Adipose tissue estrogen production and metabolism in premenopausal women.

OBJECTIVE: Although the ovaries produce the majority of estrogens in women before menopause, estrogen is also synthesized in peripheral tissues such as adipose tissue (AT). The typical female AT distribution, concentrated in subcutaneous and femoro-gluteal regions, is estrogen-mediated, but the significance of estrogen synthesis in AT of premenopausal women is poorly understood. DESIGN AND METHODS: Serum and subcutaneous and visceral AT homogenates from 28 premenopausal women undergoing non-malignant surgery were analyzed for estrone, estradiol, and serum estrone sulfate (E1S) concentrations with liquid chromatography-tandem mass spectrometry. Isotopic precursors were used to measure enzyme activities of estrone-producing steroid sulfatase and estradiol-producing 17ß-hydroxysteroid dehydrogenases (17ß-HSD). Messenger RNA (mRNA) expression levels of genes for estrogen-metabolizing enzymes were analyzed using real-time reverse transcription quantitative polymerase chain reaction. RESULTS: While estradiol was the predominant circulating active estrogen, estrone dominated in AT, with a higher concentration in visceral than subcutaneous AT (median, 2657 vs 1459 pmol/kg; P = 0.002). Both AT depots converted circulating E1S to estrone, and estrone to estradiol. Median levels of estrone were five to ten times higher in subcutaneous and visceral AT than in serum (P < 0.001) and the estradiol level in visceral AT was 1.3 times higher than in serum (P < 0.005). The local estrone concentration in visceral AT correlated positively with mRNA expression of estrone-producing enzyme aromatase (r = 0.65, P = 0.003). Waist circumference correlated positively with increased estradiol production in subcutaneous AT (r = 0.60, P = 0.039). CONCLUSIONS: Premenopausal AT demonstrated high estrogenic enzyme activity and considerable local estrogen concentrations. This may be a factor promoting female-typical AT distribution in premenopausal women.

Functional Profiling of FSH and Estradiol in Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERß) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERß protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.

Liquorice ingestion attenuates vasodilatation via exogenous nitric oxide donor but not via ß2-adrenoceptor stimulation.

We examined the effect of liquorice ingestion on haemodynamic responses to exogenous nitric oxide donor (nitroglycerin) and ß2-adrenoceptor agonist (salbutamol), and 11ß-hydroxysteroid dehydrogenase activity, in 21 volunteers and 21 reference subjects. Haemodynamic data was captured before and after sublingual nitroglycerin (0.25 mg) and inhaled salbutamol (400 µg) during orthostatic challenge utilising radial pulse wave analysis and whole-body impedance cardiography. The recordings were performed at baseline and following two weeks of liquorice intake (290-370 mg/d glycyrrhizin). Urinary cortisone and cortisol metabolites were examined. Liquorice intake elevated aortic systolic and diastolic blood pressure and systemic vascular resistance when compared with the reference group. Following research drug administration the liquorice-induced increase in systemic vascular resistance was observed in the presence of nitroglycerin (p<0.05) but no longer in the presence of salbutamol. Liquorice ingestion decreased cardiac chronotropic response to upright posture (p = 0.032) in unadjusted analysis, but when adjusted for age and sex the difference in the upright change in heart rate was no longer significant. The urinary cortisone to cortisol metabolite ratio decreased from 0.70 to 0.31 (p<0.001) after liquorice intake indicating significant inhibition of the 11ß-hydroxysteroid dehydrogenase type 2. In the reference group the haemodynamic variables remained virtually unchanged. These results suggest that liquorice exposure impaired vasodilatation in vivo that was induced by exogenous nitric oxide donor but not that induced by ß2-adrenoceptor stimulation. Trial registration: EU Clinical Trials Register 2006-002065-39 ClinicalTrials.gov NCT01742702.

Postnatal gene expression of airway epithelial sodium transporters associated with birth stress in humans.

INTRODUCTION: Lung fluid clearance is essential for successful postnatal pulmonary adaptation. The epithelial sodium channel (ENaC) and Na-K-ATPase, induced by serum- and glucocorticoid-inducible kinase 1 (SGK1) as well as aquaporins (AQP), represent key players in the switch from fetal lung fluid secretion to absorption and in early postnatal lung fluid balance. Birth stress, including a surge in catecholamines, promotes pulmonary adaptation, likely through the augmentation of epithelial sodium reabsorption. OBJECTIVES: We sought to determine the changes in the airway gene expression of molecules vital to epithelial sodium transport during early pulmonary adaptation, and the association with birth stress reflected in the norepinephrine concentration in the cord blood in humans. METHODS: We included 70 term newborns: 28 born via vaginal delivery and 42 via elective cesarean section. We determined the norepinephrine concentrations in the cord blood using tandem mass spectrometry and collected nasal epithelial cell samples at 2 min, 1 h, and 24 h postnatally to quantify ENaC, Na-K-ATPase, AQP5, and SGK1 mRNAs using RT-PCR. RESULTS: The molecular gene expression involved in airway epithelium sodium transport changed markedly within the first hour postnatally. Newborns born via elective cesarean section exhibited a lower expression of ENaC, Na-K-ATPase, and SGK1. Significant correlations existed between the expressions of ENaC, Na-K-ATPase, and SGK1, and the concentration of norepinephrine in the cord blood. CONCLUSIONS: The association of ENaC, Na-K-ATPase, and SGK1 expression with the cord blood norepinephrine concentration points to the importance of birth stress in promoting lung fluid clearance during early postnatal pulmonary adaptation.

Increased body fat mass and androgen metabolism - A twin study in healthy young women.

OBJECTIVE: Obesity may alter serum steroid concentrations and metabolism. We investigated this in healthy young women with increased body fat and their leaner co-twin sisters. DESIGN: Age and genetic background both strongly influence serum steroid levels and body composition. This is a cross-sectional study of 13 female monozygotic twin pairs (age, 23-36 years), ten of which were discordant for body mass index (median difference in body weight between the co-twins, 19 kg). METHODS: We determined body composition by dual energy X-ray absorptiometry and magnetic resonance imaging, serum androgens by liquid chromatography-tandem mass spectrometry, and mRNA expression of genes in subcutaneous adipose tissue and adipocytes. RESULTS: The heavier women had lower serum dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and sex hormone-binding globulin (SHBG) (P < 0.05 for all) compared to their leaner co-twins with no differences in serum testosterone or androstenedione levels. Serum DHEA correlated inversely with %body fat (r = -0.905, P = 0.002), and DHT positively with SHBG (r = 0.842, P = 0.002). In adipose tissue or adipocytes, expressions of STS (steroid sulfatase) and androgen-related genes were significantly higher in the heavier compared to the leaner co-twin, and within pairs, correlated positively with adiposity but were not related to serum androgen levels. None of the serum androgen or SHBG levels correlated with indices of insulin resistance. CONCLUSIONS: Serum DHEA levels were best predicted by %body fat, and serum DHT by SHBG. These or other serum androgen concentrations did not reflect differences in androgen-related genes in adipose tissue. General or intra-abdominal adiposity were not associated with increased androgenicity in young women.

Testicular Function and Bone in Young Men with Severe Childhood-Onset Obesity.

BACKGROUND: Previous studies suggest increased risk for hypoandrogenism and fractures in men with obesity. We aimed to describe the effects of severe childhood-onset obesity on the cross talk between metabolic state, testes, and skeleton at late puberty. METHODS: A cohort of adolescent and young adult males with severe childhood-onset obesity (n = 21, mean age 18.5 years) and an age-matched control group were assessed for testicular hormones and X-ray absorptiometry-derived bone mass. RESULTS: Current median body mass indexes for the obese and control subjects were 37.4 and 22.9. Severe early-onset obesity manifested with lower free testosterone (median [interquartile range] 244 [194-332] vs. 403 [293-463] pmol/L, p = 0.002). Lower insulin-like 3 (1.02 [0.82-1.23] vs. 1.22 [1.01-1.46] ng/mL, p = 0.045) and lower ratio of testosterone to luteinizing hormone (2.81 [1.96-3.98] vs. 4.10 [3.03-5.83] nmol/IU, p = 0.008) suggested disrupted Leydig cell function. The degree of current obesity inversely correlated with free testosterone (τ = -0.516, p = 0.003), which in turn correlated positively with bone area at all measurement sites in males with childhood-onset obesity. CONCLUSIONS: Severe childhood-onset obesity is associated with impaired Leydig cell function in young men and lower free testosterone may contribute to impaired skeletal characteristics.

The relationship between diurnal cortisol secretion and climacteric-related symptoms.

OBJECTIVES: Chronic stress, also associated with climacteric-related symptoms, may influence cortisol secretion. We studied cortisol metabolism in peri- and postmenopausal women with diverse climacteric-related symptoms. STUDY DESIGN AND MAIN OUTCOME MEASURES: The study population was 35 women, aged 45-70 years. Plasma cortisol levels were measured from blood samples collected every 20 min over 24 h. Urinary cortisol was analysed from 24-hour urine collections. Climacteric-related symptoms (vasomotor, sleep, depressive, anxiety, cognitive, sexual, menstrual, and somatic) were evaluated with the Women's Health Questionnaire (WHQ). Associations between cortisol variables (24-hour, night, day, maximum, minimum, morning baseline, cortisol awakening response (CAR), area under the curve, slope, and 24-hour urinary cortisol) and the symptoms were first examined with a correlation analysis. Then, the women were divided into two groups according to their climacteric symptomatology, and differences in cortisol variables between the groups were investigated. Diurnal cortisol curves by symptomatology were also analyzed visually. RESULTS: In the correlation analysis, more frequent vasomotor symptoms were associated with a higher CAR (rs = 0.37, p = 0.039) and lower 24-hour urinary cortisol excretion (rs= -0.45, p = 0.012), and more frequent depressive symptoms were associated with a higher minimum cortisol level (rs = 0.33, p = 0.0498). When the women were divided into two groups, women with more frequent vasomotor (p = 0.012) or somatic symptoms (p = 0.021) had a lower 24-hour urinary cortisol excretion than less symptomatic women. CONCLUSIONS: Although previous studies have reported associations between climacteric-related symptoms and cortisol secretion, these two factors were not substantially interrelated in our study.

Estrogen biosynthesis in breast adipose tissue during menstrual cycle in women with and without breast cancer.

Circulating estrogens fluctuate during the menstrual cycle but it is not known whether this fluctuation is related to local hormone levels in adipose tissue. We analyzed estrogen concentrations and gene expression of estrogen-regulating enzymes in breast subcutaneous adipose tissue in premenopausal women with (n = 11) and without (n = 17) estrogen receptor-positive breast cancer. Estrone (E1) was the predominant estrogen in premenopausal breast adipose tissue, and E1 and mRNA expression of CYP19A1 in adipose tissue correlated positively with BMI. Adipose tissue estradiol (E2) concentrations fluctuated during the menstrual cycle, similarly to the serum concentrations. In women with breast cancer median adipose tissue E1 (1519 vs. 3244, p < .05) and E2 (404 vs. 889 pmol/kg, p < .05) levels were lower in the follicular than in the luteal phase whereas in control women no significant differences were observed. In the follicular phase, mRNA expressions of HSD17B1 (median 0.06; interquartile range 0.05-0.07 vs. 0.17; 0.03-0.2, p = .010) and CYP19A1 (0.08; 0.07-0.14 vs. 0.22; 0.09-0.54, p = .025) were lower in women with breast cancer than in controls. In conclusion, the changes in adipose tissue E1 and E2 concentrations and the estrogen-regulating CYP19A1 and HSD17B1 during the menstrual cycle may be related to dysfunctional local estrogen metabolism in women with breast cancer.

Estrogen Metabolism in Abdominal Subcutaneous and Visceral Adipose Tissue in Postmenopausal Women.

Context: In postmenopausal women, adipose tissue (AT) levels of estrogens exceed circulating concentrations. Although increased visceral AT after menopause is related to metabolic diseases, little is known about differences in estrogen metabolism between different AT depots. Objective: We compared concentrations of and metabolic pathways producing estrone and estradiol in abdominal subcutaneous and visceral AT in postmenopausal women. Design, Setting, Patients, and Interventions: AT and serum samples were obtained from 37 postmenopausal women undergoing surgery for nonmalignant gynecological reasons. Serum and AT estrone, estradiol, and serum estrone sulfate (E1S) concentrations were quantitated using liquid chromatography-tandem mass spectrometry. Activity of steroid sulfatase and reductive 17ß-hydroxysteroid dehydrogenase enzymes was measured using radiolabeled precursors. Messenger RNA (mRNA) expression of estrogen-converting enzymes was analyzed by real-time reverse transcription quantitative polymerase chain reaction. Results: Estrone concentration was higher in visceral than subcutaneous AT (median, 928 vs 706 pmol/kg; P = 0.002) and correlated positively with body mass index (r = 0.46; P = 0.011). Both AT depots hydrolyzed E1S to estrone, and visceral AT estrone and estradiol concentrations correlated positively with serum E1S. Compared with visceral AT, subcutaneous AT produced more estradiol from estrone (median rate of estradiol production, 1.02 vs 0.57 nmol/kg AT/h; P = 0.004). In visceral AT, the conversion of estrone to estradiol increased with waist circumference (r = 0.65; P = 0.022), and estradiol concentration correlated positively with mRNA expression of HSD17B7 (r = 0.76; P = 0.005). Conclusions: Both estrone and estradiol production in visceral AT increased with adiposity, but estradiol was produced more effectively in subcutaneous fat. Both AT depots produced estrone from E1S. Increasing visceral adiposity could increase overall estrogen exposure in postmenopausal women.

Metabolism of sex steroids is influenced by acquired adiposity-A study of young adult male monozygotic twin pairs.

Obesity and ageing are associated with lower serum testosterone levels in men. How fat distribution or adipose tissue metabolism, independent of genetic factors and age, are related to sex steroid metabolism is less clear. We studied the associations between adiposity and serum sex hormone concentrations, and mRNA expression of genes regulating sex hormone metabolism in adipose tissue in young adult male monozygotic (MZ) twin pairs. The subjects [n=18 pairs; mean age, 32 years; individual body mass indexes (BMIs) 22-36kg/m2] included 9 male MZ twin pairs discordant for BMI [intra-pair difference (Δ) in BMI ≥3kg/m2]. Sex steroid concentrations were determined by liquid chromatography-tandem mass spectrometry, body composition by dual-energy X-ray absorptiometry and magnetic resonance imaging, and mRNA expressions from subcutaneous adipose tissue by Affymetrix. In BMI-discordant pairs (mean ΔBMI=5.9kg/m2), serum dihydrotestosterone (DHT) was lower [mean 1.9 (SD 0.7) vs. 2.4 (1.0) nmol/l, P=0.040] and mRNA expressions of DHT-inactivating AKR1C2 (P=0.021) and cortisol-producing HSD11B1 (P=0.008) higher in the heavier compared to the leaner co-twins. Serum free 17ß-estradiol (E2) was higher [2.3 (0.5) vs. 1.9 (0.5) pmol/l, P=0.028], and in all twin pairs, serum E2 and estrone concentrations were higher in the heavier than in the leaner co-twins [107 (28) vs. 90 (22) pmol/l, P=0.006; and 123 (43) vs. 105 (27) pmol/l, P=0.025]. Within all twin pairs, i.e. independent of genetic effects and age, 1) the amount of subcutaneous fat inversely correlated with serum total and free testosterone, DHT, and sex hormone-binding globulin (SHBG) concentrations (P<0.01 for all), 2) intra-abdominal fat with total testosterone and SHBG (P<0.05), and 3) liver fat with SHBG (P=0.006). Also, 4) general and intra-abdominal adiposity correlated positively with mRNA expressions of AKR1C2, HSD11B1, and aromatase in adipose tissue (P<0.05). In conclusion, acquired adiposity was associated with decreased serum DHT and increased estrogen concentrations, independent of genetic factors and age. The reduction of DHT could be linked to its increased degradation (by AKR1C2 and HSD11B1) and increased estrogen levels to increased adiposity-related expression of aromatase in adipose tissue.

Food and Nutrient Intake and Nutritional Status of Finnish Vegans and Non-Vegetarians.

BACKGROUND: Vegetarian and vegan diets have become more popular among adolescents and young adults. However, few studies have investigated the nutritional status of vegans, who may be at risk of nutritional deficiencies. OBJECTIVE: To compare dietary intake and nutritional status of Finnish long-term vegans and non-vegetarians. METHODS: Dietary intake and supplement use were estimated using three-day dietary records. Nutritional status was assessed by measuring biomarkers in plasma, serum, and urine samples. Vegans' (n = 22) data was compared with those of sex- and age-matched non-vegetarians (n = 19). RESULTS: All vegans adhered strictly to their diet; however, individual variability was marked in food consumption and supplementation habits. Dietary intakes of key nutrients, vitamins B12 and D, were lower (P < 0.001) in vegans than in non-vegetarians. Nutritional biomarker measurements showed lower concentrations of serum 25-hydroxyvitamin D3 (25(OH)D3), iodine and selenium (corrected for multiple comparisons, P < 0.001), Vegans showed more favorable fatty acid profiles (P < 0.001) as well as much higher concentrations of polyphenols such as genistein and daidzein (P < 0.001). Eicosapentaenoic acid proportions in vegans were higher than expected. The median concentration of iodine in urine was below the recommended levels in both groups. CONCLUSIONS: Long-term consumption of a vegan diet was associated with some favorable laboratory measures but also with lowered concentrations of key nutrients compared to reference values. This study highlights the need for nutritional guidance to vegans.

Steroid sulfatase activity in subcutaneous and visceral adipose tissue: a comparison between pre- and postmenopausal women.

OBJECTIVE: Adipose tissue is an important extragonadal site for steroid hormone biosynthesis. After menopause, estrogens are synthesized exclusively in peripheral tissues from circulating steroid precursors, of which the most abundant is dehydroepiandrosterone sulfate (DHEAS). Our aim was to study activity of steroid sulfatase, an enzyme hydrolyzing DHEAS, and expression of steroid-converting enzyme genes in subcutaneous and visceral adipose tissue derived from pre- and postmenopausal women. DESIGN: Serum and paired abdominal subcutaneous and visceral adipose tissue samples were obtained from 18 premenopausal and seven postmenopausal women undergoing elective surgery for non-malignant reasons in Helsinki University Central Hospital. METHODS: To assess steroid sulfatase activity, radiolabeled DHEAS was incubated in the presence of adipose tissue homogenate and the liberated dehydroepiandrosterone (DHEA) was measured. Gene mRNA expressions were analyzed by quantitative RT-PCR. Serum DHEAS, DHEA, and estrogen concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Steroid sulfatase activity was higher in postmenopausal compared to premenopausal women in subcutaneous (median 379 vs 257 pmol/kg tissue per hour; P=0.006) and visceral (545 vs 360 pmol/kg per hour; P=0.004) adipose tissue. Visceral fat showed higher sulfatase activity than subcutaneous fat in premenopausal (P=0.035) and all (P=0.010) women. The mRNA expression levels of two estradiol-producing enzymes, aromatase and 17ß-hydroxysteroid dehydrogenase type 12, were higher in postmenopausal than in premenopausal subcutaneous adipose tissue. CONCLUSIONS: Steroid sulfatase activity in adipose tissue was higher in postmenopausal than in premenopausal women suggesting that DHEAS, derived from the circulation, could be more efficiently utilized in postmenopausal adipose tissue for the formation of biologically active sex hormones.

Quantitative determination of estrone by liquid chromatography-tandem mass spectrometry in subcutaneous adipose tissue from the breast in postmenopausal women.

Estrone is the most abundant estrogen after the menopause. We developed a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for determination of estrone in adipose tissue. Subcutaneous adipose tissue from the breast was collected during elective surgery in postmenopausal women undergoing mastectomy for treatment of breast cancer (n=13) or reduction mammoplasty (controls, n=11). Homogenized adipose tissue was extracted with organic solvents and the estrone fraction was purified by LH-20 column chromatography from the excess of lipids. The concentration of estrone was analyzed by LC-MS/MS. The method was accurate with an intra-assay variation of 8% and an interassay variation of 10%. The median concentration of estrone in subcutaneous adipose tissue from the breast did not differ between breast cancer and control women, 920 pmol/kg and 890 pmol/kg, respectively. In breast cancer patients but not in the controls, breast adipose tissue estrone levels correlated positively with the serum estrone concentration. In conclusion, the new method provides a reliable means to measure estrone concentrations in adipose tissue in postmenopausal women.

Preanalytical validation and reference values for a mass spectrometric assay of serum vanillylmandelic acid for screening of catecholamine secreting neuroendocrine tumors.

BACKGROUND: Urinary vanillylmandelic acid (VMA) is used to diagnose and monitor catecholamine secreting neuroendocrine tumors (NETs). We developed and validated a new liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for determination of serum VMA. METHODS: We used serum samples from healthy volunteers (n=314) and patients suspected for NET (n=36). Deuterated VMA as an internal standard was added to samples before solid phase extraction (SPE) and LC-MS/MS analysis. We studied the effects of sample storage, sampling device and a meal on serum VMA and metanephrine concentrations. Diurnal variation and age-dependent reference intervals were established. The diagnostic performance was compared with a urinary HPLC assay for VMA and metanephrines and a serum metanephrine LC-MS/MS assay. RESULTS: Serum VMA is stable at least for one day at +4°C, seven days at room temperature and 98 days at -20°C. Type of sampling device was not critical, but elevated serum VMA occurs after a meal (p = 0.031). Serum VMA increased with age. Therefore, we suggest clinical cut-off values of 62 nmol/L, 80 nmol/L and 108 nmol/L for age groups 18-50 yrs, 51-70 yrs and > 70 yrs, respectively. Comparison between a urinary VMA HPLC assay and serum VMA LC-MS/MS assay showed good correlation. CONCLUSIONS: Our LC-MS/MS assay is fast and sensitive and suits well for use in a clinical laboratory. Compared to 24-h urine collection our serum assay enables well controlled sampling and convenient preanalytical steps.

Mitochondrial coenzyme Q10 determination via isotope dilution liquid chromatography tandem mass spectrometry.

Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria. In the assay, mitochondrial suspensions are spiked with CoQ10-[(2)H6] internal standard, extracted with organic solvents, and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).

Duration of gestation and mode of delivery affect the genes of transepithelial sodium transport in pulmonary adaptation.

BACKGROUND: Respiratory distress due to inadequate lung liquid clearance is a significant problem in infants delivered late preterm or early term, especially by elective cesarean delivery (CD). Lung liquid clearance depends on epithelial ion transport and in animals is induced by glucocorticoids. OBJECTIVES: In newborn late preterm and term infants to study airway epithelial gene expressions of epithelial sodium channel (ENaC), and the serum and glucocorticoid-inducible kinase 1 (SGK1), and their association with cortisol, mode of delivery, and gestational age (GA). METHODS: Infants were delivered at 35(0/7)-41(6/7) weeks. Cortisol in umbilical cord plasma was analyzed with liquid chromatography-tandem mass spectrometry. ENaC and SGK1 mRNAs in airway epithelial cells obtained within 3 h and at 1 day postnatally were quantified with real-time PCR. RESULTS: ENaC and SGK1 mRNAs were significantly lower in late preterm and early term infants than in those ≥ 39(0/7) weeks. Significant correlations existed between both ENaC and SGK1 and cord cortisol and GA. In term infants, SGK1 mRNA at 1.5 h was higher after vaginal delivery than elective CD. CONCLUSIONS: In late preterm and early term infants, low expression of ENaC and SGK1 may parallel insufficient lung liquid clearance predisposing to respiratory distress. Lower SGK1 expression after term CD could translate into insufficient sodium and lung liquid absorption. The findings demonstrate a central role for cortisol in regulation of ENaC and potentially perinatal sodium and lung liquid clearance.

Breast adipose tissue estrogen metabolism in postmenopausal women with or without breast cancer.

CONTEXT: It has been shown that breast tumor actively produces and metabolizes steroid hormones. However, little is known about the possible mechanisms through which the nonmalignant adipose tissue contributes to steroid hormone metabolism. OBJECTIVE: We compared the metabolic pathways producing active estradiol in breast sc adipose tissue of postmenopausal women with or without breast cancer. DESIGN AND SETTING: Serum and adipose tissue samples were obtained during elective surgery. PATIENTS: We studied postmenopausal women undergoing mastectomy due to an estrogen receptor-positive breast tumor (n = 14) and women undergoing breast reduction mammoplasty (n = 14). INTERVENTIONS: Estrone, estradiol, and estradiol fatty acyl ester concentrations were determined by liquid chromatography-tandem mass spectrometry. mRNA expression levels of estrogen-converting enzymes were analyzed by quantitative RT-PCR. RESULTS: Estradiol concentration in breast sc adipose tissue was lower in women with cancer than in controls (median 33 vs 62 pmol/kg; P = .002), whereas the serum concentrations did not differ. Also, the mRNA expression for 17ß-hydroxysteroid dehydrogenase type 12 was lower in the adipose tissue of women with cancer compared with controls (0.19 ± 0.10 vs 0.37 ± 0.21, P = .018). CONCLUSIONS: Estrogen metabolism is differentially regulated in the adipose tissue of women with or without cancer. In the sc adipose tissue proximal to breast tumor 17ß-hydroxysteroid dehydrogenase type 12 expression is lower than in controls, which could indicate that the conversion of estrone to estradiol is decreased. Further studies are needed to establish the clinical significance of our findings in the development and growth of breast cancer in postmenopausal women.

Combination of LC-MS/MS aldosterone and automated direct renin in screening for primary aldosteronism.

BACKGROUND: Aldosterone to renin ratio (ARR) is used in screening for primary aldosteronism (PA). However, there are only few studies on the influence of assay methods on ARR and its cut-off limits. METHODS: Plasma direct renin immunoreactivity by chemiluminescence immunoassay (DR) was compared to renin activity assay (PRA), and a specific liquid chromatography-mass spectrometric method (LC-MS/MS) to radioimmunoassay (RIA) for plasma aldosterone. There were 75 samples for renin assays, and 42 samples of 39 patients for both renin and aldosterone assays. PA screening was considered positive if ARR by the aldosterone RIA:PRA was ≥800pmol/L:µg/L/h or by LC-MS/MS:DR≥44pmol/L:ng/L. RESULTS: The correlation between the DR and PRA methods (n=75, r(2)=0.845) and between LC-MS/MS and RIA (n=42, r(2)=0.973) was high in general, but low between the renin methods (n=49, r(2)=0.435) at low PRA values. When ARR was used in screening for PA, there were three divergent cases (positive only by alternative methods), but when applied in combination with criteria for elevated aldosterone, the methods showed good agreement, resulting in eight positive and 31 negative screening results. CONCLUSIONS: The automated DR assay combined with LC-MS/MS method for aldosterone provides a rapid, reliable, and specific method for screening of PA.

Postnatal ovarian activation has effects in estrogen target tissues in infant girls.

CONTEXT: Shortly after birth, pituitary gonadotropin secretion transiently activates in both sexes, and this surge is more robust in preterm (PT) than in full-term (FT) infants. In boys, the gonadotropin surge is associated with testicular activity and is considered an important part of normal reproductive development. In contrast, gonadal activation and its consequences in infant girls are poorly understood. OBJECTIVE: Our objective was to evaluate the association of postnatal ovarian activity with simultaneous changes in estrogen target tissues in FT and PT girls. PATIENTS AND METHODS: We measured urinary estradiol (E2) levels in 29 FT and 34 PT girls using a mass spectrometric method from 1 week (D7) to 6 months of age (M1-M6). To assess the contribution of ovarian E2 on urinary E2 levels, the levels in girls were compared with the levels of boys of similar cohorts (29 FT and 33 PT boys). E2 levels were compared with simultaneous changes in estrogenic target tissues including mammary glands in both sexes and uterus and vulvar epithelium in girls. RESULTS: Median urinary E2 levels increased after D7 in girls, but not in boys. Mammary gland diameter was larger in girls than in boys from M4 in FT (P < .001) and M2 in PT infants (P < .0001). In PT girls, E2 levels increased at term and were then higher than those in FT girls (P < .0001). Urinary E2 levels in PT girls were positively associated with mammary gland and uterine growth. CONCLUSIONS: These findings indicate that gonadal steroidogenesis activates during the postnatal gonadotropin surge in girls. In addition, the resulting elevated E2 levels affect target tissues, suggesting that postnatal pituitary-ovarian activation plays a role in normal female reproductive development.

Determination of cortisol in serum, saliva and urine.

Cortisol is quantitatively the major glucocorticoid product of the adrenal cortex. The main reason to measure cortisol is to diagnose human diseases characterised by deficiency of adrenal steroid excretion in Addison's disease or overproduction in Cushing's syndrome (CS). In both cases a sensitive, accurate and reproducible assay of cortisol is required. Several methods have been described for the quantitative measurement of cortisol in both serum and urine. The most widely used methods in routine clinical laboratories are immunoassays (IA) and enzyme immunoassays (EIA), luminescence and fluorescence assays, which are available in numerous commercial kits and on automated platforms. However, there remains a number of problems in the so-called direct immunoassays if extraction and prepurification are not carried out before the assay. Recently, more specific chromatographic methods have been introduced, such as high pressure liquid chromatographic (HPLC) or liquid chromatography tandem mass spectrometric assays (LC-MS/MS). The high specificity especially of LC-MS/MS facilitates reliable measurement of cortisol both in plasma, urine and saliva samples.