Steven J. Soldin

Timing of Levothyroxine Administration Affects Serum Thyrotropin Concentration

CONTEXTnPatients treated with levothyroxine typically ingest it in a fasting state to prevent food impairing its absorption. The serum thyrotropin concentration is the therapeutic index of levothyroxine action.nnnOBJECTIVEnThe study objective was to determine the effect of the timing of levothyroxine administration in relationship to food on serum thyrotropin levels.nnnDESIGNnParticipants were randomized to one of six sequences, each consisting of three 8-wk regimens in a three-period crossover design. These regimens were in a fasting state, at bedtime, and with breakfast. The concentrations of TSH, free T(4), and total T(3) during each of the three timing regimens were documented. The primary outcome was the difference between serum TSH concentrations under fasting conditions compared with concentrations during the other 8-wk regimens.nnnSETTINGnThe study was conducted in an academic medical center.nnnPARTICIPANTSnStudy participants were receiving levothyroxine for treatment of hypothyroidism or thyroid cancer.nnnRESULTSnSixty-five patients completed the study. The mean thyrotropin concentration was 1.06 +/- 1.23 mIU/liter when levothyroxine was administered in the fasting state. When levothyroxine was taken with breakfast, the serum thyrotropin concentration was significantly higher (2.93 +/- 3.29 mIU/liter). When levothyroxine was taken at bedtime, the serum TSH concentration was also significantly higher (2.19 +/- 2.66 mIU/liter).nnnCONCLUSIONnNonfasting regimens of levothyroxine administration are associated with higher and more variable serum TSH concentrations. If a specific serum TSH goal is desired, thereby avoiding iatrogenic subclinical thyroid disease, then fasting ingestion of levothyroxine ensures that TSH concentrations remain within the narrowest target range.

Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: altered pathways in neuronal development and steroid biosynthesis.

Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ∼4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.

Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization

OBJECTIVESnThe steroids estradiol (E2), estrone (E1), and estriol (E3) are the major estrogens. E1/E2 and their metabolite 16-hydroxyestrone (16-OHE1, known to be carcinogenic) could be involved in the development of many cancers including human breast cancer. The aim of the current study was to develop a rapid and simple high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to simultaneously measure E1, E2, E3 and 16-OHE1 in human serum without the need for solid phase extraction or derivatization.nnnMETHODSnAn API-5000 triple-quadrupole mass spectrometer coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with deuterium-labeled internal standard (IS) for each analyte. Quantitation by multiple reaction monitoring (MRM) analysis was performed in negative ion mode.nnnRESULTSnThe limits of detection were 1.0 pg/mL for E1 and 16-OHE1 and 2.0 pg/mL for E2 and E3. Within-day CVs were <6.5% for all analytes tested and between-day CVs ranged from 4.5% to 9.5%. Recovery ranged from 88% to 108%.nnnCONCLUSIONnThis method allows for the simultaneous measurement of four estrogens in human serum within 8 min. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing, micro sample requirement, and high throughput.

Osmotic and Nonosmotic Regulation of Arginine Vasopressin during Prolonged Endurance Exercise

CONTEXTnAlthough the primary cause of exercise-associated hyponatremia (EAH) is relative overconsumption of fluids beyond the kidneys ability to excrete excess fluid, the mechanisms limiting maximum renal excretory ability during exercise remain to be elucidated.nnnOBJECTIVEnThe objective of the study was to: 1) perform a comprehensive evaluation of the endocrine secretion of pituitary, natriuretic and adrenal steroid hormones, and cytokines immediately before and after running an ultramarathon; and 2) evaluate the relationship between osmotic and nonosmotic stimuli to arginine vasopressin (AVP) secretion within the overall context of assessing the hormonal regulation of fluid balance during prolonged endurance exercise.nnnDESIGNnThis was an observational study.nnnSETTINGnThe study setting was a 56-km ultramarathon.nnnPARTICIPANTSnEighty-two runners participated in the study.nnnINTERVENTIONSnThere were no interventions.nnnMAIN OUTCOME MEASURESnPlasma sodium concentration [Na(+)] and plasma volume [(AVP)(p)] were measured.nnnRESULTSnFluid homeostasis during exercise (356 +/- 4 min) was maintained with ad libitum fluid intakes. [Na(+)] was maintained from before the race (139.3 +/- 0.3 mmol/liter) to after the race (138.1 +/- 0.4 mmol/liter) with a significant decrease in plasma volume (-8.5 +/- 0.1%, P < 0.01). Increases in the plasma (AVP)(p) (3.9-fold), oxytocin (1.9-fold), brain natriuretic peptide (4.5-fold), and IL-6 (12.5-fold) were highly significant (P < 0.0001). Changes in brain natriuretic peptide, oxytocin, and corticosterone were associated with 47% of the variance noted in (AVP)(p) and 13% of the variance in plasma [Na(+)] in pathway analyses.nnnCONCLUSIONSn(AVP)(p) was markedly elevated after the ultramarathon despite unchanged plasma [Na(+)](.) Therefore, an inability to maximally suppress (AVP)(P) during exercise as a result of nonosmotic stimulation of AVP secretion may contribute to the pathogenesis of exercise-associated hyponatremia if voluntary fluid intake were to exceed fluid output.

Steroid hormone levels associated with passive and active smoking

CONTEXTnCigarette tobacco smoke is a potent environmental contaminant known to adversely affect health including fertility and pregnancy.nnnOBJECTIVEnTo examine the associations between second-hand cigarette tobacco-smoke exposure, or active smoking and serum concentrations of steroid hormones using tandem mass spectrometry.nnnDESIGNnHealthy women (18-45 y) from the general community in the Metropolitan Washington, DC were recruited at the follicular stage of their menstrual cycle. Participants were assigned to one of three study groups: active smokers (N=107), passive smokers (N=86), or non-smokers (N=100). Classifications were based on a combination of self-reporting and serum cotinine concentrations.nnnMETHODSnSerum androgens, estrogens, progestins, androstenedione, aldosterone, cortisol, corticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 11-deoxycortisol and 25-hydroxy-vitamin D3 (25-OHVitD3) and cotinine were measured by isotope dilution tandem mass spectrometry (LC/MS/MS) (API-5000). Kruskal-Wallis tests were used to assess median differences among the three groups, with Dunns multiple comparison test for post hoc analysis.nnnRESULTSnSerum estrone, estradiol, and estriol concentrations were lower in active and passive smokers than in non-smokers. The three study groups differed significantly in serum concentrations of 16-OHE1, aldosterone and 25-OHVitD3, as well as in the ratios of many of the steroids. Pair-wise comparison of the groups demonstrated significant differences in hormone concentrations between (i) smokers and non-smokers for aldosterone: (ii) passive smokers and non-smokers for aldosterone, progesterone and estriol. Moreover, for smokers and passive smokers, there were no significant differences in these hormone concentrations.nnnCONCLUSIONSnSmoke exposure was associated with lower than normal median steroid hormone concentrations. These processes may be instrumental in explaining some adverse effects of tobacco smoke on female health and fertility.

Thyroid hormone testing by tandem mass spectrometry

In the euthyroid person, absolute free thyroxine concentrations remain constant and correlate with the tissue hormone level, its biologic effect and the metabolic status of the patient. However, most circulating thyroid hormone is bound to plasma proteins and only a minute amount is in the unbound free form. Studies have shown that current free thyroxine immunoassays are binding protein dependent. Novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) methods have successfully dealt with problems inherent in many immunoassays for thyroid hormones and afforded improved specificity and accuracy in thyroid hormone measurements. We emphasize problems with thyroid hormone testing employing immunoassays including direct and indirect thyroid hormone immunoassays, sample processing, methods of free hormone separation and review the emerging role of liquid chromatography-tandem mass spectrometry in thyroid hormone testing. The latest generation of tandem mass spectrometers has superior limits of quantification, permitting omission of previously employed derivatization steps. Liquid chromatography-tandem mass spectrometry affords the specificity, precision, and limits of quantification necessary for the reliable measurement of thyroid hormones, enhancing diagnostic capabilities, and affording the profiles of the iodothyronines and thyronamines. These methods are especially important in states of disease and during pregnancy when protein binding is a factor that interferes with other methods for thyroid hormone analysis.

Age and CYP3A5 genotype affect tacrolimus dosing requirements after transplant in pediatric heart recipients.

BACKGROUNDnTacrolimus is one of the commonly used immunosuppressive drugs for pediatric heart transplants. Large variation exists in pharmacokinetics during the direct post-transplant period, resulting in an increased risk of adverse events. Limited data are available on the interaction of age, CYP3A5 and ABCB1 genotype, and disease severity on the variation in disposition and outcome in pediatric heart transplant recipients.nnnMETHODnWe studied the relationship between age and CYP3A5 and ABCB1 genotype and the Pediatric Risk of Mortality (PRISM) score on tacrolimus dose (mg/kg), steady-state trough concentrations, and concentration/dose ratio, as well as rejection and renal function for 14 days after heart transplant in children.nnnRESULTSnTacrolimus was administered to 39 children (median age, 6.0 years) after transplant. A correlation was found between the age at the time of transplant and the tacrolimus dosing requirements (r(s) = -0.447, p = 0.004) and the concentration/dose ratio (r(s) = 0.351, p = 0.029). CYP3A5 expressors required median (interquartile range) higher doses of tacrolimus (0.14 [0.09] vs 0.06 [0.04] mg/kg/12 hours, p = 0.001), and had lower concentration/dose ratios (45.34 [44.54] vs 177.78 [145.38] ng/ml per mg/kg/12 hours, p < 0.0001). This relationship was not seen with the ABCB1 genotype. Age and CYP3A5 genotype predicted the tacrolimus dosing requirements as well as the concentration/dose ratio (R(2) = 0.351, p = 0.001 and R(2) = 0.521, p < 0.001). No relationship was found between any of the CYP3A5 or ABCB1 genotypes and the estimated glomerular filtration rate.nnnCONCLUSIONnYounger age and CYP3A5 expressor genotype were independently associated with higher dosing requirements and lower tacrolimus concentration/dose ratios.

Acute changes in endocrine and fluid balance markers during high-intensity, steady-state, and prolonged endurance running: unexpected increases in oxytocin and brain natriuretic peptide during exercise.

Maintenance of fluid homeostasis during periods of heightened physical stress can be best evaluated in humans using exercise as a model. Although it is well established that arginine vasopressin (AVP), aldosterone and atrial natriuretic peptide (ANP) are the principle hormones regulating fluid balance at rest, the potential contributions of other related endocrine factors, such as oxytocin (OT) and brain natriuretic peptide (BNP), have not been well described during exercise. Seven endurance-trained runners completed three separate running trials: a maximal test to exhaustion (high intensity), a 60-min treadmill run (steady state), and a 56 km ultramarathon (prolonged endurance exercise). Statistically significant pre- to post-run increases were found only following the ultramarathon in [AVP](p) (1.9 vs 6.7 pg/ml; P<0.05), [OT](p) (1.5 vs 3.5 pg/ml; P<0.05), [NT-proBNP](p) (23.6 vs 117.9 pg/ml; P<0.01), [interleukin 6](p) (4.0 vs 59.6 pg/ml; P<0.05), [cortisol](p) (14.6 vs 32.6 microg/ml; P<0.01), [corticosterone](p) (652.8 vs 3491.4 ng/ml; P<0.05) and [11-deoxycortisol](p) (0.1 vs 0.5 microg/ml; P<0.05) while a significant post-run increase in [aldosterone](p) was documented after high-intensity (4.9 vs 12.5 ng/ml; P<0.05), steady-state (6.1 vs 16.9 ng/ml; P<0.05) and prolonged endurance running (2.6 vs 19.7 ng/ml; P<0.05). Similarly, changes in fluid balance parameters were significantly different between the ultramarathon versus high-intensity and steady-state running with regard to plasma volume contraction (less % contraction), body weight loss (increased % weight loss), plasma [Na(+)] Delta (decreased from baseline), and urine osmolality Delta (increase from baseline). Hypothetically driven relationships between [OT](p) and [AVP](p) (r=0.69; P<0.01) and between [NT-proBNP](p) Delta and plasma [Na(+)] Delta (r=-0.79; P<0.001)--combined with the significant and unexpected pre- to post-race increases after prolonged endurance exercise--allows for possible speculation that OT and BNP may assist their better known companion hormones (AVP and ANP) in the regulation of fluid balance during conditions of extreme physical stress.

Use of Steroid Profiles in Determining the Cause of Adrenal Insufficiency

HYPOTHESISnA cortisol response to adrenocorticotropin injection is the standard test for diagnosing adrenal insufficiency. Multiple steroid hormones can now be accurately measured by tandem mass spectrometry in a single sample. The study objective was to determine whether a steroid profile, created by simultaneous measurement of 10 steroid hormones by tandem mass spectrometry, would help determine the cause of adrenal insufficiency.nnnDESIGNnA 10-steroid profile was measured by tandem mass spectrometry during the performance of a standard high dose cortrosyn stimulation test. The steroids were measured at baseline, 30, and 60min following synthetic adrenocorticotropin injection. Adrenal insufficiency was defined as a peak cortisol level of less than 20microg/dL. Testing was conducted in the general clinical research center of a university medical center. Normal volunteers, patients suspected of having adrenal insufficiency, and patients with known adrenal insufficiency participated.nnnRESULTSnOur results showed that adrenal insufficiency of any cause was adequately diagnosed using the response of 11-deoxycortisol, dehydroepiandrosterone, or these analytes combined in a two-steroid profile. A three-steroid profile yielded a test with 100% accuracy for discriminating primary adrenal insufficiency from normal status. Primary adrenal insufficiency was well separated from secondary adrenal insufficiency using only a single aldosterone value. 11-Deoxycortisol, dehydroepiandrosterone, and a two-steroid profile each provided fair discrimination between secondary adrenal insufficiency and normal status.nnnCONCLUSIONSnWe conclude that stimulated levels of aldosterone, 11-deoxycortisol, dehydroepiandrosterone, and a two- or three-steroid profile provided additional discrimination between states of adrenal sufficiency and insufficiency. It is proposed that a steroid profile measuring cortisol, aldosterone, 11-deoxycortisol, and dehydroepiandrosterone would potentially improve the ability to determine the cause of adrenal insufficiency.

Pediatric reference intervals for aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, testosterone and 25-hydroxy vitamin D3 using tandem mass spectrometry.

OBJECTIVESnTo determine age and sex-specific pediatric reference intervals for aldosterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, testosterone, and 25-hydroxy vitamin D(3).nnnBACKGROUND AND DESIGNnReference intervals were determined for neonates and children 0-18 years of age. The study was conducted at both Childrens National Medical Center and Georgetown University using outpatient blood samples obtained between January 1, 2004 and June 30, 2008.nnnMETHODSnSerum samples were analyzed using isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with deuterium-labeled internal standards at Childrens National Medical Center and Georgetown University Medical Center Bioanalytical Core Laboratory.nnnRESULTS AND CONCLUSIONSnThis is the first study to provide pediatric reference intervals of steroid hormones for children from birth to 18 years of age using LC/MS/MS. Reference intervals were established for aldosterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, testosterone, and 25-hydroxy vitamin D(3). All the analytes exhibited at least some age dependence. Sex differences between early and late childhood and adolescence were found for 17alpha-hydroxyprogesterone and testosterone. Seasonal differences were apparent for 25-hydroxy vitamin D(3).