healthy Obese Cushing’s Ruled out Cushing’s Syndrome
Laudat (13) 1988 Inpatient Salivette RIA 2.1 ± 1.1 (101) n.d. n.d. 16.1 ± 7.8 (14) 100 100
Barrou (68) 1996 Outpatient 2 days 2 mg dex Salivette RIA n.d. 0.8 – 2.8 (64) n.d. 3.3 – 34 (27) 100 100 Martinelli (63) 1999 In-patient, children spitting RIA n.d. 1.8 ± 0.3 (21) n.d. 30.1 ± 24.4 (11) 100 100
CONCLUSIONS
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