Plasma Steroid Profiling and Response to Trophins to Illustrate Intra

F MCMANUS and others Changes in plasma steroids 224:2 149–157 Research measured by LC–MS

Plasma steroid proﬁling and response to trophins to illustrate intra-adrenal dynamics

F McManus, R Fraser, E Davies, J M C Connell1 and E M Freel Correspondence should be addressed Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow Cardiovascular Research Centre, to F McManus 126 University Place, Glasgow G12 8TA, UK Email 1College of Medicine, Dentistry and Nursing, Ninewells Hospital and Medical School, University of Dundee, Frances.McManus@ Dundee DD1 9SY, UK glasgow.ac.uk

Abstract

The importance of corticosteroids in cardiovascular and other chronic disease is recognised. In Key Words addition, plasma steroid precursor-to-product ratios are useful and convenient indirect " adrenal indicators of efﬁciency of key steroidogenic enzymes (aldosterone synthase, 11b-hydroxylase " corticosteroid and 17a-hydroxylase). The use of liquid chromatography–tandem mass spectrometry " enzyme (LC–MS/MS) has enabled measurement of numerous corticosteroid compounds simultaneously. However, normal responses to trophins and variation in salt intake are not well described. This study examined these parameters in a large group of healthy volunteers. Sixty normotensive volunteers were recruited and underwent infusion of angiotensin II (AngII) and ACTH, following low- and high-salt diet. Measurement of plasma steroids at baseline and 30 min after infusion of

Journal of Endocrinology trophin was carried out by LC–MS. As expected, plasma mineralocorticoid levels increased in response to salt restriction and were suppressed with salt loading; ACTH infusion increased all corticosteroids, while AngII increased mineralocorticoids and suppressed glucocorticoid production. ACTH increased S:F but decreased DOC:B, thus the S:F ratio is a more appropriate index of 11b-hydroxylase efficiency. The B:F ratio increased following ACTH treatment and salt restriction. A larger proportion of plasma B than generally accepted may be derived from the zona glomerulosa and this ratio may be most informative of 17a-hydroxylase activity in salt- replete subjects. Although DOC:aldosterone, B:aldosterone and 18-hydroxyB:aldosterone should provide indices of aldosterone synthase efficiency, responses of individual compounds to trophins suggest that none of them accurately reflect this. Based on these data, aldosterone

synthase activity is most accurately reﬂected by aldosterone concentration alone. Journal of Endocrinology (2015) 224, 149–157

Introduction

Corticosteroids are key regulators of the cardiovascular of aldosterone results in hypertension with an excess of system. It has increasingly been accepted that aberrant left ventricular hypertrophy, renal dysfunction and blood pressure regulation and other cardiovascular con- arrhythmia (Milliez et al. 2005), while cortisol excess, or sequences can be associated with altered production of Cushing’s syndrome, causes, among other features, high the end products of the corticosteroid pathways, aldoster- blood pressure, obesity, coagulopathy and dysglycaemia one and cortisol. For example, autonomous production (Arnaldi et al. 2003).

http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JOE-14-0561 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 01:01:13AM via free access Research F MCMANUS and others Changes in plasma steroids 224:2 150 measured by LC–MS

Cortisol and aldosterone are the final products of To date, most studies demonstrate variations in specific pathways (Fig. 1), each comprising a series of corticosteroid metabolism analysed using urinary metab- enzyme-catalysed reactions. The efficiency of each enzyme olites and there are many metabolites for each compound. may vary, for example, in a genetically determined Plasma concentrations afford a more direct index of manner, so that each step in the pathway may become secretion and allow characterisation of short-term rate limiting to a greater or lesser extent. This phenom- changes, for example, the response to acute alteration in enon is well documented in a series of rare monogenic angiotensin II (AngII) or adrenocorticotrophic hormone disorders of corticosteroid synthesis (congenital adrenal (ACTH) drive, which may alter both absolute secretion hypertrophy (CAH)), where abnormal levels of intermedi- rates of aldosterone and cortisol, respectively, and their ate compounds can be both pathogenic and diagnostic relationship to their respective precursors. Indeed, the (Miller & Auchus 2011). Importantly, qualitatively similar short-term response to stimulation may be the only way to but more subtle variations in corticosteroid production discern a potentially altered enzyme efficiency and steroid have been detected in normal and hypertensive subjects pattern (as is the case with the plasma 17a-hydroxypro- (Imrie et al. 2006, Freel et al. 2007, Freel et al. 2008) and gesterone response to ACTH in ‘non-classic’ CAH; White & related to polymorphic variation within the genes Speiser 2000). To assess the clinical significance of profile encoding enzymes that catalyse the final steps in the differences, reliable normal levels and responses to biosynthesis of aldosterone (aldosterone synthase, stimulants of glucocorticoids and mineralocorticoid pro- CYP11B2 and 11b-hydroxylase, CYP11B1). In these duction are necessary: in recent years, the technique of studies, secretion of the pathway end products, aldoster- liquid chromatography–tandem mass spectrometry one and cortisol, was normal and only analysis of the (LC–MS/MS) has largely replaced multiple RIA and has pattern of intermediate steroid levels revealed these made larger studies of plasma steroid concentrations interesting and potentially important differences. more achievable, thus making such an approach feasible.

Pregnenolone 17α-pregnenolone 17α-hydroxylase β 3 -HSD 3β-HSD Journal of Endocrinology Progesterone 17α-progesterone 17α-hydroxylase 21α-hydroxylase 21α-hydroxylase Deoxycorticosterone 11-deoxycortisol 11β-hydroxylase and Aldo synthase 18-hydroxyDOC 11β-hydroxylase

Corticosterone 11β-HSD Cortisol 11β-hydroxylase and 11-dehydrocorticosterone β Aldo synthase 11 -HSD

18-hydroxycorticosterone Cortisone

Aldo synthase

Aldosterone

Figure 1 Biosynthetic pathway of mineralocorticoids and glucocorticoids within the adrenal cortex. The dashed line denotes zona glomerulosa activity and the solid line, biosynthetic reactions occurring within the zona fasciculata. 11b-HSD, 11b-hydroxysteroid dehydrogenase; 18-hydroxyDOC, 18-hydroxydeoxycorticosterone.

A full spectrum of compounds can be assessed simul- Plasma measurements of adrenal corticosteroids taneously with high precision accuracy and specificity using LC–MS/MS (McDonald et al. 2011). The following study examined Samples were stored at room temperature and centrifuged at the modulation of relative corticosteroid product flow 25 8C for 15 min at 3000 g. Plasma samples containing 60 ng through three pathways, namely, the aldosterone path- of internal standard (6b-methylprednisolone) were applied way in the zona glomerulosa and the 17a-hydroxysteroid to Chem Elut SPE cartridges (Varian, Inc., Palo Alto, CA, USA) and 17-deoxysteroid pathways in the zona fasciculata, and the corticosteroids eluted with dichloromethane (HPLC in a large group of normal subjects under different grade). The eluates were evaporated to dryness under nitrogen physiologically relevant environmental conditions. Its and the residue re-dissolved in 10% acetonitrile (60 ml; principal aim was to provide a normal basis for investi- HPLC grade). An aliquot (20 ml) was applied to an HPLC gating the clinical significance of steroid-related enzyme column (Polaris, 5 m 18-A, 150!20 mm) coupled to a mass polymorphisms. spectrometer (Varian 1200L with triple quadropole detector). The corticosteroids were serially eluted using an acetonitrile: Materials and methods water gradient containing 2 mmol ammonium acetate. The performance criteria and normal ranges for each cortico- Patient selection and protocols steroid are summarised in Table 1. The internal standard was 16b-methylprednisolone (Ingram & Fraser 2010). Normal volunteers were recruited by advertising in the Plasma renin concentration was measured by the local media and were required to be in good health, Diasorin Liaison immunochemiluminometric analyser between the ages of 18–70 and not on any antihyper- (DiaSorin Ltd, Wokingham, Berkshire, UK). tensive or steroid-containing medication at the time of recruitment. All subjects underwent dietary manipulation to standardise their salt intake to either ‘high-salt’ or Statistical analysis ‘low-salt’ states. Briefly, subjects adhered to a modest Statistical analysis was performed using Minitab (State salt-restricted diet for 5 days and were randomised to College PA, USA, version 15). Where steroid data could be receive supplementation with either slow-sodium tablets, normalised by transformation (log ), they were analysed in order to achieve a salt intake of w200 mmol/day 10 using Student’s t-test or paired t-test where appropriate; (200 mmmol diet), or placebo, in order to achieve a salt Journal of Endocrinology alternatively, nonparametric methods of hypothesis intake of w100 mmol/day (100 mmol diet). On day 3, 24 h testing were utilised as described. urine was collected for determining urinary sodium excretion to assess compliance and salt status. In the morning of day 4, subjects attended the Clinical Research Facility of the University of Glasgow where they were Results cannulated via a peripheral vein. After 30 min of recumbent Baseline characteristics and efficacy of dietary salt rest, blood was drawn for steroid measurements. I.v. ACTH manipulation. was administered at a rate of 1 ng/kg per min and, after 30 min, the infusion was stopped for further blood samp- Sixty subjects (33 women and 27 men) participated with ling. The volunteers returned on day 5 to be infused with amean(GS.D.) blood pressure at recruitment of 126.7 AngII, administered at a rate of 3 ng/kg per min (BAChem, Table 1 Values, normal range and coefficient of variation (CV) Weil am Rhein, Germany) under the same conditions. data in normal human plasma (nZ60) After a period of at least 2 weeks, the process was repeated

so that all subjects were studied under both salt conditions. Mean morning Between batch CV Infusions were prepared by the Western Inﬁrmary level (normal (% (concentration; Pharmacy Production Unit, Glasgow. Blood pressure Compound range) (nmol/l) nmol/l)) was monitored at 10 min intervals throughout both 18OHB 4.1 (0.04–13.85) 18.7 (0.02) infusions. Blood samples were drawn using the Vacutainer Aldosterone 0.22 (0.04–0.94) 13.4 (0.1) Cortisol 248.6 (30.3–933.7) 9.1 (182.3) System (BD, Oxford Science Park, Oxford, UK) at baseline Corticosterone (B) 6.28 (0.27–45.6) 8.2 (6.6) before the infusion and at completion of the infusion. 11-deoxycortisol (S) 0.3844 (0–2.02) 14.5 (0.49) This study was approved by the West of Scotland Research Deoxycorticosterone 0.0212 (0–0.07) 21.1 (0.02) (DOC) Ethics Committee.

Table 2 Plasma corticosteroids measured under variations in salt intake. Data shown are medians and interquartile ranges (IQR) (nZ60)

Median (IQR) P Wilcoxon’s 200 mmol diet 100 mmol diet signed rank

18OHB (nmol/l) 0.03 (0.01–0.05) 0.05 (0.01–0.09) !0.01 Aldosterone (nmol/l) 0.07 (0.02–0.13) 0.12 (0.06–0.18) !0.01 Cortisol (F) (nmol/l) 201.4 (139.9–274.8) 199.2 (150.0–301.6) NS 18OHDOC (nmol/l) 0.10 (0.04–0.25) 0.10 (0.04–0.26) NS Corticosterone (B) (nmol/l) 3.17 (2.14–6.26) 4.41 (2.61–8.15) !0.05 11-deoxycortisol (S) (nmol/l) 0.33 (0.17–0.53) 0.32 (0.21–0.56) NS Deoxycorticosterone (nmol/l) 0.02 (0.01–0.04) 0.02 (0.01–0.34) NS

(G13.9)/76 (G10.3) mmHg and a mean age of 50.1 (G17.1) to highlight the variation in results with different years. There was no significant blood pressure or age methodologies. difference between men and women. Compliance to the Precursor-to-product ratios according to salt status are sodium-rich/restricted diet was checked by the urine sodium summarised in Table 3. Data were not normally distrib- excretion rate and plasma renin concentration measure- uted and so were log transformed (after ratios were ments. Following the 100 mmol/day diet, the mean urinary calculated) to allow for parametric hypothesis testing sodium excretion rate was 97.1G39.5 mmol/24 h and using a paired t-test. The ratios of 11-deoxycortisol to plasma renin concentration 19.1G13.9 mU/ml. Following cortisol (S:F) and 11-deoxycorticosterone to B (DOC:B) the 200 mmol/day diet, the mean urinary sodium excretion were not significantly affected by salt intake. The ratio of rate was 199.8G64.6 mmol/24 h and plasma renin con- 18OHB to aldosterone was significantly lower during the centration 9.7G5.9 mU/ml. (P!0.001). lower salt intake regime. B:F, in contrast, was significantly lower during higher salt intake. DOC:18OHDOC was unaffected by salt regime. Effect of variation in salt intake on corticosteroid production Influence of ACTH on corticosteroid production Journal of Endocrinology Individual corticosteroid concentrations in the two dietary regimes are compared in Table 2. Predictably, aldosterone The effects of ACTH on individual steroid concentrations concentration was significantly lower during the higher according to dietary sodium intake are summarised in salt intake. In addition, concentrations of aldosterone Table 4. Predictably, the levels of all compounds increased precursors, corticosterone (B) and 18-hydroxycortico- significantly during ACTH infusion, but the proportionate sterone (18OHB) were slightly but significantly suppressed responses were different. The highest fold-increase (Table 4) by the higher salt intake. The levels of glucocorticoid was observed in B concentration and the fold increases compounds were unaffected. Under both conditions, the in S and 18OHDOC all exceeded those in F. Alteration in concentration of aldosterone and 18OHB are surprisingly sodium status did not affect the pattern of steroid response. low when compared with previously published results, Log- transformed precursor-to-product ratios accor- and while the reasons for this are not clear, it does serve ding to salt intake are shown in Table 5.S:Frose

Table 3 Log10-transformed corticosteroid ratios (indirect marker of enzyme efﬁciencies) under conditions of high and low dietary salt intake (nZ60)

200 mmol diet 100 mmol diet

Log ratio Enzyme action Mean S.D. Mean S.D. Paired t-test

S:F 11b-hydroxylase K2.83 0.37 K2.78 0.33 NS DOC:B 11b-hydroxylase/aldosterone K2.23 0.55 K2.39 0.58 NS synthase 18OHB:Aldo Aldosterone synthase K0.18 0.54 K0.45 0.63 0.03 B:F 17a-hydroxylase K1.74 0.24 K1.64 0.26 0.006 DOC:18OHDOC 11b-hydroxylase K1.55 1.24 K1.46 1.40 NS

signiﬁcantly in response to ACTH, but DOC:B was

IQR) signiﬁcantly reduced while DOC:18OHDOC was slightly

G lower (this did not reach statistical signiﬁcance in the high salt group). There was a large increase in B:F while Median fold

change ( 18OHB:aldosterone was unaffected by ACTH. a P 0.001 2.12 (1.23–4.46) 0.001 4.56 (2.93–10.43) 0.001 8.93 (4.53–18.07) 0.001 3.55 (2.10–6.92) 0.001 2.11 (1.47–2.77) 0.001 2.71 (1.64–4.55) 0.001 2.16 (1.19–4.87) Effect of AngII on corticosteroid production during ! ! ! ! ! ! ! different salt intakes

The effects of AngII infusion on plasma steroids according to salt intake are shown in Table 6. In the low dietary salt

High salt group, plasma aldosterone and 18OHB concentrations rose signiﬁcantly, while those of the glucocorticoids, F and

IQR) S, fell signiﬁcantly. The corticosteroid response to AngII

G stimulation in the higher salt regimen cohort was similar to that in the lower salt regimen cohort (Table 6). The only

Median ( difference in steroid response to AngII according to sodium intake was in the corticosterone concentration, which did not significantly change under low-salt con- ditionsbutwassignificantlylowerunderhigh-salt conditions, and 18OHDOC, which demonstrated no significant change under high-salt conditions but fell

IQR) slightly, but signiﬁcantly, under low-salt conditions. G Precursor-to-product ratios according to salt intake in response to AngII are shown in Table 7. Some differ- Median fold change ( ences in the response of these ratios to AngII stimulation according to salt intake were observed. Most notably, a

P the ratio of B:F rose slightly in the low salt group (PZ0.05), 0.001 2.43 (1.41–5.42) 0.02 (0.01–0.04) 0.06 (0.03–0.08) 0.001 4.82 (2.91–8.70) 0.33 (0.17–0.53) 1.65 (1.06–2.31) 0.001 8.99 (4.94–13.45) 3.17 (2.15–6.27) 31.51 (23.3–46.1) 0.001 3.32 (1.94–7.44) 0.09 (0.04–0.25) 0.37 (0.25–0.54) 0.001 2.07 (1.61–2.80) 201.4 (139.9–274.8) 399.0 (361.9–473.7) 0.001 2.14 (1.22–3.36) 0.07 (0.02–0.13) 0.22 (0.09–0.28) 0.001 2.11 (1.32–5.70) 0.04 (0.02–0.06) 0.09 (0.05–0.16) Journal of Endocrinology ! ! ! ! ! ! ! but this was not replicated under high-salt conditions. In addition, during higher salt intake, the ratio of S:F fell in response to AngII (PZ0.02), while no signiﬁcant difference in this ratio was found during low salt

Low salt intake. Under both salt intake conditions, the ratio 18OHB:aldosterone fell signiﬁcantly and there was no IQR) change in the DOC:18OHDOC or DOC:B ratios. G 60). Z n Median ( Discussion

Basal Post ACTHThe data generated Basal from Post ACTH this study of normotensive, healthy volunteers provide a valuable reference resource detailing plasma steroid proﬁles (as measured by LC– MS/MS) at baseline and in response to major agonists of adrenal corticosteroid production. Variation in the

efﬁciency (that is, Km and Vmax) of the individual enzymes comprising the biosynthetic pathways to the principal

(nmol/l) bioactive corticosteroids, cortisol, corticosterone and

Plasma corticosteroids measured before and after ACTH stimulation separated according to dietary salt status aldosterone, will alter the overall efﬁciency of their synthesis, response to agonists and the concurrent levels of their precursor compounds (Fig. 1) in the plasma. Data compared by the Wilcoxon’s signed rank test. Deoxycorticosterone (DOC) 0.02 (0.01–0.03) 0.05 (0.03–0.08) 11-deoxycortisol (S) 0.32 (0.21–0.56) 1.82 (1.24–2.56) Corticosterone (B) 4.42 (2.61–8.15) 37.07 (27.00–46.21) 18OHDOC 0.09 (0.04–0.26) 0.38 (0.26–0.53) Cortisol (F) 199.2 (150.0–301.6) 429.6 (379.3–479.7) Aldosterone 0.12 (0.06–0.18) 0.22 (0.16–0.37) Table 4 Corticosteroid 18 OHBLow salt, 100a mmol/day; high 0.05 salt, (0.01–0.10) 200 mmol/day ( 0.13 (0.06–0.20) Efﬁciency is determined genetically through inherited

Table 5 Corticosteroid ratios (log-transformed) and associated enzyme activity before and after i.v. infusion of ACTH separated according to dietary salt consumption. Data compared by Student’s t-test (nZ60)

Low salt High salt

Mean (GS.D.) Mean (GS.D.)

Log ratio Enzyme action Basal Post ACTHP Basal Post ACTH P

S:F 11b-hydroxylase K2.78 (0.33) K2.41 (0.29) !0.001 K2.82 (0.37) K2.43 (0.25) !0.001 DOC:B 11b-hydroxylase/ K2.39 (0.58) K2.85 (0.34) !0.001 K2.23 (0.55) K2.79 (0.31) !0.001 aldosterone synthase 18OHB:Aldo Aldosterone synthase K0.44 (0.63) K0.30 (0.41) NS K0.18 (0.54) K0.24 (0.27) NS B:F 17a-hydroxylase K1.64 (0.26) K1.08 (0.14) !0.001 K1.74 (0.24) K1.11 (0.17) !0.001 DOC:18OHDOC 11b-hydroxylase K1.55 (1.63) K1.63 (0.14) !0.001 K0.67 (0.54) K0.85 (0.40) NS

differences in protein structure. Major changes from DOC:aldosterone could indicate that of aldosterone normal activity are associated with the rare but well- synthase (Kater & Biglieri 1994). This simple study, characterised metabolic disruptions of congenital which used zone-specific stimulation at intensities com- adrenal hyperplasia, but there is abundant evidence that mensurate with quotidian experience, has attempted to much less severe polymorphic variations within CYP11B1 select the most reliable indices of enzyme efficiency and (11b-hydroxylase), CYP11B2 (aldosterone synthase) and these are discussed in more detail later in this study. CYP17A1 (17a-hydroxylase) also affect efficiency to an extent that is probably clinically significant (Barr et al. 11b-hydroxylase 2007, Connell et al. 2008, Freel et al. 2008) (L Diver, J M C Connell & E Davies 2014, unpublished observations). 11b-hydroxylase catalyses the conversion of S to F and DOC Thus, a robust and convenient means of assessing the to B. The activity of an exclusive zona fasciculata enzyme quantitative effect and clinical relevance of a modest should be ACTH dependent and salt status/AngII indepen- polymorphic variation in steroid biosynthetic genes as dent (Nishimoto et al. 2010). Therefore, these agonists well as the acute changes in response to conventional should affect S:F and DOC:B similarly, if they convincingly trophins of adrenal steroid synthesis would be valuable. represent 11b-hydroxylation in the 17a-hydroxysteroid Journal of Endocrinology Plasma steroid profiling by means of LC–MS provides and 17-deoxysteroid pathways respectively. This was not such a means and the study of a large group of normal the case. ACTH increased S:F but decreased DOC:B, subjects described herein provides a yardstick with reflecting the much greater relative response of B which clinical cases can be compared and the influence than F, perhaps reflecting superior kinetics for the DOC to of genetic variation assessed. B conversion (Barr et al. 2007). Moreover, some plasma B The use of the ratio of the concentration of an end and DOC are derived from the zona glomerulosa aldo- product corticosteroid in plasma to that of its precursor is sterone pathway, although this zone probably has a lower an established index of enzyme deficiency and has most synthetic capacity (Miller & Auchus 2011). The results recently been evaluated in neonates (Hicks et al. 2014). In also indicated that B levels significantly responded to the presence of efficiency-altering polymorphisms, basal mild salt restriction and AngII (under high-salt-intake hormone levels will be within the normal range but conditions), whereas DOC levels were unaffected by kinetics demand that, to achieve this, precursor (i.e. these conditions. Thus, S:F remains the index of choice to substrate) levels will vary. The lower the efficiency of the reflect 11b-hydroxylase efficiency in the zona fasciculata. enzyme, the higher the ratio of precursor to product. The balance of 11b- and 18-hydroxylation activities Adrenal stimulation will emphasise this difference, as the of this enzyme may be different in different forms of reaction becomes rate limiting. experimental hypertension and this has been explored in For each of the key adrenal biosynthetic enzymes, rat models of hypertension (Rapp & Dahl 1971, 1976). As there is more than one possible index ratio. Thus, both far as we are aware, this has not been examined in relation ratios of S:F and DOC:B could be used to assess 11b- to human 11b-hydroxylase polymorphisms. In this study, hydroxylase efficiency (Connell et al. 1996). Similarly, ACTH slightly altered DOC:18OHDOC in favour of the either B:F or DOC:S could indicate the efficiency of 17a- latter compound (in the low salt group only), but DOC:B hydroxylase, and 18-OHB:aldosterone, B:aldosterone or was even more markedly reduced and uninfluenced by

dietary sodium (Table 5). The signiﬁcance of the DOC:

IQR) B:18OHDOC relationship requires more study. G

Median fold 17a-hydroxylase change ( 17a-hydroxylase determines the relative production rates

a of 17a-hydroxysteroids and 17-deoxycorticosteroids. It P 0.001 0.67 (0.47–0.89) 0.001 0.80 (0.71–0.87) 0.001 0.78 (0.60–1.02) 0.001 2.56 (1.51–4.19) 0.001 1.30 (0.98–2.45) ! ! ! ! ! also functions as a lyase, releasing C19 compounds; this aspect was not studied herein (Zuber et al. 1986). The ratios B:F and DOC:S should equally represent its activity and, as it is expressed only in the zona fasciculata, ACTH but

High salt not salt status or AngII should increase the ratios as the enzyme efﬁciency becomes limiting. There was a marked

IQR) increase in B:F following ACTH treatment (Table 5), G suggesting that, even under basal conditions, the enzyme’s capacity is close to the limiting point. However, it is striking Median ( that mild salt restriction also signiﬁcantly increased B:F (Table 3), although the effect of AngII was marginal or absent (Table 7). This suggests that a larger proportion of plasma B than generally accepted may be derived from the zona glomerulosa in this circumstance and that the ratio may be most informative in salt-replete subjects. IQR) G Aldosterone synthase Median fold change ( Aldosterone synthase catalyses a three-stage conversion of DOC to aldosterone and is expressed exclusively in the a

P zona glomerulosa (Kawamoto et al. 1992). Intermediate 0.001 0.71 (0.77–1.20) 0.26 (0.01–0.42) 0.20 (0.13–0.29) 0.001 0.82 (0.70–0.90) 197.8 (137.3–267.3) 159.7 (107.1–200.8) 0.001 2.32 (1.56–3.53) 0.09 (0.03–0.14) 0.21 (0.12–0.35) Journal of Endocrinology ! ! ! stages are B and 18-hydroxyB, but DOC is the most efﬁcient substrate in vitro (Fisher et al. 1998). All these precursor compounds also originate from the zona fasciculata. Salt status and AngII are speciﬁc agonists but Low salt aldosterone levels also respond acutely to ACTH (Arvat et al. 2000). In theory, DOC:aldosterone, B:aldosterone IQR)

G and 18-hydroxyB:aldosterone should provide indices of efﬁciency. ACTH did not alter 18-hydroxyB:aldosterone 60).

Z under either dietary regime (Table 5), possibly because this Median ( n agonist acts primarily on the early aldosterone pathway

Basal Post AngIIwhile salt status Basal and AngII Post AngII act more specifically on the late aldosterone pathway (Lantos et al. 1967). Mild salt restriction (Table 3) and AngII (Table 7) under both dietary regimes significantly lowered this ratio, although both its individual components increased. This may suggest that under baseline conditions, this enzyme is operating significantly below its limits of efficiency and is easily

(nmol/l) responsive to stimulation. However, the relative increase

Plasma corticosteroids measured before and after AngII stimulation separated according to dietary salt status in plasma 18-hydroxyB concentration was much greater during ACTH stimulation than during salt status/AngII stimulation suggesting signiﬁcant zona fasciculata origin. Data compared by the Wilcoxon’s signed rank test. Deoxycorticosterone (DOC) 0.02 (0.01–0.04) 0.02 (0.01–0.05) NS 1.04 (0.47–1.29) 0.03 (0.01–0.04) 0.02 (0.02–0.03) NS 0.99 (0.78–1.21) 18OHDOCCorticosterone (B)11-deoxycortisol (S) 3.68 (2.42–8.04) 0.30 (0.16–0.46) 0.10 (0.04–0.24) 3.32 (2.42–5.91) 0.27 (0.15–0.32) 0.07 (0.04–0.24) NS 0.01 0.89 (0.65–1.22) 0.88 (0.59–1.10) 3.55 (2.06–6.13) 0.09 (0.03–0.24) 2.72 (1.91–4.73) 0.08 (0.02–0.22) NS 0.80 (0.55–1.20) Cortisol (F) 194.6 (135.0–256.9) 160.6 (114.6–210.6) Table 6 Corticosteroid 18 OHBLow salt, 100a mmol/day; high 0.06 salt, (0.02–0.10) 200 mmol/day ( 0.08 (0.04–0.15) 0.006 1.46 (0.80–2.27) 0.04 (0.1–0.07) 0.06 (0.02–0.11) Aldosterone 0.13 (0.06–0.23) 0.33 (0.21–0.44) The highly signiﬁcant changes in DOC:aldosterone and

Table 7 Corticosteroid ratios (log10-transformed) and associated enzyme activity before and after i.v. infusion of AngII separated according to dietary salt consumption (nZ60). Data compared by Student’s t-test

Low salt High salt

Mean (GS.D.) Mean (GS.D.)

Log ratio Enzyme action Basal Post AngIIP Basal Post AngII P

S:F 11b-hydroxylase K2.82 (0.33) K2.85 (0.38) NS K2.78 (0.34) K2.86 (0.40) 0.02 DOC:B 11b-hydroxylase/ K2.30 (0.54) K2.31 (0.60) NS K2.15 (0.53) K2.07 (0.56) NS aldosterone synthase 18OHB:Aldo Aldosterone synthase K0.37 (0.37) K0.56 (0.31) 0.001 K0.28 (0.49) K0.51 (0.30) 0.004 B:F 17a-hydroxylase K1.63 (0.21) K1.57 (0.30) 0.05 K1.69 (0.24) K1.69 (0.28) NS DOC:18OHDOC 11b-hydroxylase K1.57 (1.15) K1.44 (1.27) NS K0.56 (0.56) K0.59 (0.60) NS

B:aldosterone may be misleading, as the concentrations of the lack of a placebo infusion arm makes this theory difficult neither DOC nor B changed across AngII treatment. Taken to disprove and is an acknowledged weakness of this study. together, these data indicate that none of these three Whatever the influence of AngII on glucocorticoid ratios is an unambiguous index of aldosterone synthase production, our data demonstrate the advantage of the use efficiency; aldosterone concentration itself, possibly under of LC–MS technology and human volunteers to measure mild salt restriction, is to be preferred. plasma corticosteroids and explore the influence of variation in hypothalamic–pituitary–adrenal axis activity Effect of agonists on corticosteroid production in order to demonstrate short-term, but immediate, alterations in plasma concentrations. The principal agonists of adrenal corticosteroid A potential weakness of our study is the possibility production (ACTH, AngII and sodium) demonstrated of poor adherence of study subjects to dietary sodium predictable effects; ACTH increased production of all manipulation, particularly among a population in whom steroids (Table 4) and salt restriction increased mineralo- there is often a high baseline dietary salt intake. Although corticoid levels (Table 2). As expected, AngII infusion there was a clear difference in mean urinary sodium stimulated aldosterone and its precursors, but gluco- excretion according to dietary intake, the relatively large corticoid production fell significantly (Table 6). The effect Journal of Endocrinology S.D. in each case introduces the potential for ‘overlap’ of AngII on glucocorticoid production is controversial and in sodium status between groups and obfuscation of these results appear to be at odds with in vitro and animal results. To address this, we reanalysed to include only data. For example, previous studies have shown that AngII patients with extremes of sodium excretion (!100 and stimulates cortisol production from bovine adrenal cells O150 mmol/24 h) and were reassured to find no major and that this effect is mediated through a phospholipase changes in levels of plasma steroids or precursor:product C-coupled angiotensin type 1 (AT1) receptor (Rabano et al. ratios but a considerable reduction in subject numbers 2004). In addition, earlier cell-based studies demonstrated (22 per group). Furthermore, alteration in dietary salt that AngII could both stimulate and suppress B production produced the appropriate alteration in plasma renin con- from adrenal cells depending upon the origin of such centration (which was unaltered when limited to the cells; the stimulatory effect was observed in ‘fasciculata-rich’ smaller cohort) as well as plasma mineralocorticoid levels. cells and not in total adrenal cells (Spinedi et al.1989). In summary, the results from this study illustrate that However, a previous small study of human volunteers infused with AngII demonstrated temporary ACTH S:F and B:F remain the indices of choice for monitoring b a suppression, which could be in keeping with our results the efficiencies of 11 -hydroxylase and 17 -hydroxylase (Semple et al.1979). However, in the latter study, ACTH respectively, and their usefulness is enhanced by low-dose levels eventually recovered and thus we might have ACTH stimulation and salt repletion. Aldosterone synthase, observed a rise in glucocorticoid levels in our subjects if because of its complex triple reaction sequence and we had extended the sampling time after cessation of confinement to a single zone, is best monitored by aldo- infusion. This is worthy of further investigation. However, sterone concentration alone, if possible with mild AngII we acknowledge that the apparent ‘suppressive’ effect of stimulation/salt restriction. These data, from a large group of AngII could simply reflect an initial stress response of healthy human volunteers, should provide robust baseline glucocorticoids to cannulation, which settled with time; information to help inform subsequent studies exploring

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Received in ﬁnal form 16 November 2014 Accepted 19 November 2014 Accepted Preprint published online 20 November 2014