Diurnal variation of steroid hormones and their reference intervals using mass spectrometric analysis.

OBJECTIVE: Accurate measurement of steroid hormones remains challenging. Mass spectrometry affords a reliable means for quantitating steroid profiles accurately. Our objective was to establish and define (1) the extent of diurnal fluctuations in steroid concentrations that potentially necessitate strict adherence to time of sample acquisition and (2) time-dependent steroid reference intervals. DESIGN: Nine steroid markers were examined in couplets in males and females. METHODS: Using isotope dilution high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis, we developed a multi-steroid profile requiring only a minimal volume of serum (0.1 mL). Couplet (AM and PM) measurements of steroid hormones for 120 healthy females (F) and 62 healthy males (M) were obtained. Patients were recruited from several participating centers. RESULTS: The following diurnal values were noted to be significantly different in both females and males: cortisone, cortisol, corticosterone, 11 deoxycortisol (11 DOC), androstenedione, 17a-hydroxyprogesterone (17 OHP) and dehydroepiandrosterone (DHEA). Testosterone was only found to have significant diurnal variance in males. Progesterone showed no significant difference in AM and PM values for either groups and thus may provide an internal control. CONCLUSIONS: When diagnosing endocrine disorders, it is imperative to acknowledge the 24-h diurnal variation of the biochemical steroid markers. We highlight the importance of standardization of collection times and appropriate implementation of reference intervals. PRECIS: We identify diurnal fluctuations in steroid concentrations with time of day and emphasize the importance of adhering to firm time of sample acquisition.

Does TSH Reliably Detect Hypothyroid Patients?

OBJECTIVES: To evaluate the reliability of normal Thyroid Stimulating Hormone (TSH) as a thyroid function test and assess the effect of Adrenocorticotropic Hormone (ACTH) on serum TSH concentration. DESIGN AND METHODS: Patients presenting to the National Institutes of Health Department of Endocrinology outpatient clinic with symptoms consistent with hypothyroidism were identified. Thyroid hormone concentrations were measured by liquid chromatography/tandem mass spectrometry and immunoassay. Patients with normal TSH concentrations were assessed for both clinical and biochemical hypothyroidism.We evaluated the effect of ACTH stimulation (performed on patients for assessment of adrenal function) on TSH concentration. RESULTS: Patients with symptoms consistent with hypothyroidism but with normal TSH values in the range of 1-4 IU/mL and normal free T4 (FT4) values by immunoassay measurements were confirmed to be biochemically hypothyroid following measurements of thyroid hormones by mass spectrometry. We present case studies of two patients, a 76-year-old male and a 58-year-old female. Improvement in the male patient's hypothyroid symptoms, including afternoon fatigue, constipation, alopecia, dry skin and high cholesterol, was documented after initiating thyroid hormone replacement.ACTH stimulation resulted in an average decrease of 17% in TSH between time 0 and 60 minutes post stimulation. CONCLUSION: Although measurement of TSH is a convenient screen for thyroid function, it is influenced by many factors which may affect its overall reliability. We believe thyroid function should be assessed by more than a single test. We recommend measurement of thyroid hormone concentrations by mass spectrometry if the patient's clinical presentation is discordant with their TSH levels.

Novel biomarker SYT12 may contribute to predicting papillary thyroid cancer outcomes.

AIM: To investigate biomarkers for predicting papillary thyroid cancer outcomes. MATERIALS & METHODS: The expression of biomarkers (ITGA2, SYT12 and CDH3) was studied in a prospective cohort of patients with papillary thyroid cancer. Three outcomes of initial metastases, baseline status and longitudinal status were analyzed and correlated with the biomarkers. RESULTS: SYT12 provided the best prediction of initial metastasis (sensitivity: 72%; specificity: 54%). SYT12 had the highest accuracy for predicting longitudinal status (sensitivity: 100%; specificity: 47%). The best performance for longitudinal status resulted from combining SYT12 with American Thyroid Association risk stratification, with sensitivity and specificity of 88 and 73%, respectively. CONCLUSION: SYT12 has some prognostic significance in papillary thyroid cancer. Further validation studies in larger populations are warranted.

The impact of age and gender on papillary thyroid cancer survival.

CONTEXT: Thyroid cancer predominately affects women, carries a worse prognosis in older age, and may have higher mortality in men. Superimposed on these observations is the fact that most women have attained menopause by age 55 yr. OBJECTIVE: The objective of the study was to determine whether men contribute disproportionately to papillary thyroid cancer (PTC) mortality or whether menopause affects PTC prognosis. DESIGN: Gender-specific mortality was normalized using age-matched subjects from the U.S. population. Multivariate Cox proportional hazard regression models incorporating gender, age, and National Thyroid Cancer Treatment Cooperative Study Group stage were used to model disease-specific survival (DSS). PARTICIPANTS AND SETTING: Patients were followed in a prospective registry. MAIN OUTCOME MEASURE: The relationships between gender, age, and PTC outcomes were analyzed. RESULTS: The unadjusted hazard ratio (HR) for DSS for women was 0.40 [confidence interval (CI) 0.24-0.65]. This female advantage diminished when DSS was adjusted for age at diagnosis and stage with a HR encompassing unity (HR 0.72, CI 0.44-1.19). Additional multivariate models of DSS considering gender, disease stage, and various age groupings showed that the DSS for women diagnosed at under 55 yr was improved over men (HR 0.33, CI 0.13-0.81). However, the HR for DSS increased to become similar to men for women diagnosed at 55-69 yr (HR 1.01, CI 0.42-2.37) and at 70 yr or greater (HR 1.17, CI 0.48-2.85). CONCLUSIONS: Although the overall outcome of women with PTC is similar to men, subgroup analysis showed that this composite outcome is composed of two periods with different outcomes. The first period is a period with better outcomes for women than men when the diagnosis occurs at younger than 55 yr; the second is a period with similar outcomes for both women and men diagnosed at ages greater than 55 yr. These data raise the question of whether an older age cutoff would improve current staging systems. We hypothesize that older age modifies the effect of gender on outcomes due to menopause-associated hormonal alterations.

Trimester-specific changes in maternal thyroid hormone, thyrotropin, and thyroglobulin concentrations during gestation: trends and associations across trimesters in iodine sufficiency.

OBJECTIVES: To describe the interrelationships of thyroid functions based on trimester-specific concentrations in healthy, iodine-sufficient pregnant women across trimesters, and postpartum. METHODS: Circulating total 3,5,3'- triidothyronine (T(3)) and thyroxine (T(4)) concentrations were determined simultaneously using liquid chromatography tandem mass-spectrometry (LC/MS/MS). Free thyroxine (FT(4)), thyroid-stimulating hormone (TSH), and thyroglobulin (Tg) were measured using immunoassay techniques. Linear mixed effects models and correlations were calculated to determine trends and associations, respectively, in concentrations. RESULTS AND CONCLUSIONS: Trimester-specific T(3), FT(4), TSH, and Tg concentrations were significantly different between the first and third trimesters (all p < 0.05); second and third trimester values were not significantly different for FT(4), TSH, and Tg (all p > 0.25) although T3 was significantly higher in the third, relative to the second trimester. T(4) was not significantly different at any trimester (all p > 0.80). With two exceptions, analyte concentrations tended not to be correlated at each trimester and at 1-year postpartum. One exception was that T(3) and T(4) tended to be associated (all p < 0.05) at all time points except the third trimester (rho = 0.239, p > 0.05). T(4) and FT(4) concentrations tended to correlate positively during pregnancy (rho 0.361-0.382, all p < 0.05) but not postpartum (rho = 0.179, p > 0.05). Trends suggest that trimester-specific measurements of T(3), FT(4), Tg, and possibly TSH are warranted.

Prolonged, symptomatic hypocalcemia with pamidronate administration and subclinical hypoparathyroidism.

A 62-yr-old woman with thyroid carcinoma metastatic to bone, and a history of subclinical hypoparathyroidism was admitted to the hospital in hypocalcemic crisis 5 wk after receiving iv pamidronate. The patient had tetany and laryngospasm. An electrocardiogram showed junctional rhythm with QT segment prolongation. The patient had previously maintained a low-normal serum calcium on 500-750 mg of calcium carbonate and 600 IU of vitamin D daily. One week after pamidronate administration the patient's calcium and vitamin D supplementation were inadvertently discontinued. She continued to take daily intranasal calcitonin. At the time of her hospitalization for hypocalcemia, the patient's serum calcium was 4.3 mg/dL. The patient received aggressive calcium and vitamin D supplementation. However, her serum calcium remained below 6 mg/dL for a 2-wk period, and took another week to return to the normal range. In this article, we discuss the counterregulatory responses necessary to maintain calcium homeostasis following osteoclast inhibition by bisphosphonates. We also review the risk factors for hypocalcemia following bisphosphonate administration. Pamidronate and other bisphosphonates are becoming an integral part of the management of normocalcemic patients with malignant bone disease. Therefore, awareness of risk factors for hypocalcemia and familiarity with avenues available for protection from potentially catastrophic hypocalcemia are both crucial.

Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement.

We have analyzed the domain structure of the mouse glucocorticoid receptor by expression of in vitro mutated receptor in COS-7 cells. The receptor consists of a core domain rich in Cys, Lys, and Arg amino acids which can bind specific DNA sequences (glucocorticoid response elements) and activate transcription. The activity of this centrally located domain is modulated by the activity of the other two domains. The N-terminal domain of the receptor plays a role in decreasing nonspecific DNA binding and may therefore improve the ability of the protein to discriminate between specific and nonspecific DNA binding sites. This activity maps to a small, highly acidic region of the N-terminal domain. The C-terminal domain of the receptor contains the glucocorticoid binding site and in addition represses the transcriptional activity of the receptor in the absence of hormone. Hormone binding relieves the repression allowing transcription activation. The C-terminal domain contains a short sequence conserved among steroid receptors; its deletion yields a receptor that activates transcription in the absence of hormone.

Angiotensin-estrogen central interaction: localization and mechanism.

Intracerebroventricular (ICVT) administration of estradiol benzoate (EB) to ovariectomized female rats decreased drinking and pressor responses to central injections of angiotensin II (AII). Estrogen treatment does not have this effect in male rats. As EB given ICVT reaches many brain areas, the site of action of EB was localized using crystalline implants of EB in the medial preoptic area or the ventromedial nucleus of the hypothalamus. These areas were chosen as they have a high density of estrogen receptors. Only medial preoptic area application of estrogen decreased angiotensin II-induced drinking. Angiotensin receptor binding was examined in homogenates from different brain regions to determine if the mechanism through which estrogen decreases central responses to AII involves altered receptor function. Systemic EB did not affect AII receptor binding in several brain regions but binding was decreased in homogenates from the preoptic area and septum-thalamus blocks which encompassed structures (median preoptic nucleus, organum vasculosum, and subfornical organ) implicated in central actions of AII. The sex specificity of the effect of estrogen was dependent on sexual differentiation of the brain. Manipulation of the neonatal hormone environment, which alters this brain differentiation, also altered the characteristic responses of the two sexes to estrogen. Neonatal androgenization of females, which causes masculinization and defeminization, resulted in animals which as adults no longer responded to EB with decreased drinking. On the other hand, preventing the development of a male brain by neonatal castration produced animals which as adults tended to decrease their drinking following estrogen. In summary, this study found that EB acts in the preoptic area to depress AII-induced responses by a site specific modulation of central AII receptors. Alteration of early brain development changed the responses of the two sexes to estrogen, perhaps by altering sexual differentiation of the preoptic area.

Angiotensin-estrogen interaction in female brain reduces drinking and pressor responses.

Water intake fluctuates over the estrous cycle in rats, with daily intake lowest on the day of estrus. Since estrogen levels are highest preceding estrus and since the brain integrates thirst behavior, a possible central action of estrogen on fluid regulatory mechanisms was investigated in adult male and ovariectomized female rats. Ad libitum drinking was not changed the day of intracerebroventricular injection of estradiol benzoate (EB) but was significantly depressed the following day in females only. Drinking induced by intracerebroventricular angiotensin but not carbachol or cellular dehydration was also depressed in females the day after central EB treatment; again, no change occurred in males. To determine whether central EB depressed other centrally mediated responses to angiotensin, pressor responses to intracerebroventricular injections of angiotensin, carbachol, and hypertonic NaCl were also assessed. Only angiotensin-induced pressor responses in females were attenuated by central EB treatment, with a time course similar to that observed for the depression of drinking. We conclude that central regulatory responses to angiotensin are modulated by estrogen in female but not male brains.

Sodium appetite decreased by central angiotensin blockade.

Disturbances in body water and electrolytes that trigger sodium appetite, such as sodium depletion or hypovolemia, are potent activators of the renin-angiotensin system. In the absence of an actual deficit in body fluids, angiotensin injections are adequate to stimulate increased sodium ingestion. To assess whether angiotensin is a significant mediator of sodium appetite induced by acute alterations in body fluids, sodium intake was examined in rats during central or peripheral angiotensin blockade. Central blockade of angiotensin receptors by intracerebroventricular (ICVT) injection of the analogue antagonist saralasin decreased (but did not eliminate) sodium intake after polethylene glycol-induced hypovolemia or sodium depletion resulting from dialysis against glucose. Conversely, peripheral blockade of angiotensin converting enzyme with orally active captopril potentiated rather than decreased sodium appetite and stimulated water intake after sodium depletion. This increased water and salt intake after peripheral inhibition of converting enzyme was reversed, however, by concurrent central blockade of angiotensin receptors. These data support the hypothesis that angiotensin participates in sodium appetite associated with acute alteration in body fluids. Furthermore, the brain is the site at which angiotensin exerts its influence on sodium appetite. While the involvement of angiotensin of brain origin is not ruled out, the change in sodium appetite after peripheral blockade of converting enzyme suggests that circulating angiotensin derived from renal renin may interact with central angiotensin receptors regulating sodium appetite.