Late-night salivary cortisol measurement in the diagnosis of Cushing's syndrome

Making a definite diagnosis of Cushing's syndrome is a challenging problem. Unsuspected Cushing's syndrome occurs in 2-3% of patients with poorly controlled diabetes, 0.5-1% with hypertension, 6-9% with incidental adrenal masses, and 11% with unexplained osteoporosis and vertebral fractures. The increasing recognition of this syndrome highlights the need for a simple, sensitive, and specific diagnostic test. Patients with Cushing's syndrome consistently do not reach a normal nadir of cortisol secretion at night. The measurement of late-night salivary cortisol levels might, therefore, provide a new diagnostic approach for this disorder. Salivary cortisol concentrations reflect those of active free cortisol in plasma and saliva samples can easily be obtained in a nonstressful environment (e.g. at home). Late-night salivary cortisol measurement yields excellent overall diagnostic accuracy for Cushing's syndrome, with a sensitivity of 92-100% and a specificity of 93-100%. Several factors can, however, make interpretation of results difficult; these factors include disturbed sleep-wake cycles, contamination of samples (particularly by topical corticosteroids), and illnesses known to cause physiologic activation of the pituitary-adrenal axis. In this Review, we discuss the methods and value of measuring salivary cortisol for the diagnosis of Cushing's syndrome, and put forward some recommendations to maximize accuracy of results.     

Late-night salivary cortisol as a screening test for the diagnosis of Cushing's syndrome in Japan

Measurement of late-night and/or midnight salivary cortisol currently used in US and European countries is a simple and convenient screening test for the initial diagnosis of Cushing's syndrome (CS). Unfortunately, this test has not been widely used in Japan. The purpose of this study was to evaluate the usefulness of the measurement of late-night salivary cortisol as a screening test for the diagnosis of CS in Japan. We studied 27 patients with various causes of CS, consisting of ACTH-dependent Cushing's disease [5] and ectopic ACTH syndrome [4] and ACTH-independent adrenal CS [11] and subclinical CS [7]. Eleven patients with type 2 diabetes and obesity and 16 normal subjects served as control group. Saliva samples were collected at late-night (23:00) in a commercially available device and assayed for cortisol by radioimmunoassay. There were highly significant correlations (P<0.0001) between late-night serum and salivary cortisol levels in normal subjects (r = 0.861) and in patients with CS (r = 0.788). Late-night salivary cortisol levels in CS patients (0.975 +/- 1.56 microg/dl) were significantly higher than those in normal subjects (0.124 +/- 0.031 microg/dl) and in obese diabetic patients (0.146 +/- 0.043 microg/dl), respectively. Twenty-five out of 27 CS patients had late-night salivary cortisol concentrations greater than 0.21 microg/dl, whereas those in control group were less than 0.2 microg/dl. Receiver operating characteristic curve (ROC) analysis showed that the cut-off point of 0.21 microg/dl provides a sensitivity of 93% and a specificity of 100%. Therefore, it is concluded that the measurement of late-night salivary cortisol is an easy and reliable noninvasive screening test for the initial diagnosis of CS, especially useful for large high-risk populations, such as diabetes and obesity.     

Evaluation of salivary cortisol measurements for the diagnosis of subclinical Cushing's syndrome

Late-night salivary cortisol (NSC) has been recognized as a sensitive and easy-to-perform screening test for the diagnosis of overt Cushing's syndrome (CS). However, there have been few reports on the diagnostic utility of salivary cortisol (SC) measurement in the diagnosis of subclinical Cushing's syndrome (SCS). Therefore, the present study was designed to evaluate the usefulness of SC measurements at late-night and after overnight 1 mg dexamethasone suppression test (DST) for the diagnosis of SCS in 42 patients with adrenal incidentaloma. We evaluated 16 patients with SCS, 12 with nonfunctioning adenoma (NFA), 8 with primary aldosteronism (PA), and 6 with pheochromocytoma (Pheo). NSC levels in SCS patients (0.238 ± 0.106 μg/dL) were significantly (P < 0.05) higher than those in NFA patients (0.154 ± 0.104 μg/dL); the cutoff value (0.11 μg/dL) by ROC analysis gave high sensitivity (100%) with low specificity (50%). Post DST SC levels in SCS patients (0.238 ± 0.116 μg/dL) were significantly (P = 0.0081) higher than those in NFA patients (0.136 ± 0.110 μg/dL); the cutoff value (0.12 μg/dL) by ROC analysis gave high sensitivity (93.8%) with somewhat improved specificity (58.3%). Both NSC and post DST SC levels were comparable between NFA, PA, and Pheo patients. In conclusion, our study revealed that measurements of NSC and/or post DST SC among patients with adrenal incidentaloma prove to have high sensitivities, but low specificities for the diagnosis of SCS from NFA, suggesting its possible alternative option before the screening tests for SCS currently employed in Japan.     

Midnight salivary cortisol for the initial diagnosis of Cushing's syndrome of various causes

We assessed the value of midnight salivary cortisol for the initial diagnosis of Cushing's syndrome. Sixty-three patients with various causes of Cushing's syndrome (37 with Cushing's disease, 17 with adrenal Cushing's syndrome, and nine with ectopic ACTH syndrome) and 54 control subjects with simple obesity were studied. All patients with Cushing's syndrome excreted more than 90 microg urinary free cortisol (UFC)/d (248 nmol/d), and all controls excreted less than 90 microg/d UFC. All patients with Cushing's syndrome had a midnight salivary cortisol concentration above 2.0 ng/ml (5.52 nmol/liter), whereas only three controls did so [2.0 ng/ml (5.52 nmol/liter); 2.05 ng/ml (5.66 nmol/liter); and 3.6 ng/ml (9.96 nmol/liter)]. This cut-off provides a sensitivity of 100% and a specificity of 96%. In patients with Cushing's syndrome, midnight salivary cortisol concentrations were correlated with UFC collected over the same period of time (0800-0800 h). Salivary cortisol measurements taken every 4 h showed a typical lack of circadian variation. The daily measurement of midnight salivary cortisol concentrations for 2 wk or more in five other out-patients (with obvious Cushing's disease, subclinical adrenal Cushing's syndrome, suspected Cushing's syndrome, pituitary incidentaloma, and prolactinoma) demonstrated the clinical utility of this factor. Measuring midnight salivary cortisol is an easy and noninvasive means of diagnosing hypercortisolism. Its diagnostic accuracy is identical to, if not better than, that of previously described gold standards.     

Nighttime salivary cortisol: a useful test for the diagnosis of Cushing's syndrome

Clinical features such as weight gain, depression, hypertension, and menstrual irregularities, although common in the general population, may raise the possibility of Cushing's syndrome. Up to 30% of urine cortisol and dexamethasone suppression screening tests may return an incorrect result, suggesting that better tests are needed. This study evaluated the utility of nighttime salivary cortisol measurement as a screening test for Cushing's syndrome. We evaluated 139 inpatients and 4 outpatients with possible Cushing's syndrome, 16 inpatients and 7 outpatients with other nonadrenal disorders, and 34 healthy outpatients. Using cut points that excluded all subjects without Cushing's syndrome, we compared the sensitivity for the detection of Cushing's syndrome of nighttime salivary cortisol levels (2330 and 2400 h for inpatients and bedtime for outpatients), simultaneous inpatient serum cortisol levels, and urine glucocorticoid excretion. An assay- specific inpatient 2400-h salivary cortisol or an outpatient bedtime salivary cortisol greater than 550 ng/dl (15.2 nmol/liter) identified 93% of patients with Cushing's syndrome (confidence interval, 89-98%) and excluded all individuals without the disorder. Salivary cortisol measurements worked as well as plasma measurements and better than urine glucocorticoid excretion. We concluded that bedtime salivary cortisol measurement is a practical and accurate screening test for the diagnosis of Cushing's syndrome.     

唾液皮质醇诊断库欣综合征的临床意义

近年来多项研究显示唾液皮质醇对于诊断库欣综合征是一个敏感和方便的筛查指标。午夜唾液皮质醇的测定可明确诊断大部分的库欣综合征,排除假性库欣综合征;结合小剂量地塞米松唾液皮质醇抑制试验能提高诊断敏感性。唾液皮质醇的稳定性尤其适用于周期性库欣综合征的诊断和随访。     

Measurement of free urinary cortisol and cortisone using liquid chromatography associated with tandem mass spectrometry method

Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation, based on automated preparative methods and tandem mass spectrometry, described in our country.     

基于多维液相色谱质谱组合分析的痢疾杆菌蛋白质基因组学

目的 应用多维液相色谱质谱组合体系为基础的蛋白质组学方法对福氏痢疾杆菌基因组注释进行完善。方法 痢疾杆菌福氏2a型301株(Sf2a301)的全菌蛋白经胰酶消化,二维液相色谱分离后进行MALDI-TOF/TOF和ESI-MS/MS组合鉴定,质谱数据分别应用MASCOT和SEQUEST软件检索基于Sf2a301全基因组构建的6个读码框数据库,完成对原基因组注释的验证和补充。结果 研究表明多维液相色谱质谱组合体系能够增加鉴定蛋白的覆盖率,共鉴定Sf2a301的1 231个蛋白编码基因产物,涵盖了COGs 数据库22个功能分类组中的20个,包含306个功能未知的假定蛋白。发现了9个未注释的基因,得到RT-PCR和Northern blot的进一步验证。新基因大多数是重叠基因,包含3个嵌套基因。结论 多维液相色谱质谱组合体系相对于单一的串联质谱技术能够更加有效验证、补充痢疾杆菌的基因组注释,更新后的基因组注释库为今后开展痢疾杆菌功能研究提供更多的靶点。     

液相串联质谱法定量检测高密度脂蛋白中脂质类化合物

目的本文旨在建立一种快速提取高密度脂蛋白(HDL)中脂质并结合液相串联质谱定性及快速定量检测的方法。方法采用酸化甲醇沉淀结合超声萃取的方法抽提脂质,高速离心去除蛋白颗粒,取上清液,直接采用液相串联质谱在多反应监测模式下进行定量分析。结果该分离提取脂质的方法简单迅速,采用多反应监测模式可同时定量监测30多种具有标准品的脂质成份,包括鞘氨醇类、鞘磷脂、神经酰胺和氧化型的卵磷脂。结论酸化甲醇超声抽提离心法可简便迅速地获得HDL中的脂质成份,结合液相串联质谱可对HDL中的多种脂质进行定量检测,该方法具有高效、重现性好等优点,适用于大量液体生物样本的分析。     

The establishment of a method for serum glycated albumin based on liquid chromatography/tandem mass spectrometry

正Objective To establish a candidate reference method for the determination of serum glycated albumin based on isotope dilution liquid chromatography/tandem mass spectrometry(ID-LC/MS).Methods According to the Japan Society of Clinical Chemistry(JSCC)reference method,the serum albumin was firstly purified by SDS-     

Diagnostic value of salivary cortisol in Cushing's syndrome (CS)

Objective The diagnosis of Cushing's syndrome (CS) remains a challenge in clinical endocrinology. The aim of this study was to determine the reproducibility and diagnostic value of late‐night salivary cortisol (SAF₂₃) for CS and its utility along the follow‐up of treated patients. In addition, using the same radioimmunoassay reactives, the cut‐off values for saliva and serum cortisol, assessed synchronically after the overnight 1 mg dexamethasone suppression test (DST), were defined. Design Twenty‐one patients with confirmed CS and 121 volunteers were studied. All the subjects collected 24‐h urine for cortisol (UFC). On the same day whole saliva was obtained from the subjects at 23 h for SAF₂₃. The intraclass coefficient of correlation (ICC) of SAF₂₃ was estimated in 47 subjects (21 CS and 26 C). At 8 h, after DST, simultaneous saliva and serum samples for cortisol (SAF_dex and F_dex, respectively) were obtained in 51 subjects (17 CS and 34 C). After specific therapy, 18 patients with CS were followed with SAF₂₃ measurements. SAF and F were expressed as nm . Results The intraclass coefficient of correlation of SAF₂₃ was 0·89 in CS and 0·83 in C. SAF₂₃ > 3·8 nm showed a sensitivity and specificity of 100% and 97·5%, respectively, for diagnosing CS. SAF₂₃ correlated positively with UFC (r = 0·685; P = 0·0001). After DST, SAF_dex significantly correlated with F_dex (r = 0·61, P < 0·0001). A cut‐off value of SAF_dex > 2·0 nm and F_dex > 50·0 nm detected CS with 100% sensitivity and specificity. After successful surgical therapy, 13 patients with CS had SAF₂₃ levels < 3·8 nm (1·4 ± 0·8 nm ). Conclusions SAF₂₃ and SAF_dex seem to be good screening tools based on their noninvasive nature, remarkable reproducibility and diagnostic performances.     

Midnight salivary cortisol versus urinary free and midnight serum cortisol as screening tests for Cushing's syndrome

The diagnosis of Cushing's syndrome (CS) is often a challenge. Recently, the determination of late night salivary cortisol levels has been reported to be a sensitive and convenient screening test for CS. However, no studies have included a comparison with other screening tests in a setting more closely resembling clinical practice, i.e. few patients with CS to be distinguished from patients with pseudo-Cushing states (PC), including the large population of obese patients. The aim of this study was to compare the diagnostic performance of midnight salivary cortisol (MSC) measurement with that of midnight serum cortisol (MNC) and urinary free cortisol (UFC) in differentiating 41 patients with CS from 33 with PC, 199 with simple obesity, and 27 healthy normal weight volunteers. Three patients with CS had MSC levels lower than the cut-off point derived from receiver operator characteristic analysis (9.7 nmol/liter), yielding a sensitivity for this parameter of 92.7%. In the whole study population, no statistically significant differences in terms of sensitivity, specificity, diagnostic accuracy, and predictive values were observed among tests. In particular, the overall diagnostic accuracy for MSC (93%; 95% confidence interval, 90.1-95.9%) was similar to those of UFC (95.3%; 94.1-96.5%) and MNC (95.7%; 93.4-98%; both P = NS). The diagnostic performance of MSC was superimposable to that of MNC also within the area of overlap in UFC values (< or =569 nmol/24 h) between CS and PC. In conclusion, MSC measurement can be recommended as a first-line test for CS in both low risk (simple obesity) and high-risk (i.e. PC) patients. Given its convenience, this procedure can be added to tests traditionally used for this purpose, such as UFC and MNC.     

Measurement of salivary cortisol with liquid chromatography-tandem mass spectrometry in patients undergoing dynamic endocrine testing

Objective  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) eliminates cross-reactivity, which is a major limitation of immunoassays used for the measurement of salivary cortisol (SalC). We aimed to evaluate the potential of SalC measured by LC-MS/MS in patients undergoing assessment of the HPA axis.
Design and patients  Cross-sectional study of 78 patients admitted for routine testing in a specialized endocrine unit.
Measurements  Matched serum and saliva samples were collected from 68 patients who had a short synacthen test (SST, 250 mcg im) and 10 patients who had an insulin tolerance test (ITT, insulin 0·15 U/kg iv). Serum cortisol (SerC) was measured with an automated immunoassay and SalC with LC-MS/MS. Adequate SerC responses were >500 nmol/l.
Results  In all patients with adequate responses, the relative increase in SalC was significantly higher than that in SerC [6·4(0·3–26·1) vs. 1·0(0·3–4·9), P  <   0·0001)]. The SerC–SalC relationship was better explained by an exponential rather than a linear model ( R ² = 0·83 vs. R ² = 0·65, both P  <   0·0001). Based on 59 patients with adequate SerC responses to an SST, an adequate SalC response was defined as 8·3 nmol/l. Seven patients following an SST and three patients following an ITT showed inadequate responses in both SerC and SalC, but two patients with CBG deficiency showed a low SerC with normal SalC.
Conclusions  We have shown an excellent diagnostic sensitivity and specificity of LC-MS/MS SalC in the assessment of the HPA axis and superiority over SerC when CBG levels are altered. The exponential relationship between SerC and SalC supports the concept of CBG binding capacity saturation.     

唾液皮质醇检测在皮质醇增多症诊断中的应用

本研究运用竞争性放射免疫分析法，对40名健康成人，58例单纯性肥胖患者和23例经病理证实的库欣综合症患者进行唾液皮质醇测定，发现单次午夜唾液皮质醇测定诊断库欣综合征的敏感性和特异性可达100．0％和91．4％，提示唾液皮质醇检测是一种高诊断价值的无创性检查方法，可作为库欣综合征的首选筛查指标。     

Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry

The hepatic peptide hormone hepcidin is considered the central regulator of iron metabolism. Characterizing the circulating levels of this peptide is critical to understanding its role in the development of clinically relevant syndromes, such as anemia of inflammation/chronic disease, and may provide insight into potential clinical interventions. While quantitative methods have been published for the determination of urinary hepcidin and serum prohepcidin, no definitive methods have been published for the determination of hepcidin in serum. In this report, we describe a quantitative method for the determination of both human and mouse hepcidin in serum and plasma. The method employs protein precipitation and solid-phase extraction followed by liquid chromatographic separation and tandem mass spectrometry detection. The method has a quantitative range of 0.25 ng/mL to 500 ng/mL serum for mouse hepcidin and 1 ng/mL to 500 ng/mL serum for human hepcidin. The method uses small sample volumes (50 microL for mice and 100 microL for humans) and 96-well formats for rapid sample processing. The method was used to establish baseline serum and plasma concentrations of hepcidin in normal C57Bl/6 mice and healthy human volunteers.     

Salivary cortisol measurement--a reliable method for the diagnosis of Cushing's syndrome.

The measurement of cortisol in saliva is becoming more widely accepted as a screening test for the diagnosis of hypercortisolism. Since 1986, cortisol measurement in saliva has been continuously used in our department. In this study we compared salivary cortisol profiles from proven Cushing's disease patients with profiles from healthy subjects and obese children. The purpose was to evaluate the predictive value of the method for the diagnosis of hypercortisolism and to define cut-off levels to exclude or identify hypercortisolism. Cortisol in saliva was measured in 150 Cushing's disease patients (30 children, 120 adults, ranging from age 4-70), 100 healthy subjects (55 children, 45 adults, ranging from age 6-60), and 31 children (age 7-15) with an age-related body-mass-index above the 90th percentile. Generally, five saliva samples were taken over the day at 6:00-8:00 a.m., 11:00-12:00 a.m., 4:00-6:00 p.m., 7:00-8:00 p.m., and 10:00 p.m. The samples were measured using a radioimmuno-assay (INCSTAR Corporation, Stillwater, Minnesota, USA). For healthy subjects, morning levels of cortisol in saliva between 3-19 microg/l were found. These levels dropped to levels in between <1-11 microg/l at 11:00-12:00 a.m., <1-6 microg/l at 4:00-6:00 p.m., <1-4.5 microg/l at 7:00-8:00 p.m., and <1-2.9 microg/l at 10:00 p.m. The measured values showed a correlation with age, height, and weight. In Cushing's disease patients, the circadian salivary cortisol rhythm was missing, compared to healthy subjects. There was no significant difference in salivary cortisol levels or circadian rhythm between healthy or obese children. We found a high sensitivity for the detection of hypercortisolism at the 10:00 p.m. salivary cortisol measurement. The following, age dependent cut-off levels for salivary cortisol at 10:00 p.m. were calculated for the exclusion of hypercortisolism. Age 6-10: 1.0 microg/l (specificity 100%, sensitivity 87.5%); age 11-15: 1.7 microg/l (specificity 100%, sensitivity 100%); age 16-20: 1.6 microg/l (specificity 100%, sensitivity 76.2%); age 21-60: 1.6 microg/l (specificity 100%, sensitivity 90.9%) [corrected] For the proof of Cushing's syndrome, the following age-dependent cut-off levels at 10:00 p.m. were found: age 6-10: 1.9 microg/l (specificity 100%, sensitivity 80%); age 11-15: 1.7 microg/l (specificity 100%, sensitivity 100%); age 16-20: 2.5 microg/l (specificity 100%, sensitivity 84.2%); age 21-60: 1.9 microg/l (specificity 100%, sensitivity 97.6 %) [corrected] The cortisol assessment in saliva is a sensitive and reliable method to discriminate normocortisolemic from hypercortisolemic patients. From our view, the major advantages of this method are the reliability, non-invasiveness, and use in ambulatory patients.