Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test.

CONTEXT: Adrenal insufficiency (AI) is usually diagnosed by low plasma cortisol levels following a short Synacthen test (SST). Most plasma cortisol is bound to corticosteroid-binding globulin, which is increased by estrogen in combined estrogen-progestin oral contraceptives (COCs). Women with AI using COCs are therefore at risk of having an apparently normal plasma cortisol level during SST, which would not adequately reflect AI. OBJECTIVE: This work aimed to test whether salivary cortisol or cortisone during SST is more robust against the COC effect and to calculate the lower reference limits (LRLs) for these to be used as tentative diagnostic cutoffs to exclude AI. METHODS: Forty-one healthy women on COCs and 46 healthy women without exogenous estrogens underwent an SST with collection of plasma and salivary samples at 0, 30, and 60 minutes after Synacthen injection. The groups were compared using regression analysis with age as covariate and the LRLs were calculated parametrically. RESULTS: SST-stimulated plasma cortisol levels were significantly higher in the COC group vs controls, while mean salivary cortisol and cortisone levels were slightly lower in the COC group. Importantly, COC use did not significantly alter LRLs for salivary cortisol or cortisone. The smallest LRL difference between groups was seen for salivary cortisone. CONCLUSION: Salivary cortisol and especially salivary cortisone are considerably less affected by COC use than plasma cortisol during SST. Due to similar LRLs, a common cutoff for salivary cortisol and cortisone during SST can be used to exclude AI in premenopausal women irrespective of COC use.

Salivary cortisol and cortisone in diagnosis of Cushing's syndrome - a comparison of six different analytical methods.

OBJECTIVES: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing's syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS. METHODS: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves. RESULTS: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4-3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7-1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5-16.6 nmol/L at 23:00 h and 3.0-3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96. CONCLUSIONS: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

Confounding effects of liquorice, hydrocortisone, and blood contamination on salivary cortisol but not cortisone.

Objective: To determine the effects of liquorice consumption, topical hydrocortisone, and blood contamination on salivary cortisol and cortisone concentrations. Design and methods: Thirty healthy volunteers were randomized to a low, medium, or high dose of liquorice. Late-night saliva samples were collected using a Salivette® collection device at baseline, during 1 week of daily liquorice consumption, and during 4 weeks' washout. Saliva sampling was also performed before and after the application of topical hydrocortisone on the skin. Furthermore, in a subgroup (n = 16), saliva and venous blood were collected from each individual and mixed to achieve graded blood contamination in saliva. Salivary cortisol and cortisone were analyzed with liquid chromatography-tandem mass spectrometry. Results: Significant increases in salivary cortisol concentrations were observed during medium- (+49%) and high-dose (+97%) liquorice intake, which returned to baseline 4 days after liquorice withdrawal. Topical hydrocortisone on fingers holding the collection swab increased salivary cortisol concentrations >1000-fold with concomitant pronounced elevation of the cortisol:cortisone ratio. Salivary cortisol increased significantly after contamination with blood ≥0.5%. Visual examination could safely detect these samples. Salivary cortisone concentrations were unaffected by liquorice consumption and blood contamination, and only marginally affected by topical hydrocortisone. Conclusion: Liquorice, topical hydrocortisone, and blood contamination may all cause elevated salivary cortisol concentrations. Improved sampling instructions and visual examination of the sample may minimize these risks. Salivary cortisone is essentially unaffected by the different preanalytical confounders and may be used as a first-line screening test for Cushing's syndrome.

Reference intervals of salivary cortisol and cortisone and their diagnostic accuracy in Cushing's syndrome.

OBJECTIVE: The challenge of diagnosing Cushing's syndrome (CS) calls for high precision biochemical screening. This study aimed to establish robust reference intervals for, and compare the diagnostic accuracy of, salivary cortisol and cortisone in late-night samples and after a low-dose (1 mg) dexamethasone suppression test (DST). DESIGN AND METHODS: Saliva samples were collected at 08:00 and 23:00 h, and at 08:00 h, after a DST, from 22 patients with CS and from 155 adult reference subjects. We also collected samples at 20:00 and 22:00 h from 78 of the reference subjects. Salivary cortisol and cortisone were analysed with liquid chromatography-tandem mass spectrometry. The reference intervals were calculated as the 2.5th and 97.5th percentiles of the reference population measurements. Diagnostic accuracies of different tests were compared, based on areas under the receiver-operating characteristic curves. RESULTS: The upper reference limits of salivary cortisol and cortisone at 23:00 h were 3.6 nmol/L and 13.5 nmol/L, respectively. Using these reference limits, CS was detected with a sensitivity (95% CI) of 90% (70-99%) and specificity of 96% (91-98%) for cortisol, and a 100% (84-100%) sensitivity and 95% (90-98%) specificity for cortisone. After DST, cortisol and cortisone upper reference limits were 0.79 nmol/L and 3.5 nmol/L, respectively. CS was detected with 95% (75-100%) sensitivity and 96% (92-99%) specificity with cortisol, and 100% (83-100%) sensitivity and 94% (89-97%) specificity with cortisone. No differences in salivary cortisol or cortisone levels were found between samples collected at 22:00 and 23:00 h. CONCLUSION: Salivary cortisol and cortisone in late-night samples and after DST showed high accuracy for diagnosing CS, salivary cortisone being slightly, but significantly better.

20α- and 20ß-dihydrocortisone may interfere in LC-MS/MS determination of cortisol in saliva and urine.

Background LC-MS/MS methods offer high selectivity in cortisol determinations. However, endogenous steroid metabolites may still interfere and compromise the results, for example in the diagnosis of Cushing's syndrome. Erroneously elevated cortisol may, in particular, be misleading at the low concentrations found in salivary samples obtained at late night and after dexamethasone suppression. Methods Interferences in our LC-MS/MS method used for determination of cortisol in saliva and urine were identified by comparing their retention times and mass spectra with those of pure candidate substances. The chromatographic conditions used in our LC-MS/MS method, including column and mobile phase gradient, were varied in order to separate the target compound from the interferences. Results Two interferences, which were co-eluting or eluting close to cortisol in our original method, were successfully separated from cortisol by adjustment of the chromatographic conditions. These interferences were found in both urine and saliva and were identified as the two endogenous cortisol isomers 20α- and 20ß-dihydrocortisone. The isomers share molecular mass and mass spectrometric fragmentation pattern with cortisol using electrospray ionization in the positive-ion mode. Both give rise to the transitions m/z 363.1>121.1, 363.1>115.1 and 363.1>97.1. In our original LC-MS/MS setup, the 20ß-dihydrocortisone co-eluted with cortisol in the chromatography step resulting in false high determinations. Conclusions Cortisol determination by LC-MS/MS may suffer from erroneously elevated results unless 20α- and 20ß-dihydrocortisone are chromatographically separated from cortisol.

Automated white blood cell counts in cerebrospinal fluid using the body fluid mode on the platform Sysmex XE-5000.

BACKGROUND: The Sysmex XE-5000 offers automated quantification of red blood cells and white blood cells (WBCs) in body fluids, with differentiation of polymorphonuclear cells (PMNs) and mononuclear cells (MNCs). METHODS: We evaluated automated WBC counting in cerebrospinal fluid (CSF) using the body fluid mode on the Sysmex XE-5000, comparing it with flow cytometry as the reference method, and also with manual counting by microscopy. Experimental analysis for linearity and limit of detection was performed by diluting isolated WBCs in cell-free CSF. To study the ability to discriminate between PMNs and MNCs, samples were spiked using MNCs separated from peripheral blood. Comparison of WBC counts between a counting chamber and the XE-5000 was performed for 198 CSF samples. RESULTS: In the experimental set-up, within-run (CV 19%) and between-day imprecision (CV 15.3%) in quantitating total number of WBC on XE-5000 was acceptable for WBC counts ≥ 25 × 10(6)/L. Compared with expected cell counts, mean bias was + 2.6% for flow cytometry, + 5.5% for XE-5000 and - 73.2% for manual counting. Differentiation between PMNs and MNCs was in concordance with flow cytometry. In comparisons of clinical CSF samples, overall agreement between the XE-5000 and manual counting was observed in 81% of the samples, but mean difference in WBC differentiation was higher for PMN (51.1 × 10(6)/L) than for MNC (7.95 × 10(6)/L). CONCLUSION: Despite limited precision at low WBC counts, XE-5000 could be a favourable alternative to the labour-intensive, time-consuming and less reliable manual counting and cuts turnaround times in routine CSF-based diagnosis.

Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations.

The microtubule-associated protein 4 (MAP4) has recently been shown to counteract destabilization of interphase microtubules caused by catastrophe promotion but not by tubulin sequestering. To address how MAP4 discriminates between destabilization of microtubules by these two mechanisms, we have evaluated the combined phenotypes of MAP4 coexpressed with Op18/stathmin family member derivatives with either catastrophe-promoting or sequestering activities. This approach relies on the finding that overexpression of MAP4 alone stabilizes microtubules during all phases of the cell cycle in human leukemia cells, and causes a potent mitotic block and a dramatic, previously unobserved, phenotype characterized by large monoastral spindles. Coexpression of either catastrophe-promoting or tubulin-sequestration-specific Op18 derivatives was found to modulate the activity of ectopic MAP4 during mitosis, but with differential functional outcome. Interestingly, the tubulin-sequestering derivative suppressed the monoastral mitotic phenotype of MAP4 (i.e. coexpression facilitated the formation of functional spindles). To evaluate whether this phenotypic suppression could be explained by tubulin-sequestration-dependent modulation of MAP4 activity, a plasma-membrane-targeted, tubulin-sequestering chimera was constructed to decrease the cytosolic free tubulin concentration substantially. This chimera likewise suppressed the monoastral phenotype caused by overexpression of MAP4, suggesting a direct downregulation of MAP4 activity by reduced free tubulin concentrations.

MAP4 counteracts microtubule catastrophe promotion but not tubulin-sequestering activity in intact cells.

Microtubules are polar polymers that continually switch between phases of elongation and shortening, a property referred to as dynamic instability. The ubiquitous microtubule associated protein 4 (MAP4) shows rescue-promoting activity during in vitro assembly of microtubules (i.e., promotes transitions from shortening to elongation), but its regulatory role in intact cells is poorly defined. Here, we demonstrate that ectopic MAP4 promotes outgrowth of extended MTs during beta1-integrin-induced cell spreading. An inducible cotransfection protocol was employed to further analyze the regulatory role of MAP4 in human leukemia cells with microtubules partially destabilized by either ectopic tubulin-sequestering proteins or proteins that promote catastrophes (i.e., transitions from elongation to shortening). Coexpression of proteins that sequester free tubulin heterodimers with different efficiencies was found to abolish microtubule stabilization by MAP4. In contrast, however, the microtubule-stabilizing activity of MAP4 was found to suppress the activities of two distinct and specific catastrophe promoters, namely, XKCM1 and a nonsequestering truncation derivative of Op18/stathmin. These observations reveal specificity in the microtubule-stabilizing activity of MAP4 that differentiates between two mechanistically distinct types of MT destabilization.