A new derivative for oxosteroid analysis by mass spectrometry.

Here we report a new method for oxosteroid identification utilizing "tandem mass tag hydrazine" (TMTH) carbonyl-reactive derivatisation reagent. TMTH is a reagent with a chargeable tertiary amino group attached through a linker to a carbonyl-reactive hydrazine group. Thirty oxosteroids were analysed after derivatisation with TMTH by electrospray ionization mass spectrometry (ESI-MS) and were found to give high ion-currents compared to underivatised molecules. ESI-tandem mass spectrometry (MS/MS) analysis of the derivatives yielded characteristic fragmentation patterns with specific mass reporter ions derived from the TMT group. A shotgun ESI-MS method incorporating TMTH derivatisation was applied to a urine sample.

Matrix-assisted laser desorption/ionisation time of flight spectrometry for the fast screening of oxosteroids using aromatic hydrated hydrazines as versatile probes.

To make the use of MALDI-based mass spectrometry feasible for the fast analysis of oxosteroids, three new aromatic probes have been designed to be used simultaneously as derivatisation agents and MALDI matrices. This concept brings a number of benefits: the sample handling is reduced, the workflow is less time consuming allowing high throughput and the interferences caused by the MALDI matrix are avoided. Identification was successfully attained for all oxosteroids used in this study. As proof-of-concept, the identification of oxosteroids in urine was performed to evaluate the robustness of the new methodology. The oxosteroids 17-α-methyltestosterone, nandrolone, boldenone, 17-α-Trenbolone, fluoxymesterolone, mesterolone and bolasterone were identified in human urine at the minimum concentration level recommended by the world anti-doping agency, 2 ng/mL.

Androgen imbalance in premenopausal women with benign breast disease and breast cancer.

OBJECTIVE: The alteration of steroid hormonal status in premenopausal breast disease (benign and malignant) were investigated by comparing the urinary profile of androgens and corticoids. METHODS: The urinary concentrations of 25 androgens and corticoids were quantitatively determined by a gas chromatographymass spectrometry system in patients with benign breast disease (35 cases, 20-54 years), breast cancer (34, 27-54), and healthy controls of similar age (25, 22-51). RESULTS: In premenopausal patients with breast cancer, a significantly lower rate of excretion of 11-deoxy-17-ketosteroids and their metabolites was found in comparison with normal females. These levels were also inversely associated with benign breast disease. No significant differences were found between the three groups for the concentration of 11-oxy-17-ketosteroids, 17-hydroxy-corticoids and their metabolites. The urinary ratio of adrenal androgen metabolites to cortisol metabolites [(11-DOKS & M)/11-OKS] declined in the order of normal female control (4.04 +/- 0.72; mean +/- SD), breast benign mass (2.29 +/- 0.42) and breast cancer (0.94 +/- 0.27). CONCLUSION: Our data suggest that the hormonal imbalance of androgen deficiency and/or corticoid sufficiency is closely associated with the benign and malignant conditions of premenopausal breast disease and the ratio of (11-DOKS & M)/11-OKS may be an effective discriminant factor of these groups.

The psychological and physiological effects of an intensive-care unit environment on healthy individuals.

OBJECTIVE: The ideal inpatient environment would be one in which patient stress and anxiety are alleviated, but current inpatient hospital settings often do not seem to take this fact into consideration. To date, the effects of the actual hospital environment itself on patients is poorly understood. The purpose of the present study was to investigate the types of psychological and physiological changes that people undergo in response to being placed in an inpatient setting. DESIGN AND SETTING: Ten healthy volunteers were admitted to an intensive-care unit (ICU) for 4 days and 3 nights. The psychological and physiological changes before and after admission were examined. A qualitative assessment was also performed. RESULTS: Most healthy individuals at times develop feelings of depression, which can be measured by the Profile of Mood States and the Zung Self-Rating Depression Scale (P < .05). In this study, participants' fatigue and confusion increased (P < .05) and vigor decreased (P < .01) as measured by the Profile of Mood States. There was also an increase in the General Health Questionnaire scores (P < .01). However, no significant physiological effects were apparent, as measured by peripheral lymphocyte counts, natural killer cells activity, and urinary 17-kerosteroid and 17-hydroxycorticosteroid levels. Subjects often engaged in introspection during hospitalization and often had a negative attitude towards the hospital environment. CONCLUSIONS: The findings of increased feelings of depression attributable solely to being in an ICU setting are inconsistent with the type of environment generally considered necessary to alleviate patient anxiety and tension in a critical-care-ward environment. The negative perception of this environment strongly suggests room for improvement. Efforts in this regard should focus on improving the five senses, particularly sight, sound, and taste within the ICU.

Comparative rates of androgen production and metabolism in Caucasian and Chinese subjects.

Clinically apparent prostate cancer occurs more commonly among Caucasians living in Western countries than in Chinese in the Far East. Prior studies demonstrated diminished facial and body hair and lower levels of plasma 3 alpha-androstanediol glucuronide and androsterone glucuronide in Chinese than in Caucasian men. Based upon these findings, investigators postulated that Chinese men could have diminished 5 alpha-reductase activity with a resultant decrease in prostate tissue dihydrotestosterone levels and clinically apparent prostate cancer. An alternative hypothesis suggests that decreased 3 alpha-androstanediol glucuronide and androsterone glucuronide levels might reflect reduced production of androgenic ketosteroid precursors as a result of genetic or environmental factors. The present study examined 5 alpha-reductase activity, androgenic ketosteroid precursors, and the influence of genetic and environmental/dietary factors in groups of Chinese and Caucasian men. We found no significant differences in the ratios of 5 beta-:5 alpha-reduced urinary steroids (a marker of 5 alpha-reductase activity) between Chinese subjects living in Beijing, China, and Caucasians living in Pennsylvania. To enhance the sensitivity of detection, we used an isotopic kinetic method to directly measure 5 alpha-reductase activity and found no difference in testosterone to dihydrotestosterone conversion ratios between groups. Then, addressing the alternative hypothesis, we found that the Caucasian subjects excreted significantly higher levels of individual and total androgenic ketosteroids than did their Chinese counterparts. To distinguish genetic from environmental/dietary factors as a cause of these differences, we compared Chinese men living in Pennsylvania and a similar group living in Beijing, China. We detected a reduction in testosterone production rates and total plasma testosterone and sex hormone-binding levels, but not in testosterone MCRs in Beijing Chinese as a opposed to those living in Pennsylvania. Comparing Pennsylvania Chinese with their Caucasian counterparts, we detected no significant differences in total testosterone, free and weakly bound testosterone, sex hormone-binding globulin levels, and testosterone production rates. Taken together, these studies suggest that environmental/dietary, but not genetic, factors influence androgen production and explain the differences between Caucasian and Chinese men.

The short-term effect of dietary pectin on plasma levels and renal excretion of dehydroepiandrosterone sulfate.

Studies specifically investigating the effects of single dietary components on plasma levels of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are rare. Especially no data is available with regard to specific dietary fibers. Therefore, the impact of pectin (a representative fiber that affects the enterohepatic recirculation of bile acids) was studied in a randomized crossover trial consisting of three diet periods characterized by the same food supply and daily doses of 0 g, 15 g or 30 g pectin. Blood and 24-h-urine samples were collected at the end of each 4-day diet period from 6 healthy male volunteers. Plasma levels of DHEA, cortisol and the major binding protein of DHEAS albumin remained unchanged with the varying pectin supplements. Also, no changes were observed for several urinary analytes including urinary DHEAS. However, effects of pectin intake (30, 15 versus 0 g/d) were seen for plasma DHEAS (9.3 +/- 2.8, 9.2 +/- 2.6, 8.0 +/- 3.1 mumol/L, p < 0.01) and total plasma cholesterol (4.4 +/- 0.7, 4.5 +/- 0.7, 4.7 +/- 0.8 mmol/L, p = 0.1). Obviously, the altered intake of fiber in the form of pectin affects plasma concentrations of DHEAS and cholesterol in an opposite direction. The reason for this is not known but a dietetically induced modulation of the binding properties of plasma albumin for DHEAS appears possible. Our findings suggest that the target tissue-available, not protein-bound fraction of circulating DHEAS (as reflected by the renal DHEAS output) is not necessarily altered when total plasma concentrations of DHEAS vary.

Daily excretion levels of an unidentified ketosteroid in the urine of patients with Cushing's syndrome and healthy subjects measured by a new method.

We developed a new method for measuring an unidentified ketosteroid glucuronide (US-G) detected by the method of Iwata et al. for measuring 17-ketosteroid glucuronides by reversed phase HPLC on a Capcell-Pak C8 column with three kinds of mobile phase solutions (Iwata method; Clin Chem 35: 795-799, 1989). The Iwata method inadequately separated US-G and two hydroxy 17-ketosteroides, 11 beta-hydroxyetiocholanolone and 11 beta-hydroxyandrosterone, and it exhibits insufficient sensitivity for measuring traces of US-G in the urine of healthy subjects. We solved these problems by developing a new method which measures US-G in urine, as a free type by hydrolyzing the glucuronide type enzymatically, by normal phase HPLC on a Capcell-Pak Silica column with one kind of mobile phase solution. By this method, the levels of US excreted as a glucuronide in the urine of healthy subjects and of patients with Cushing's syndrome were determined as proportions of the levels of 11 beta-hydroxyandrosterone. The average daily urinary excretion of US was 971 micrograms (125-4,995 micrograms) in patients with Cushing's syndrome (n = 22: two males and 20 females aged 26 to 65 years), and 34 micrograms (0-141 micrograms) in healthy subjects (n = 63: 49 males, and 14 females aged 21 to 54 years), and the differences were clearly significant. However, there were no differences between the urinary US levels of patients with pituitary adenoma and patients with adrenal adenoma. Furthermore, no US was detected in the urine of patients with aldosteronism (two males and eight females aged 34 to 61 years).(ABSTRACT TRUNCATED AT 250 WORDS)

Síndrome de Cushing: Estudo comparativo de métodos diagnósticos / Cushing's syndrome: A comparative study of diagnostic methods

Comparamos as respostas dos corticosteróides urinários:17-hidroxicorticosteróides (17-OHCS), 17-cetosteróides (17-KS) e cortisol livre (FC) à supressäo com dexametasona (baixa dose - 2 mg e alta dose - 8mg) em 71 pacientes com síndrome de Cushing (45 com doença de Cushing, 12 com carcinoma adrenal, oito com adenoma adrenocortical e seis com hiperplasia nodular da córtex adrenal). Os pacientes com doença de Cushing e hiperplasia nodular da córtex adrenal näo apresentam supressäo suficiente com 2 mg de dexametasona,porém o fazem com 8 mg. Os tumores adrenais näo demonstram supressäo adequada. A resposta hiperativa dos 17-OHCS, 17-KS e FC ao hormônio adrenocorticotrópico (ACTH) exógeno foi comparada na doença de Cushing. Concluimos que a determinaçäo urinária dos 17-OHCS, 17-KS e FC säo parâmetros diagnósticos úteis, embora a dosagem do FC mostrou ser um índice mais sensível da hiperfunçäo adrenocortical

Variable hormonogenesis in Cushing's syndrome.

Four patients with Cushing's syndrome and variable cyclic hormonogenesis are reported and 40 other cases from the literature are reviewed. These cases were divided into four categories depending on regular or irregular cyclic adrenal hypersecretion and presence or absence of concomitant fluctuations in the clinical course. The manifestations of cyclic adrenal hypersecretion in these patients varied from daily to yearly intervals. Cyclic activity persisted for as long as 25 years, with cycle lengths varying from 12 hours to 85 days. Some patients demonstrated complex biochemical cyclic patterns. Clinical presentations varied from a single outstanding symptom, such as recurring oedema, to a complex clinical syndrome. The aetiology in these patients varied: 12 appeared to be pituitary dependent, 11 had corticotropin-producing tumours and another eight were described as showing 'adrenal hyperplasia'. A hypothalamic disorder was found in four, a benign adrenal adenoma in two, and an adrenal 'mass' and adrenocortical nodular dysplasia in single patients. Evaluation during the intercyclic phase may reveal normal pituitary function. Inconsistent responsiveness to administration of dexamethasone in different phases of cyclic activities may suggest the presence of cyclic Cushing's syndrome.

Role of prolonged excessive estrogen stimulation in the pathogenesis of endometrial sarcomas: two cases and a review of the literature.

Two cases of endometrial sarcoma that developed many years after exposure to unopposed exogenous and endogenous estrogens, with no previous pelvic irradiation, are described. The literature is reviewed and the possible effect of prolonged unopposed estrogen stimulation is suggested as an etiologic factor in such extremely rare conditions. These cases add additional support to the assumption that prolonged and excessive endogenous or exogenous unopposed estrogen stimulation of the uterus may increase the risk of endometrial sarcoma.

Serum 3 alpha-androstanediol glucuronide measurements in sexually mature women with congenital adrenal hyperplasia during therapy.

Serum 3 alpha-androstanediol glucuronide (3 alpha-diol G) measurements may indicate the extent of androgen metabolism and action in target tissues. To test this supposition we measured serum 3 alpha-diol G concentrations in 23 women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, including 13 with the salt-losing and 10 with the simple virilizing form, while they were receiving glucocorticoid and, in some cases, mineralocorticoid therapy. Their mean age was 28.3 yr (range, 17.9-38.7 yr). Twenty-four-hour urinary 17-ketosteroid excretion, plasma androstenedione and testosterone levels, and serum 3 alpha-diol G levels were measured during the follicular phase. The values were within or below the normal range in 87%, 78%, 70%, and 91% of the patients, respectively. By contrast, plasma 17-hydroxyprogesterone levels were normal in only 12% of the patients. Serum 3 alpha-diol G levels correlated well with 24-h urinary 17-ketosteroid excretion (r = 0.75) and plasma 17-hydroxyprogesterone (r = 0.77), androstenedione (r = 0.84), and testosterone (r = 0.93) levels. The serum 3 alpha-diol G levels were not significantly different in the women with the salt-losing form and those with the simple virilizing form. However, they were significantly lower (P less than 0.05) in the women with normal menses compared to those with abnormal menses. This finding underscores the validity of serum 3 alpha-diol G measurements as indicators of androgen production and metabolism in women. The excellent correlation between the serum 3 alpha-diol G levels and standard measures of control indicates that the former measurement may be a useful adjunct in the management of women with congenital adrenal hyperplasia.

Application of high-performance liquid chromatography to the measurement of cortisol secretion rate.

After administration of [3H] cortisol, the specific radioactivity of the urinary cortisol metabolites is determined by chemical conversion of the 17-hydroxycorticosteroid conjugates to 17-ketosteroids, extraction with Sep-Pak C18 cartridges, derivatisation with 2,4-dinitrophenylhydrazine and isocratic reversed-phase high-performance liquid chromatography. This allows on-line measurement of ultra-violet absorbance; fractions are collected and 3H is measured by scintillation counting. Alternatively extraction and chromatography are done after enzymic hydrolysis of the steroid conjugates, and the major metabolites are determined colorimetrically in the fractions. The results from the two methods were consistent with each other and with published data.

Cushing's syndrome resulting from primary pigmented nodular adrenocortical disease.

In a 10-year-old boy with Cushing's syndrome, the dexamethasone suppression test, the metyrapone test, and both basal and corticotropin-releasing factor-stimulated corticotropin levels all indicated a primary adrenal disorder. However, a computed tomographic scan failed to detect an adrenal tumor. At surgery, the adrenal glands were not enlarged but were studded with small pigmented nodules composed of enlarged nonmalignant adrenocortical cells. This unusual abnormality, referred to as primary pigmented nodular adrenocortical disease, is associated with autonomous hypersecretion of cortisol primarily in children and young adults. Our patient was cured by total bilateral adrenalectomy and corticosteroid replacement therapy, the treatment of choice for this condition.

The effect of acute non-fatal haemorrhage on adrenocortical function.

The effect of acute non-fatal haemorrhage on adrenocortical function was assessed in 19 Burmese subjects. The results indicated that acute non-fatal haemorrhage as produced by blood donation stimulated the adrenal cortex to increase its secretion.

Comparison of 20-oxosteroids and cortisol in urine as parameters of adrenocortical function.

The coefficient of correlation between values for urinary 20-oxosteroids and urinary cortisol was 0.75 in 106 24-hours urines. Urinary cortisol was usually the more specific and sensitive parameter for the evaluation of adrenocortical function. With an analysis time of 4-5 hours for 100 samples, the method for cortisol was also faster and cheaper. Furthermore, the intra- and interassay variances of 2.7 and 8.2%, respectively, indicate high precision.

Panthenol and glucocorticoids.

Urinary metabolites of glucocorticoids have been measured in man before and after administration of panthenol in high doses. The quantitative assay was performed according to the technique of Porter and Silber which measures only cortisol and some of its metabolites. In all panthenol treated subjects a significant increase in the urinary excretion of 17-alpha-21-dihydroxy-20-keto steroids has been observed and it appeared to be higher in male ageing between 18 and 25. The results obtained confirm that a statistically significant increase in glucocorticoid production after panthenol administration in high doses is present also in man.

Endocrine studies in a patient with functioning adrenal rest tumor of the liver.

We report a case of a calcified liver tumor in a 23 year old female patient who presented with virilization and a mild degree of Cushing's syndrome. Androgen levels were elevated; there was loss of cortisol circadian rhythm and marked increase in urinary 17-ketogenic and 17-ketosteroids which failed to suppress with administration of dexamethasone. Venous sampling by inferior vena cava catheterization showed that the highest steroid hormone levels were in blood from the right hepatic vein. After death, in vitro studies revealed that the tumor contained testosterone and cortisol as determined by immunofluorescence techniques. The adrenals and ovaries were atrophic. Results of metyrapone testing indicated dyshormonogenesis. To our knowledge, this is the first case of an adrenal rest tumor of the liver proved to be functionally active.

Pseudotumor cerebri, empty sella syndrome, and adrenal adenoma.

Benign intracranial hypertension (BIH) and empty sella syndrome occurred in a patient with Cushing syndrome resulting from adrenal adenoma, removal of which was followed by return of cerebrospinal fluid pressure to normal. This case does not clarify the pathophysiology of BIH, but it does raise questions about the treatment of BIH with corticosteroids, which are of unproved efficacy and may sometimes cause the same clinical syndrome.

Isolation of urinary C-20 alpha- and C-20 beta-hydroxy-C21-steroid metabolites in cases of congenital adrenal hyperplasia, postpubertal virilizing syndrome and polycystic ovary syndrome.

The proportion of 20 alpha/20 beta-epimers of the urinary C21-steroid metabolites was estimated in normal volunteers and in patients with congenital adrenal hyperplasia (CAH), postpubertal virilizing syndrome (PVS) and polycystic ovary syndrome (POS). In the normal individuals 20 alpha- and 20 beta-epimers of 5 beta-pregnane-3 alpha, 17 alpha, 20-triol (pregnanetriol) and 5-pregnene-3 beta, 17 alpha, 20-triol (5-pregnenetriol) were measured. In the other case 20 alpha- and 20 beta-epimers of the characteristic, representative metabolites were also investigated. In 26 of 27 normal persons and in all the patients with normotensive CAH, PVS and POS the 20 beta-epimer comprised 0-10% of the total pregnanetriol. The 20 beta-epimer of 5-pregnenetriol was found in only one normal case (6% of the total). The percentage of 20 beta-epimers of 3 alpha, 17 alpha, 20-trihydroxy-5 beta-pregnan-11-one, 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20-tetrol (in the normotensive CAH, PVS and POS) and 5 alpha- or 5 beta-pregnane-3 alpha, 17 alpha, 20, 21-tetrol (in the hypertensive CAH) varied from nil to 76%. The effect of functional groups and the stereochemistry of the molecule on the direction of C-20-keto group reduction is discussed; the existence of additional factors determining this reduction in certain pathological conditions is suggested.