DHEAS and Differential Blood Counts as Indirect Signs of Glucocorticoid Excess in Adrenal Non-Producing Adenomas.

The majority of incidentally discovered adrenal tumors are later characterized as non-producing adrenocortical adenomas (NPA). We asked whether laboratory abnormalities in parameters that reflect glucocorticoid action can be found in patients with NPA despite their nature of being clinically unapparent. Since glucocorticoids are potent immunosuppressants we studied blood counts and differential blood counts along with corticotropin and dehydroepiandrostenedione sulfate (DHEAS) blood concentrations, as well as cortisol values before and after an overnight 1 mg dexamethasone suppression test. We compared the results of normal individuals, of patients with adrenal adenomas and normal hormone profiles and with subclinical autonomous glucocorticoid hypersecretion, as well as overt cortisol excess. We found that almost all indices of the blood counts were significantly different between the patients groups. In particular, patients with adrenal non-producing adenomas already showed signs of glucocorticoid excess, including relative lymphocytopenia, lowered DHEAS, and ACTH concentrations than control individuals. We also found that the extent of lymphocytopenia correlated with the concentrations of DHEAS and ACTH, and DHEAS correlated well with ACTH. We conclude that the basal ACTH and DHEAS values along with the differential blood counts give good information on the extent of glucocorticoid excess and that silent adrenal adenomas seem to oversecrete glucocorticoids at concentrations that already alter these parameters.

The response of C19- and some C21-steroids during Synacthen and insulin tolerance test.

Testing of the adrenal function with ACTH 1-24 (Synacthen test) or insulin (insulin tolerance test-ITT) is commonly used. The question of ongoing debate is the dose of Synacthen. Moreover, it may be important from the physiological point of view besides measurement of cortisol levels and 17α-hydroxy-progesterone to know also the response of other steroids to these test. The plasma levels of 24 free steroids and their polar conjugates were followed after stimulation of 1 µg, 10 µg and 250 µg of ACTH 1-24 and after insulin administration in thirteen healthy subjects. The study aimed to describe a response of steroid metabolome to various doses of ACTH 1-24 and to find the equivalency of these tests. The additional ambition was to contribute to understanding of physiology of these stimulation tests and suggest an additional marker for HPA axis evaluation. No increase of most conjugated steroids and even decrease of some of them during all of the Synacthen tests and ITT at 60th min were observed. The levels of steroid conjugates decreased in ITT but did not during all of the Synacthen tests by 20 min of each test. Testosterone and estradiol did not increase during the Synacthen tests or ITT as expected. The results suggest that the conjugated steroids in the circulation can serve as reserve stock for rapid conversion into free steroids in the first minutes of the stress situation. Various doses of ACTH 1-24 used in the Synacthen tests implicate earlier or later occurrence of maximal response of stimulated steroids. The equivalent dose to ITT and standard 250 µg of ACTH 1-24 seemed to be dose of 10 µg ACTH 1-24 producing the similar response in all of the steroids in the 60th min of the test.

The Response of C19 Δ5-steroids to ACTH Stimulation and Hypoglycemia in Insulin Tolerance Test for Adrenal Insufficiency.

Studies on the time course of ACTH- or insulin-induced hypoglycemia stimulating adrenal androgens are usually limited to dehydroepiandrosterone and/or its sulphate. Our data on dehydroepiandrosterone (DHEA) and its hydroxylated metabolites clearly show that measurements of DHEA and its sulphate (DHEAS) are valuable markers of the integrity of the HPA (hypothalamus-pituitary-adrenal) axis. Assessments of HPA function should rely on measurements of baseline and/or stimulated serum cortisol concentrations, and C19 Δ5-steroids may provide additional information. The art of stimulation of 7- and 16-hydroxylated metabolites of DHEA can help our understanding of the formation sequence of these compounds.

A Comparison of Salivary Steroid Levels during Diagnostic Tests for Adrenal Insufficiency.

Numerous diagnostic tests are used to evaluate the hypothalamic-pituitary-adrenal axis (HPA axis). The gold standard is still considered the insulin tolerance test (ITT), but this test has many limitations. Current guidelines therefore recommend the Synacthen test first when an HPA axis insufficiency is suspected. However, the dose of Synacthen that is diagnostically most accurate and sensitive is still a matter of debate. We investigated 15 healthy men with mean/median age 27.4/26 (SD±4.8) years, and mean/median BMI (body mass index) 25.38/24.82 (SD±3.2) kg/m2. All subjects underwent 4 dynamic tests of the HPA axis, specifically 1 µg, 10 µg, and 250 µg Synacthen (ACTH) tests and an ITT. Salivary cortisol, cortisone, pregnenolone, and DHEA (dehydroepiandrosterone) were analysed using liquid chromatography-tandem mass spectrometry. During the ITT maximum salivary cortisol levels over 12.5 nmol/l were found at 60 minutes. Maximum cortisol levels in all of the Synacthen tests were higher than this; however, demonstrating that sufficient stimulation of the adrenal glands was achieved. Cortisone reacted similarly as cortisol, i.e. we did not find any change in the ratio of cortisol to cortisone. Pregnenolone and DHEA were higher during the ITT, and their peaks preceded the cortisol peak. There was no increase of pregnenolone or DHEA in any of the Synacthen tests. We demonstrate that the 10 µg Synacthen dose is sufficient stimulus for testing the HPA axis and is also a safe and cost-effective alternative. This dose also largely eliminates both false negative and false positive results.

Changes in the Concentrations of Corticoid Metabolites--The Effect of Stress, Diet and Analytical Method.

A precisely measured corticoid level is important for decision-making in daily clinical practice. These levels can be influenced in the pre-analytical phase, when the effect of stress, timing, and diet can be important. The aim of this study was to elucidate optimal conditions for blood sampling as well as the choice of analytical methods, which they will be used in measuring of corticoids. By studying ten women, we focused on the influences of the stress of cannulation and a large lunchtime meal on cortisol, cortisone, aldosterone and corticosterone levels. We further compared results of cortisol measurements from RIA and LC-MS/MS. Stress from cannulation caused increase of cortisol, cortisone and corticosterone already, when the cannula was being inserted. This indicates that this increase is stimulated by fear of the blood withdrawing rather, than just by the needle insertion itself. The effect of stress on corticosterone disappeared after an hour, while effect on other corticoids was still apparent. Concerning the lunchtime meal, we found an increase in all measured corticoids between 11 and 12 o'clock. After the food, there were marked decreases in cortisone and aldosterone, while declining levels of cortisol and corticosterone had rather plateaus. We compared cortisol in 90 plasma samples measured by a commercial RIA kit and the LC-MS/MS method. Results from both methods showed a strong correlation (r=0.85). When measuring corticoid metabolites, the chosen analytical method, eliminating stress factors, and precisely timed blood sampling considering the daily rhythm and food intake are critical.

How Does Energy Intake Influence the Levels of Certain Steroids?

The influence of steroid hormones on food intake is well described. However, there are only a few studies on the effect of food intake on steroid levels. The study involved eight non-smoker women (average age 29.48±2.99 years; average BMI 21.3±1.3 kg/m2); they did not use any kind of medication affecting steroidogenesis. We analysed the influence of four various stimuli on the levels of steroid hormones and melatonin. During their follicular phase of menstrual cycle, each woman had an oral glucose tolerance test (OGTT), intravenous glucose tolerance test (IVGTT), a standard breakfast and psyllium (a non-caloric fibre). Cortisol declined during each test, which is a physiological decline in the morning hours. In all tests (except of the application of the non-caloric fibre, psyllium), however, this decline was modified. After the standard breakfast there was an increase in cortisol at 40th minute. The OGTT and IVGTT tests led to a plateau in cortisol levels. Testosterone levels and those of other steroid hormones showed no relationships to tested stimulations. Oral and intravenous glucose have influenced physiological decline of melatonin levels. During the IVGTT test, melatonin levels started to increase at 20th minute, reaching a maximum at 40th minute. The OGTT test led to a delayed increase in melatonin levels, compared to IVGTT. Despite the fact that we performed the tests in the morning hours, when steroid hormone levels physiologically start to change due to their diurnal rhythm, we still found that food intake influences some of the hormone levels.

How smoking cessation influence hormonal levels in postmenopausal women?

Smoking represents the most widespread substance dependence in the world. Nicotine alters women hormonal homeostasis. Women smokers have higher testosterone and lower estradiol levels throughout life compared to nonsmokers. We monitored the effect of smoking discontinuation on steroid spectrum with 25 postmenopausal women smokers. They had been examined before discontinuation of smoking and after 6, 12, 24 and 48 weeks of abstinence. Blood was collected to determine steroid spectrum (measured by GC-MS), luteinizing hormone, follicle stimulating hormone and sex hormone binding globulin (measured by IRMA). Repeated measures ANOVA model was used for evaluation of the data. In postmenopausal women, an increase in testosterone, dihydrotestosterone, dehydroepiandrosterone and other androgens occurred. Neither nicotine replacement therapy nor weight changes nor age play a role in androgen level increase. The higher androgens levels correlated with failure in smoking cessation. Women smokers have higher androgen levels, which might play a role in smoking dependence development. Women successful in smoking cessation, compared to the non-successful ones, have lower androgen levels initially and also after smoking discontinuation.