The effects of normal aging on cortisol and adrenocorticotropin responses to hypertonic saline infusion

Psychoneuroendocrinology,Vol. 20,

NO. 6, pp. W-644,

1995

Elsevier Science Ltd

Pergamon

Printed in the USA

0306-4530(95)00004-6

THE EFFECTS OF NORMAL AGING ON CORTISOL AND ADRENOCORTICOTROPIN RESPONSES TO HYPERTONIC SALINE INFUSION Murray A. Raskind’, Elaine R. Peskind’, Marcella Pascualy’, Steven D. Edland2, Dorcas J. Dobier, Sharon Murray’, Carl Sikkema’, and Charles W. Wilkinson’ ‘Geriatric Research, Education, and Clinical Center, Seattle and American Lake Veterans Affairs Medical Centers; Alzheimer’s Disease Research Center; and Departments of Psychiatry and Behavioral Sciences, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195, USA and *Department of Biostatistics and Environmental Health, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195, USA (Received

27 September

1994; in final form 18 Januav

1995)

SUMMARY To assess the effects of aging on hypothalamic-pituitary-adrenal (HPA) axis responsivity, we compared the plasma cortisol and adrenocorticotropin (ACTS) responses to hypertonic saline infusion between normal older and young human volunteers. We administered a 90 min hypertonic saline infusion (5% sodium chloride at 0.06 mlikglmin) and a 90 min placebo infusion (0.9% sodium chloride at 0.06 ml/kg/min) to normal young subjects (n=13, age=2922 years) and normal older subjects (n=8, age=63 + 3 years). Plasma cortisol, ACTH, osmolality and arginine vasopressin (AVP) were measured before and at 30 min intervals during the infusions. The rate of increase in plasma osmolality and AVP induced by hypertonic saline infusion was similar between groups. The plasma cortisol increase during hypertonic saline infusion was greater in normal older subjects than in young subjects @=.03), but a stimulatory effect of hypertonic saline infusion on plasma ACTH was not apparent in either older or young subjects. These results suggest increased sensitivity with human aging to stimulation of cortisol release by hypertonic saline infusion at the adrenocortical level of the HPA axis. Keywords-Aging;

Cortisol; Adrenocorticotropin;

Vasopressin; Hyperosmolar.

INTRODUCTION An enhanced hypothalamic-pituitary-adrenal (HPA) axis response to environmental stimuli has been demonstrated in old rats (Sapolsky et al., 1986; Sapolsky et al., 1987, for review) and we have demonstrated enhanced cortisol and adrenocorticotropin (ACTH) responses to the cholinesterase inhibitor physostigmine in older normal humans (Raskind et al., 1990; Peskind et al., 1995). Because increased glucocorticoid concentrations appear to lower the threshold for hippocampal neuronal damage and loss in rats and primates (Landfield et al., 1981; Sapolsky, 1985; Sapolsky et al., 1990), enhanced HPA axis responsivity in older Address correspondence and reprint requests to: Dr. Murray A. Raskind, Psychiatry Service (116A), Seattle VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108, USA 637

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humans may be relevant to the hippocampal neuronal loss which occurs in human aging (Coleman & Flood, 1987) and which is a central neuropathologic feature of Alzheimer’s Disease (AD) (Ball et al., 1985). Hypertonic saline infusion has been reported to stimulate the HPA axis and increase cortisol and ACTH concentrations in normal young humans (Rittmaster et al., 1987), rats (Dohanics et al., 1991), and horses (Irvine et al., 1989). In the present study, we asked whether the cortisol and ACTH responses to hypertonic saline infusion are enhanced with normal human aging.

METHODS Subjects This study was approved by the Human Subjects Review Committee of the University of Washington and informed consent was obtained from all subjects. Subjects were 13 young normal men aged 21-44 years (29 2 2 years, mean + SE), and eight normal older adults (five men and three women), aged 52-70 years (63 + 3 years). Normal young and older subjects scored 29 or 30 on the Mini-Mental State Examination (MMSE) (Folstein et al., 1975) and had no history or other evidence of cognitive deterioration from their normal level of function. All subjects were in good general health and had no evidence of past or present renal disease, hepatic disease, thyroid disease or other diseases known to affect regulation of the HPA axis. All subjects were free of pain complaints and had no evidence of past or present major psychiatric or neurologic disorders. AR were nonsmokers and had been free of medication for at least 2 months. All subjects were normotensive (less than 135 mm Hg systolic and 90 mm Hg diastolic) and weighed within 125% of ideal body weight (1983 tables, Metropolitan Life Insurance Co.), Subject groups did not differ with respect to weight or percentage ideal body weight. Experimental Procedures Subjects underwent two studies separated by at least a 1 week interval. These studies differed only in that in the hypertonic saline condition, subjects received a 90 min infusion of hypertonic saline (5% sodium chloride at 0.06 ml/kg/min) and in the control condition, subjects received an equal volume 90 min infusion of normal saline (0.9% sodium chloride at 0.06 ml/kg/min). The order of the two studies was randomized and subjects were blind with respect to experimental condition. Studies were performed in a clinical research unit at the Seattle VA Medical Center between 0800h and 1100h. Subjects fasted from midnight prior to the study. Subjects assumed a supine position and an intravenous catheter was inserted into one antecubital vein for blood sampling; this was kept patent by a slow normal saline drip in both hypertonic saline and normal saline conditions. An intravenous catheter was inserted into the other antecubital vein and kept patent by a slow normal saline drip until the start of the infusion. Baseline blood samples for ACTH, cortisol, arginine vasopressin (AVP) and osmolality were obtained at 30 and 35 min following catheter insertion. Following the baseline sampling, infusion of hypertonic saline or normal saline began and continued for 90 min. Repeat blood samples for ACTH, cortisol, and osmolality were obtained at 30, 60, and 90 min following the start of infusion. Repeat AVP measurements were obtained at 90 min. Blood samples for ACTH and cortisol were collected in chilled glass tubes containing EDTA and aprotinin. Blood samples for AVP were collected in chilled glass tubes

Cortisol, Aging and Hypertonic Saline

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containing EDTA. Blood samples for osmolality were collected in plain glass tubes. Blood samples for ACTH, cortisol, and AVP were placed on ice immediately after collection and cold centrifuged within 1 h; plasma samples were stored at - 80°C until assay. Samples for serum osmolality were centrifuged and measured within 30 min of collection by freezing point depression.

Chemical Assays

Cortisol was measured by radioimmunoassay in unextracted plasma as previously described (Raskind et al., 1989). Samples were diluted with phosphate buffer and heated for 20 min at 80°C to denature binding globulins. Cortisol antiserum was obtained from ICN Biomedicals (Costa Mesa, CA, USA). Sensitivity of the assay was 0.1 &dl. Coefficient of variation within assays was 4% and between assays 12%. Plasma ACTH radioimmunoassay was performed with IgG ACTH-1-39 antisera (IgC Corporation, Nashville, TN, USA), 1251labeled ACTH (ICN Biomedicals, Costa Mesa, CA, USA) and human ACIH l-39 standards (Peninsula Laboratories, Belmont, CA, USA) as described previously (Radant et al., 1992). Samples were assayed in duplicate in a volume of 50 ~1. Standards were dissolved in 100 ~1 of stripped plasma. The stripped plasma is used as a diluent rather than buffer containing bovine serum albumin (BSA) because of nonspecific binding effects often attributed to the use of BSA in ACTH assay buffers. By using the stripped plasma to dilute standards and samples, parallel dilution curves are obtained with unknown plasma samples. The plasma is stripped by adding 0.3 g Dextran (70,800 MW; Sigma, St. Louis, MO, USA) and 1.2 g activated charcoal per 100 ml plasma, and mixing for 30 min at ambient temperature. The suspension is centrifuged at 20,000 g for 30 min and the supematant is collected. The addition of Dextran-coated charcoal, mixing, and centrifugation are repeated, and the supernatant is filtered with Whatman #5 filter paper and a 0.5 mm Millex-HV filter unit (Millipore Products Div., Bedford, MA, USA). The filtrate is aliquoted and 1 ml Triton X and 250 KIU aprotinin are added per ml. Other assay procedures are as recommended by IgG Corp. The detection limit for ACTH is 4 pg/ml. lntra- and interassay coefficients of variation are 11% and 16%, respectively.

Statistical Analyses

This experiment used a three factor design. There are two within subject factors, time (30, 60 and 90 min) and treatment (hypertonic saline and normal saline infusion), and one between subject factor, group membership (older or young). The primary objective was to test the null hypothesis of no difference in the cortisol and ACTH responses to hypertonic infusion between the two age groups. We tested for significance of this hypothesis by evaluating the group by treatment response interaction effects using a split-plot (each subject having received each treatment), repeated measures (responses to each treatment being measured at each 30 min interval during infusions) analysis of variance. We also evaluated treatment by time interactions within each group. If a significant within group treatment by time interaction was detected, significance of difference at each time point was evaluated by the Newman-Keuls test. Because baseline differences between older and young subjects were of interest, the significance of difference in baseline parameters (expressed as the mean of normal saline and hypertonic saline baselines) between groups was evaluated by unpaired t-test. All tests of significance were two-sided.

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RESULTS Cortisol and ACTH responses to hypertonic saline infusion and normal saline infusion are presented in Figs 1 and 2. Baseline cortisol did not differ between age groups. Of primary interest was that the cortisol response to hypertonic saline compared to normal saline was significantly greater in older subjects (group by treatment response interaction F(1,19)=5.66, p=.O3). Within group ANOVA revealed a significant treatment by time interaction for cortisol only in older subjects. Post-hoc f-tests demonstrated significant differences between hypertonic saline and normal saline in older subjects at 60 min @=.Ol) and 90 min @ < .Ol). In contrast, there was no significant difference in cortisol responses between hypertonic saline and normal saline within young subjects. Baseline ACTH did not differ between age groups. There was a significant group by treatment interaction for ACTH responses to hypertonic saline (F( 1,19)=14.45, p=.OOl), but the nature of the ACTH treatment responses was unexpected. In neither older nor young groups was a stimulatory effect of hypertonic saline on plasma ACTH apparent. Rather, in the young subjects, plasma ACTH was significantly lower in the hypertonic saline condition than in the normal saline condition at 90 min @ < .Ol). Because the older group contained three female subjects and a gender effect on cortisol responses in older persons has recently been reported (Greenspan et al., 1993), we inspected the data to determine if the enhanced cortisol responses in the older normals appeared to be

YOUNG

SUBJECTS

201

OLD 20

SUBJECTS

1

Fig. 1. Plasma cortisol response to 90 min hypertonic saline (5% NaCI) and normal saline (0.9% NaCI) infusions in eight older and 13 young subjects. *p-z .05, **p c .Ol compared to young subjects, twoway ANOVA with repeated measures followed by Newman-Keuls post-hoc tests.

YOUNG

50

SUBJECTS

1

lo-

YIWUTES

OLD 50

SUBJECTS

1

‘Oh

60

90

MINUTES

Fig. 2. Plasma adrenocorticotropic hormone (ACTH) response to 90 min hypertonic saline (5% NaCl) and normal saline (0.9% NaCl) infusions in eight older and 13 young subjects. ** p < .Ol compared to older subjects, two-way ANOVA with repeated measures followed by Newman-Keuls post-hoc test.

04 1

Cortisol, Aging and Hypertonic Saline

Table I. Serum osmolality, mean arterial pressure (MAP), and plasma arginine vasopressin (AVP) responses to hypertonic saline (HS) and normal saline (NS) in eight older and 13 young subjects 90 30 60 0 Osmolality

(mOsm/kg)

Older subjects HS* NS Young subjects HS* NS

29121 29121

296~1 291 r 1

30021 29121

304% 1 291% 1

28720 28821

29121 29921

29421 28821

297+1 28X+ I

9325 9222

9526 9425

98?6 9826

10327 99:s

9022 9022

9123 8922

9223 9123

9422 9152

MAP Older subjects HS* NS Young subjects HS* NS AVP Older subjects HS* NS Young subjects HS* NS

1.212 0.27 1.33 -c 0.37

2.35 + 0.58 1.34 + 0.38

2.9 + 0.75 1.63 2 0.32

4.5 t 1.28 1.4KzO.31

1.22 + 0.24 1.13 20.12

1.88 + 0.33 1.05 20.16

2.07 f 0.28 1.01+0.35

2.9 r 0.43 1.09 t 0.26

*Significant increase over time, p < .05.

limited to the female subjects. Cortisol response patterns were very similar and did not differ statistically between the three older female and the five older male subjects. Osmolality, AVP and MAP increased during hypertonic saline infusion in both older and young subjects (Table I). Baseline osmolality was slightly, but significantly, greater in older than in young subjects (p < .Ol). However, the plasma osmolality response to treatment did not differ between groups (F(1,19)=0.27,~=.61). Neither baseline AVP nor AVP response to treatment differed between groups. Similarly, neither baseline MAP nor the MAP response to treatment differed between groups.

DISCUSSION This study demonstrated enhanced responsivity of plasma cortisol to hypertonic saline infusion with human aging. This aging effect is consistent with our previous findings of enhanced cortisol responsivity to physostigmine in older normal humans (Raskind et al., 1990; Peskind et al., 1995). However, in these physostigmine challenge studies, we demonstrated enhanced responses of both ACTH and cortisol in older subjects, suggesting an aging effect on HPA axis responsivity at or above the pituitary level. In the current study, we could not detect a stimulatory effect of hypertonic saline on plasma ACTH

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concentrations in either young or older subjects. In fact, the only effect of hypertonic saline on ACTH was a modest suppression in the young subjects. The observed cortisol response in the absence of an ACI’H response in the older subjects suggests that hypertonic saline infusion had a stimulatory effect on cortisol release in the older subjects at the level of the adrenal cortex. A potential mechanism mediating a direct adrenocortical level cortisol response is the release of AVP during hypertonic saline infusion. AVP has a stimulatory effect on cortisol production in bovine (Bird et al., 1990) dog (Brooks & Blakemore, 1989) and rat (Hinson et al., 1987) adrenal glands. A robust stimulation by AVP of cortisol secretion from human adrenocortical tissue in vitro has also been reported (Perraudin et al., 1993). It is possible that human aging enhances adrenocortical sensitivity to vasopressin. It is also possible that the apparent lack of an aging effect on ACTH concentrations is due to an aging effect on the ratio of biological to immunological activity of ACTH. While the effect of age on post-translational processing of ACTH peptides is unknown, the post-translational processing of other anterior pituitary peptides has been reported to be altered by aging (Conn et al., 1980; Klug & Adelman, 1977). The present results differ from those of Rittmaster et al. (1987) who reported that a hypertonic saline infusion similar to the one administered in this study increased both plasma ACTH and cortisol levels in normal young subjects. The reasons for our failure to replicate their findings may be attributable to their, and our, studies having been performed during different phases of the normal HPA axis circadian rhythm. Their subjects were studied in the evening, a period of HPA axis quiescence. Our subjects were studied in the midmorning, a period of high HPA axis activity. Higher basal cortisol concentrations in the morning than the evening may have produced greater glucocorticoid feedback inhibition of ACTH release (Keller-Wood & Dallman, 1984) than in the Rittmaster et al. study, thereby obscuring any stimulatory effect of hypertonic saline infusion. Because ACTH is released into the blood in a pulsatile manner (Wilkinson, 1989) it is also possible that our 30 min sampling intervals were insensitive to transient ACTH increases. It must also be kept in mind that hypertonic saline infusion is a complex intervention which could both stimulate the HPA axis by increasing osmolality and suppress the HPA axis by increasing intravascular volume (Raff, 1988). It is therefore possible that suppression of ACTH release by hypertonic-saline-induced intravascular volume expansion counteracted any stimulation of ACTH release by increased osmolality. Higher sensitivity to volume-induced suppression of ACTH release in young than older subjects could explain the observed suppression of ACTH by hypertonic saline in the young subjects only. The results of the current study must be interpreted cautiously because of the possibility that the osmolar stimulus may not have been completely comparable between groups. Although the amount of hypertonic saline administered and the increase in plasma osmolality during hypertonic saline infusion did not differ between groups, older subjects achieved higher absolute osmolalities during hypertonic saline infusion than did young subjects. It is possible that reaching an absolute osmolar threshold is necessary before cortisol release is stimulated by increased osmolality and that the young subjects failed to reach this osmotic threshold. This latter possibility is made less likely by the fact that the osmotic threshold for AVP release was clearly achieved in the young subjects. The higher baseline plasma osmolalities in the older subjects may be attributable to an age-associated decline in renal concentrating ability (Rowe et al., 1976). Healthy older men have greater increases in plasma osmolality following water deprivation than do young men (Phillips et al., 1984). Because our subjects were water deprived from bedtime of the

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evening prior to the study, their higher plasma osmolalities are likely to be attributable to decreased renal concentrating ability compared to the young subjects. In summary, we demonstrated enhanced responsivity of cortisol but not ACTH to hypertonic saline infusion in older humans. We previously demonstrated enhanced HPA axis responsivity at or about the level of the pituitary with aging following enhancement of central cholinergic activity by physostigmine (Raskind et al., 1990; Peskind et al., 190.5). The current results are compatible with an adrenocortical mechanism for the agingassociated enhancement of the plasma cortisol response to an osmolar stimulus. These data raise the possibility that human aging increases HPA axis responsivity by multiple mechanisms. Acknowledgements: The authors wish to acknowledge Stephen A. Gait, Kristen Kling, Gail Gumhrecht, Monique Cherrier and Sally Swedine for their excellent technical assistance and Susan Martin for manuscript preparation. The authors also wish to thank Sandra Linauts and the laboratory staff of the Tacoma-Pierce County Blood Bank for providing out-of-date blood for use in the ACTH assays. This investigation was supported by the Department of Veterans Affairs and NIH Grants AG-8419 and AG-5136.

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