@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Other UBC"@en, "Non UBC"@en, "Cellular and Physiological Sciences, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:identifierCitation "BMC Medicine. 2018 Feb 12;16(1):23"@en ; dcterms:contributor "University of British Columbia. Life Sciences Institute"@en ; ns0:rightsCopyright "The Author(s)."@en ; dcterms:creator "Johnston, Zoe C"@en, "Bellingham, Michelle"@en, "Filis, Panagiotis"@en, "Soffientini, Ugo"@en, "Hough, Denise"@en, "Bhattacharya, Siladitya"@en, "Simard, Marc"@en, "Hammond, Geoffrey L"@en, "King, Peter"@en, "O’Shaughnessy, Peter J"@en, "Fowler, Paul A"@en ; dcterms:issued "2018-02-14T18:03:34Z"@en, "2018-02-12"@en ; dcterms:description """Background: Human fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone, estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention through secretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objective of this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzyme expression in normal second trimester human fetuses. Methods: This was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to 109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study design was balanced to show effects of maternal smoking. Results: Concentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and mass spectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in all adrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 and CYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-level expression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetal plasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 and NR5A1 were increased by maternal smoking. Conclusions: The human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectable aldosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen in extreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which could have knock-on effects on post-natal health."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/64638?expand=metadata"@en ; skos:note "RESEARCH ARTICLE Open AccessThe human fetal adrenal produces cortisolbut no detectable aldosterone throughoutthe second trimesterZoe C. Johnston1, Michelle Bellingham1, Panagiotis Filis2, Ugo Soffientini1, Denise Hough1, Siladitya Bhattacharya3,Marc Simard4, Geoffrey L. Hammond4, Peter King5, Peter J. O’Shaughnessy1 and Paul A. Fowler2*AbstractBackground: Human fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone,estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention throughsecretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objectiveof this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzymeexpression in normal second trimester human fetuses.Methods: This was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study designwas balanced to show effects of maternal smoking.Results: Concentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and massspectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone,dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in alladrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 andCYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-levelexpression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetalplasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 andNR5A1 were increased by maternal smoking.Conclusions: The human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectablealdosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenalregulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia duringthe second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen inextreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which couldhave knock-on effects on post-natal health.Keywords: Human, Adrenal, Fetus, Steroid, Maternal smoking* Correspondence: p.a.fowler@abdn.ac.uk2Institute of Medical Sciences, School of Medicine, Medical Sciences &Nutrition, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UKFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Johnston et al. BMC Medicine (2018) 16:23 DOI 10.1186/s12916-018-1009-7BackgroundThe human fetal adrenal gland develops initially as partof the adrenogonadal primordium and is distinct in thehuman by 7–8 weeks of gestation. The adrenals secretecortisol in response to adrenocorticotropic hormone(ACTH) as early as week 8 of gestation [1], although themain steroids produced in fetal life are dehydroepian-drosterone (DHEA) and its sulphate (DHEAS), whichact as substrates for placental estrogen production [2].Together, the fetal adrenal glands and placenta dominatehuman fetal steroid endocrinology in a manner seenonly in higher primates. Normal development and func-tion of the fetal adrenals is also essential for several pro-cesses that can affect the fetus itself or the health of theneonate. For example, disruption of the fetal adrenalscan lead to disorders of sex development [3], while fetalmisprogramming of the stress axis, through altered fetalcortisol secretion, may predispose to diseases in later life[4]. The adrenal glands are also essential for survivalearly in post-natal life through the secretion of aldoster-one, which prevents salt-wasting disorders [5], whileadequate cortisol is required to prevent adrenal insuffi-ciency in the newborn [6]. Interestingly, pre-term neo-nates are prone to salt-wasting disorders, which mayindicate a failure of aldosterone synthesis in fetal life oraltered sensitivity to the steroid [5]. Despite the import-ance of the adrenal glands for fetal and post-natal health,however, their development during fetal life in the hu-man is not well described or understood. Animal modelsare of limited relevance due to significant species differ-ences in fetal adrenal structure, steroidogenic pathwaysand endocrinology of pregnancy. However, only a limitednumber of studies have examined the human fetal adrenaldirectly ([1, 7–11] and in review [12, 13]). Consequently,we have examined human fetal adrenal steroidogenesisduring the second trimester in a large number of well-documented, normal, human fetuses. This includes thefirst comprehensive liquid chromatography (LC)/massspectrometry (MS) analysis of human fetal adrenal steroidlevels during the second trimester.Maternal smoking during pregnancy remains a signifi-cant public health issue, as it can disrupt normal fetal pro-gramming and can have irreversible effects on the post-natal life of the offspring [14]. Cigarette smoke containsmany potential toxicants (e.g. heavy metals, aldehydes, ni-trosamines and polycyclic aromatic hydrocarbons) [15]that can cross the placental barrier to reach the fetus.However, mechanisms behind the long-term effects ofsmoking on the human fetus remain largely unknown.Maternal smoking can affect fetal adrenal function anddevelopment of the hypothalamo-pituitary-adrenal (HPA)axis [16], which may be one mechanism by which smok-ing programmes later health deficits of the offspring [17,18]. We have, therefore, extended our developmentalstudy to examine the effects of maternal smoking on hu-man fetal adrenal steroidogenesis.MethodsSample collectionThe collection of fetal material involved in the adrenalstudies was approved by the National Health Service(NHS) Grampian Research Ethics Committees (REC 04/S0802/21). Human fetal kidneys were collected under aseparate, newer ethics permission as part of the ScottishAdvanced Fetal Research (SAFeR) Study. This was ap-proved by NHS Grampian Research Ethics Committees(REC 15/NS/0123). In all cases, women seeking electiveterminations of pregnancy were recruited with full writ-ten, informed consent by nurses working independentlyof the study at the Aberdeen Pregnancy Counselling Ser-vice. Maternal data, medications used and self-reportednumber of cigarettes smoked per day were recorded.Only fetuses from normally progressing pregnancies (de-termined at ultrasound scan prior to termination) fromwomen over 16 years of age and between 11 and21 weeks of gestation (7 and 20 weeks for fetal kidneys)were collected following termination by RU486 (mife-pristone) treatment (200 mg) and prostaglandin-induceddelivery, as detailed previously [19]. Fetuses were trans-ported to the laboratory within 30 min of delivery,weighed, sexed and the crown-rump length recorded.Blood samples were collected by cardiac puncture exvivo and plasma prepared in heparin-coated tubes wasstored at –80 °C. Fetal tissues were snap-frozen in liquidnitrogen and stored at –80 °C or fixed in 10% neutralbuffered formalin. Adrenal samples were analysed infour groups: control female, smoke-exposed female, con-trol male and smoke-exposed male, with groups bal-anced as far as possible for gestational age (Table 1).Maternal smoking status was confirmed by measure-ment of fetal plasma cotinine using a commercially avail-able kit (Cozart Plc, Abingdon, Kent, UK). Fifty-six fetalkidneys were analysed as a single group and as fourgroups: control female, smoke-exposed female, controlmale and smoke-exposed male.Plasma measurements of hormones and binding globulinsAssays were performed using plasma samples from 60 sec-ond trimester fetuses. Plasma ACTH levels were measured(25 μL/fetus) using a single Milliplex® MAP Human Pituit-ary Magnetic Bead Panel 1 kit (ACTH, growth hormone(GH), thyroid-stimulating hormone (TSH), ciliary neuro-trophic factor (CNTF), agouti-related protein (AGRP);Millipore Limited, Watford, UK) and analysed using a Bio-Plex200 system (Bio-Rad Laboratories Ltd, Hemel Hemp-stead, UK). Intra- and inter-assay coefficients of variationwere < 10% and < 15% respectively, sensitivity was 0.91 pg/mL and cross-reactivity was negligible. Cortisol wasJohnston et al. BMC Medicine (2018) 16:23 Page 2 of 16measured in 50 μL of plasma/fetus using a DetectX® Corti-sol Enzyme Immunoassay kit (Arbor Assays, Ann Arbor,MI, USA). Cross-reactivity with other steroids was < 8%,sensitivity was 17.3 pg/mL and inter- and intra-assay coeffi-cients of variation were 5.4% and 8.1% respectively.Corticosteroid-binding globulin (CBG) concentrations weremeasured by an established ligand-saturation assay [20].Protein, DNA and RNA extractionsTo avoid sampling error due to heterogeneous morph-ology of the adrenal gland, only whole human fetal ad-renal glands were homogenized in the presence ofprotease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich Company Ltd, Gillingham, UK). Protein, DNAand RNA were extracted from total tissue homogenateswith Qiagen AllPrep DNA/RNA/Protein mini kits(Qiagen Ltd, Crawley, UK). RNA was extracted fromwhole human fetal kidneys in the same way.Intra-adrenal steroid quantification with LC/MSSteroids were extracted from the unused, combined,flow-through fractions of the Qiagen AllPrep DNA/RNA/protein extraction protocol and quantified by LC/MS. This approach was taken to maximize the datagathered from as many fetuses as possible and reflectsthe unique nature and relative scarcity of the humanfetal samples.Steroid extractionsInitially, we determined whether intra-adrenal steroidlevels could be accurately measured from the unusedflow-through fractions of the Qiagen AllPrep DNA/RNA/protein extraction protocol. The first studies, usingTable 1 Morphological data for the mothers and fetuses involved in adrenal studiesPopulation Characteristic Female fetuses Male fetusesControl Smoke-exposed Control Smoke-exposedTotal adrenal samples N 29 19 31 30Maternal indices Age (years) 25.5 ± 1.2 23 ± 1.2 23.8 ± 1.06 26.2 ± 1Body mass index (BMI, kg/m2) 24.1 ± 0.8 26 ± 1.3 26 ± 0.95 25.8 ± 1.2Cigarettes/day 0 ± 0 9.3 ± 0.7 0.2 ± 0.2 10.2 ± 1Fetal indices Age (weeks) 14.6 ± 0.4 14.3 ± 0.8 14.6 ± 0.6 14.6 ± 0.5Weight (g) 77 ± 13.3 75.4 ± 14.1 83.4 ± 12 91.8 ± 12.9Crown-rump length (CRL, mm) 98.7 ± 5.2 95.1 ± 5 101.4 ± 4.3 105.5 ± 5.5Ponderal index (weight, g/[CRL, cm3]) 70.2 ± 6.7 72.8 ± 5.4 68.8 ± 3.3 63.2 ± 1.8Plasma cotinine (ng/mL) 6.8 ± 3.3 30.2 ± 5.4 5.3 ± 1.2 41.6 ± 6.4Sub-population: plasma steroidsa N 14 16 13 17Maternal indices Age (years) 24.3 ± 1.7 23.2 ± 1.3 24.5 ± 1.8 24.7 ± 1.2BMI (kg/m2) 23.5 ± 1.2 23.8 ± 1.3 26.2 ± 1.4 25.3 ± 1.4Cigarettes/day 0 ± 0 11 ± 1.3 0 ± 0 12.5 ± 1.1Fetal indices Age (weeks) 15.6 ± 0.6 15.2 ± 0.6 15.2 ± 0.6 15.1 ± 0.6Weight (g) 104.8 ± 19.9 93.6 ± 16.7 101.7 ± 22.5 97.1 ± 19.3CRL (mm) 109.3 ± 7.7 106.9 ± 5.7 108 ± 7.4 107.2 ± 7.4Ponderal index (weight, g/[CRL, cm3]) 64.9 ± 2.6 64 ± 2.6 73.9 ± 4.7 65 ± 2Plasma cotinine (ng/mL) 3.3 ± 1.2 41.6 ± 2.8 2.6 ± 0.9 48.6 ± 2.2Sub-population: mRNA/protein/steroidsa N 15 15 15 15Maternal indices Age (years) 24.7 ± 1.6 23.2 ± 1.4 22.9 ± 1.3 26.6 ± 1.4BMI (kg/m2) 24.2 ± 0.8 26.5 ± 1.4 26.23 ± 1.5 24.6 ± 1.6Cigarettes/day 0 ± 0 8.8 ± 0.8 0 ± 0 12 ± 1.3Fetal indices Age (weeks) 15.3 ± 0.6 15.1 ± 0.6 15.2 ± 1.2 15.2 ± 0.6Weight (g) 97 ± 19.5 84.8 ± 17 94.9 ± 18.8 94.7 ± 19.9CRL (mm) 106.4 ± 6.8 97.4 ± 6.1 104.3 ± 6.9 102.3 ± 8.7Ponderal index (weight, g/[CRL, cm3]) 66.5 ± 2.3 75.5 ± 6.7 72 ± 4.6 67 ± 3.1Plasma cotinine (ng/mL) 7.6 ± 4.6 30.2 ± 5.4 4 ± 1.5 50.9 ± 5.7aAlthough there is some overlap between the two study sub-populations, not all plasma samples were from fetuses where the adrenal glands were collected.Therefore, the two populations were not analysed togetherJohnston et al. BMC Medicine (2018) 16:23 Page 3 of 16a [3H]-testosterone recovery standard with mouse ad-renal tissue homogenate, showed that 72% of testoster-one was recovered in the discardable flow-throughfraction from the protein precipitation step (step 15 ofthe Qiagen manual) with 89.6% total recovery from thetotal combined waste fractions. In further preliminarystudies using LC/MS, the recovery of intra-adrenal ste-roids (from human fetal adrenals) was measured bycomparison of steroid levels in the initial tissue lysatewith levels in recovered fractions after RNA/DNA/pro-tein extraction. Steroids were recovered primarily in theprotein precipitation step (step 15 in the Qiagen pro-tein/DNA/RNA extraction protocol), with the exceptionof DHEAS, which was recovered in all fractions. Esti-mates of steroid recovery efficiencies from combinedfractions were as follows: cortisol 87%, androstenedione122%, 17α-hydroxyprogesterone 113%, 21-deoxycortisol87%, 11-deoxycortisol 111% and DHEAS 56%. Thesedata show that most steroids could be recovered fromtissues with high efficiency during Qiagen extraction.For subsequent studies all of the residue fractions fromthe Qiagen extraction process (steps 8, 15, 21 and 22)were combined and extracted.Three deuterated steroids (15 ng) were added as in-ternal standards (ISs) prior to steroid extraction, as de-scribed below. Steroids in the combined residuefractions were extracted twice using ethyl acetate (4:1 ra-tio to sample volume), subjected to a solid-phase extrac-tion using 1 mL Strata-X, 33 μm polymeric reversephase cartridges (Phenomenex, Torrance, CA, USA) anddissolved in a final volume of 150 μL methanol. The per-centage recovery of all steroids through ethyl acetateand solid phase extractions was > 82%, and the percentrelative standard deviation (RSD; n = 6) of all steroidmeasurements was between 1.82 and 8.75%.The standards prepared in methanol were not signifi-cantly different than those prepared using the extractionmatrix, while recovery and percent RSD showed negli-gible differences. We therefore used standards in metha-nol for further experiments.Intra-adrenal steroid quantification with LC/MSSteroids were separated by reversed phase LC on an Ul-tiMate 3000 RSLCnano (Thermo Fisher Scientific, Wal-tham, MA, USA) separation system, using an Accucore™PFP LC column with trimethyl silane (TMS) endcapping(50 mm, 2.1 mm, 2.6 μm, Thermo Fisher). For both posi-tive and negative modes, the mobile phase was a mixtureof solvents A (1% formic acid in water) and B (49:49:2methanol:acetonitrile:isopropanol). Steroids were elutedat a flow rate of 0.4 mL/min, using a linear gradientfrom 24% B to 47% B in 2.0 min, followed by a lineargradient to 66% B in 2.7 min and a subsequent lineargradient to 100% B in 0.1 min. Elution at 100% B wasmaintained for 0.9 min before lowering it to 24% B over0.1 min and equilibrating the column for 2.5 min. Thetotal run time was 8.3 min and the injection volume was5 μL.Steroids were detected with a Q Exactive Orbitrap(Thermo Fisher) mass spectrometer, using full-scan de-tection (250–500 m/z). Table 2 contains a summary ofthe limits of quantification for all steroids in this study.For each sample, one MS run was completed at full scanin positive mode and another MS run at full scan innegative mode. MS mode was found to be more suitedfor steroid profiling with the Orbitrap MS system, andthe sensitivities were similar to those achieved using LC-MS/MS with a triple quadrupole MS system [21].All instruments were controlled by Chromeleon soft-ware (Thermo Fisher), and data acquisition, peak integra-tion and quantification were performed using Xcalibur 2.2software (Thermo Fisher). Calibration curves were used toquantify the steroids, using the ratio of the steroid peakarea relative to the peak area of a specific deuterated ISthat had similar elution time and/or chemical properties.Deuterated cortisol (4-pregnen-11β,17,21-triol-3,20-dione-9,11,12,12-d4; Steraloids, Newport, RI, USA) was usedas the IS for aldosterone, cortisol, cortisone, cortico-sterone, 11-deoxycortisol, cortisone sulphate, cortico-sterone sulphate, 11-dehydrocorticosterone and Δ5-androstenediol. Deuterated progesterone (4-pregnen-3,20-dione-2,2,4,6,6,17α,21,21,21-d9; Steraloids) was usedas the IS for DHEA, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, 16α-hydroxyprogesterone, testoster-one, deoxycorticosterone, progesterone, pregnenolone andΔ4-androstenedione. Deuterated DHEAS (5-androsten-3β-ol-17-one-16,16-d2, sulphate, sodium salt; Steraloids)was used as the IS for DHEAS. Calibration curves wereconstructed from the LC/MS analyses of six to nine cali-brator samples in the range of 1–2000 ng/mL. Calibratorsamples contained all three deuterated ISs at a concentra-tion of 100 ng/mL, as well as steroid standards at relevantconcentrations from a dilution series in methanol. Ster-oid standard stocks, from which the calibrator dilutionseries were made, were all sourced from Sigma-Aldrichor Steraloids.To determine limits of detection (LODs) and limits ofquantification (LOQs), calibrator samples (n = 13) wereprepared at concentrations ranging from 0.01 ng/mL to10,000 ng/mL, containing all of the steroids and deuter-ated ISs at a fixed concentration of 100 ng/mL. Thesewere prepared in both methanol and Qiagen buffer mix-ture. Calibration curves were generated by performingleast-squares regression analysis on peak area ratios rela-tive to the IS at different concentrations, within the sensi-tivity range of each steroid. The LOD and LOQ weredefined as the lowest steroid concentration with a signal-to-noise ratio (S/N) larger than 3 and 10 respectively.Johnston et al. BMC Medicine (2018) 16:23 Page 4 of 16Table2Intra-adrenalsteroidsmeasuredbyLC/MS TotalControlfemaleSmoke-exposedfemaleControlmaleSmoke-exposedmaleSteroidLOQ(ng/mLMeOH)LOQ(adjusted)aQSb(n=60)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)Pregnenolone100.05585.019±0.49154.619±0.79134.299±0.927154.073±0.828157.087±1.13517α-Hydroxyprogesterone10.005601.291±0.182151.086±0.151151.115±0.189150.878±0.159152.087±0.624DHEAS0.10.0005601.153±0.178151.426±0.412150.782±0.16150.966±0.271151.437±0.468Progesterone10.005601.089±0.185150.993±0.194150.95±0.278151.066±0.504151.347±0.415Cortisol0.10.0005560.626±0.087140.713±0.198140.498±0.127130.471±0.142150.824±0.198Corticosterone0.10.0005570.354±0.075130.265±0.073140.213±0.08150.345±0.153150.593±0.22416α-Hydroxyprogesterone0.10.0005590.259±0.035150.259±0.036140.18±0.04150.192±0.038150.407±0.11611-Deoxycortisol0.10.0005600.119±0.017150.148±0.039150.1±0.03150.082±0.02150.146±0.042Cortisone10.005370.108±0.01890.15±0.04790.096±0.03490.075±0.023100.109±0.037Testosterone0.10.0005320.032±0.00790.062±0.0280.028±0.01370.017±0.00880.023±0.008Deoxycorticosterone0.10.0005390.023±0.003120.03±0.006100.018±0.003100.019±0.00470.025±0.009Corticosteronesulphate100.05100.018±0.0130.022±0.01920.003±0.00210.001±0.00140.045±0.03211-Dehydrocorticosterone0.10.0005120.003±0.00140.005±0.00430.001±020.002±0.00230.005±0.003Aldosterone10.0050