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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 4972-4980
Copyright © 2004 by The Endocrine Society

Alterations in Cortisol Secretory Dynamics in Adolescent Girls with Anorexia Nervosa and Effects on Bone Metabolism

Madhusmita Misra, Karen K. Miller, Cecilia Almazan, Kavitha Ramaswamy, Wannasiri Lapcharoensap, Megan Worley, Gregory Neubauer, David B. Herzog and Anne Klibanski

Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School (M.M., K.K.M., C.A., K.R., W.L., M.W., A.K.); Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School (M.M.); and Core Laboratory, GCRC (G.N.), and Eating Disorders Unit (D.B.H.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Anne Klibanski, BUL 457B, Neuroendocrine Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Anorexia nervosa (AN) is associated with low bone density in adolescents and adults. Hypercortisolemia has been reported in adults with this disorder and has been hypothesized to be a factor in bone loss. However, the secretory dynamics of cortisol in adolescents with AN and the contribution of alterations in cortisol secretion to bone metabolism in AN have not been examined. We examined the dynamics of cortisol secretion by Cluster and deconvolutional analysis in 23 girls with AN and 21 healthy adolescents of comparable age and maturity. Cortisol sampling was performed every 30 min for 12 h overnight. Twenty-four-hour urinary free cortisol (UFC) and creatinine (cr) were obtained for all subjects. The surface area (SA) of the subjects was calculated. Markers of bone turnover (type 1 procollagen, osteocalcin, and N-telopeptide) were examined. Subjects with AN were prospectively followed over 1 yr, and those who recovered weight (defined as a 10% increase in body mass index) were again studied. On Cluster analysis, girls with AN had significantly higher mean cortisol (8.6 ± 2.0 vs. 5.9 ± 1.1 µg/dl; P < 0.0001), nadir cortisol (5.5 ± 2.3 vs. 3.4 ± 1.2 µg/dl; P = 0.0008), valley mean cortisol (7.0 ± 2.7 vs. 4.7 ± 1.5 µg/dl; P = 0.001), peak amplitude (12.6 ± 4.4 vs. 7.8 ± 3.0 µg/dl; P = 0.0004), peak area (652 ± 501 vs. 340 ± 238 µg/dl; P = 0.02), and total area under the curve (6112 ± 1467 vs. 4117 ± 802 µg/dl; P < 0.0001) than healthy adolescents. On deconvolutional analysis, the frequency of nocturnal secretory bursts (7.0 ± 1.2 vs. 5.8 ± 1.3 /12 h; P = 0.001), total nocturnal pulsatile cortisol secretion (69.3 ± 14.7 vs. 53.9 ± 11.1 µg/dl; P = 0.0003), and total cortisol secretion (89.6 ± 18.8 vs. 71.2 ± 17.6 µg/dl; P = 0.002) were significantly higher in girls with AN than in healthy controls. Cortisol half-life trended higher in girls with AN. However, basal cortisol secretion and approximate entropy did not differ between the groups. UFC/cr and UFC/cr.SA were significantly higher in girls with AN than in controls [0.050 ± 0.028 vs. 0.036 ± 0.017 (P = 0.04) and 0.035 ± 0.020 vs. 0.023 ± 0.012 (P = 0.03)]. Six of 23 girls with AN had UFC/cr.SA values that were more than 2 SD above those in healthy controls. An inverse correlation was noted between measures of cortisol concentration as well as pulsatile secretion and measures of nutritional status (body mass index, fat mass, leptin, insulin, and IGF-I). An oral glucose load suppressed cortisol levels in healthy adolescents, but not in AN patients. Weight recovery was associated with a significant decrease in the number of secretory bursts. In girls with AN, strong inverse correlations were noted between levels of cortisol (mean, nadir, and total area under the curve) and levels of markers of bone formation (C-terminal propeptide of type 1 procollagen and osteocalcin). Conversely, in healthy controls, cortisol values did not predict levels of markers of bone turnover. Adolescent girls with AN have significantly higher serum cortisol concentrations and UFC/cr.SA values than healthy adolescents. This increased cortisol concentration is a function of increased frequency of secretory bursts, resulting in increased pulsatile secretion. Hypercortisolemia appears to be a direct consequence of undernutrition and is associated with a decrease in markers of bone formation. Therefore, high cortisol values in AN may contribute to the low bone density observed in adolescents with this disorder by decreasing bone formation.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
WE HAVE PREVIOUSLY demonstrated that adolescent girls with anorexia nervosa (AN) have low bone density associated with a low bone turnover state (1). Although hypogonadotropic hypogonadism occurs in AN, estradiol values do not predict bone density in this disorder, and oral estrogen replacement has not proven effective in improving bone density in adults with AN (2). Other hormones that may affect bone density include GH and cortisol. GH is an endogenous anabolic trophic hormone for bone (3), and in a recent report we described a nutritionally acquired resistance to GH in girls with AN (4). We showed that GH levels predicted markers of bone turnover in healthy adolescents, but not in AN, suggesting a role for GH resistance in low bone density in this condition.

Hypercortisolemia is deleterious to bone (5, 6, 7, 8, 9) and has been described in adult women with AN (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Cortisol excess results in low bone density in conditions of endogenous hypercortisolism, such as Cushing’s syndrome (5, 6, 8), and also in conditions associated with exogenous administration of glucocorticoids (7, 9). However, urinary free cortisol (UFC) values have been reported to be normal in adolescent girls with AN, with no difference compared with healthy adolescents (1, 24, 25). We recently demonstrated that although UFC values were not elevated in girls with AN, UFC values corrected for creatinine (UFC/cr), and for creatinine and surface area (UFC/cr.SA), were significantly higher in this group compared with healthy adolescents (4). This correction was based on a report by Legro et al. (26), which suggested that correcting for cr and SA may be necessary when assessing cortisol values in adolescents. UFC values increase with increasing age through the pubertal years, and standardizing cortisol values for creatinine and surface area results in a fairly constant value in the 12–17 yr age group. Cortisol secretory patterns have not been reported in healthy adolescents or in AN, and the effects of maturity on cortisol secretion are unclear. Given the important role of pubertal maturation on bone density (27), this is important to determine.

We therefore investigated cortisol secretory patterns in adolescent girls with AN compared with a group of healthy adolescents using Cluster and deconvolutional analyses, and the effects of alterations in these patterns on bone turnover. In addition, effects of pubertal maturity on cortisol secretory patterns were examined in both AN and healthy adolescents.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subject selection

We enrolled 44 Caucasian subjects, 23 girls with AN (diagnosed by DSM-IV criteria), 12.2–18.8 yr old [chronological age (CA), 16.2 ± 1.6 yr; bone age (BA), 15.8 ± 1.5 yr], and 21 healthy girls of comparable CA (15.4 ± 1.8 yr) and maturity (BA, 15.7 ± 2.1 yr). Clinical characteristics (including body composition and bone turnover markers), data regarding some hormonal parameters (IGF-I, estradiol, leptin, and urinary free cortisol), and methods of recruitment have been previously reported (4). Five girls with AN and four controls were premenarchal. All other girls with AN had been amenorrheic for at least 3 months at study initiation. No healthy control had a present or past history of an eating disorder. Informed assent and consent were obtained for all subjects, and the institutional review board of Partners Health Care approved the study.

Experimental protocol

Eligibility was determined at a screening visit, which included a history, physical examination, and blood draw to rule out hyperthyroidism, hypergonadotropic hypogonadism, and hyperprolactinemia. Subjects were admitted overnight to the General Clinical Research Center (GCRC) of the Massachusetts General Hospital. A 24-h urine collection was completed on the day of the study visit for UFC and cr, and BA was determined. Body composition was determined by dual energy x-ray absorptiometry and has been reported (4). Subjects had dinner before 1930 h and then had an iv catheter placed for frequent sampling for cortisol, every 30 min, overnight between 2000 h on the night of admission and 0800 h the next morning. Fasting blood samples were drawn for measurements of free and total T4, total T3, IGF-I, leptin, bone formation markers [C-terminal propeptide of type 1 procollagen (PICP) and osteocalcin (OC)]. All healthy girls and 18 girls with AN had cortisol values measured before, and 30 and 60 min after administration of a 100-g oral glucose load. A second morning 2-h urine sample was collected for N-telopeptide (NTX), a bone resorption marker, and creatinine. Subjects were examined at 3, 6, and 12 months after the baseline visit, and girls with AN who recovered weight (i.e. recovered 10% of their baseline body mass index (BMI) were studied again with frequent sampling (n = 10).

Anthropometric measurements

A single stadiometer at the GCRC was used for measurements of height, which were obtained in triplicate and averaged. Weight was measured on an electronic scale. BMI was determined as the ratio of weight in kilograms to height in centimeters squared. SA was calculated using the following formula (square root of [(weight in kg x height in cm)/3600]). The standards of Greulich and Pyle were used to determine BA from an x-ray of the wrist and hand (28).

Biochemical assessment

UFC over 24 h was measured by the hospital laboratory using the GammaCoat 125I RIA (Diasorin, Inc., Stillwater, MN; detection limit, 1 µg/dl; coefficient of variation, 7%) and has been previously reported (4). UFC was standardized for cr (UFC/cr) by dividing the UFC by cr excretion over the 24-h period and for cr and SA (UFC/cr.SA), based on the recommendations of Legro et al. (26). Glucose levels were measured by the hospital laboratory using previously described methods (29). We measured serum cortisol with an RIA (Diagnostic Products Corp., Los Angeles, CA; limit of detection, 1 µg/dl; sensitivity, 0.21 µg/dl; coefficient of variation, 2.5–4.1%). Although this assay measures steroid metabolites other than cortisol, it remains the standard assay for this steroid hormone. Samples were sent to Quest Diagnostics (Norwich, NY) for analysis of free T4 by equilibrium dialysis and for determination of total T4 levels by standard methods. Total T3 was measured by RIA (Diasorin, Inc.; sensitivity, 9.0 ng/dl; coefficient of variation, 3.1–7.9%).

RIA was used to determine levels of serum leptin (Linco Diagnostics, Inc., St. Charles, MO; sensitivity, 0.5 µg/liter; coefficient of variation, 3.4–8.3%), insulin (Linco Research, Inc.; sensitivity, 2 µU/ml; coefficient of variation, 2.2–4.4%), estradiol (ultrasensitive assay; Diagnostic Systems Laboratories, Inc., Webster, TX; detection limit, 2.2 pg/ml; coefficient of variation, 6.5–8.9%), and PICP (Diasorin, Inc.; limit of detection, 25 ng/ml; coefficient of variation, 1.3–3.8%). We used an immunoradiometric assay to determine levels of OC (Nichols Institute Diagnostics, Inc., San Juan Capistrano, CA; sensitivity, 0.5 ng/ml; coefficient of variation, 3.2–5.2%) and an ELISA to measure NTX (Ostex International, Inc., Seattle, WA; detection limit, 20 nmol bone collagen equivalent; coefficient of variation, 5–19%).

Analysis of cortisol secretion and concentration

Cluster analysis (using a 1 x 2 Cluster configuration; one sample in the test nadir and two in the test peak) of the data obtained from frequent sampling overnight was used to determine cortisol concentration parameters [mean, nadir, number of concentration peaks, peak width, peak amplitude, peak area, number of valleys, valley mean cortisol, and total area under the curve (AUC) for cortisol] (30). We then performed deconvolutional analysis (31) on these data using previously published methods to obtain measures of cortisol half-life, basal cortisol secretion (basal secretion rate x duration of sampling), number of secretory bursts over the sampling period, interval between secretory bursts, burst amplitude, burst mass (area under bursts), pulsatile secretion (burst frequency over 12 h x mean burst mass), and total secretion (basal + pulsatile secretion). Approximate entropy, a measure of the degree of disorderliness of cortisol secretion, was determined (32, 33).

Body composition

Lean body mass and fat mass were determined using dual energy x-ray absorptiometry (QDR 4500, Hologic, Inc., Waltham, MA) (34, 35).

Statistical methods

All data are presented as the mean ± SD. All data were analyzed using the JMP program (version 4, SAS Institute, Inc., Cary, NC). The t test was used to calculate differences between means. Where data were not normally distributed, nonparametric tests (Wilcoxon rank sum) were performed. Univariate and multiple regression analyses were used to determine predictors of bone turnover markers and of cortisol concentration and secretion.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical characteristics

A number of clinical characteristics for 20 controls and 22 AN have been previously reported (4), and data for this group are summarized along with newer findings in Table 1Go. Subjects with AN had significantly lower weights, BMIs, and percent fat mass than controls. Levels of PICP trended lower in AN. Levels of fasting insulin and leptin were markedly lower in AN. Girls with AN had lower levels of estradiol than healthy controls measured in the early follicular phase of their cycles. Total T4 and T3 values were lower in AN vs. controls, whereas free T4 values trended lower.


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  		TABLE 1. Clinical characteristics of adolescent girls with AN and healthy adolescents

 
Cortisol values

Urinary cortisol. UFC/cr (0.050 ± 0.028 vs. 0.036 ± 0.017 x 10–3; P = 0.04) and UFC/cr.SA (0.035 ± 0.020 vs. 0.023 ± 0.012 x 10–3/m2; P = 0.03) were significantly higher in girls with AN compared with controls. Twenty-six percent (six of 23) girls with AN had UFC/cr.SA values that were more than 2 SD above the mean for controls. Uncorrected UFC values did not differ between the groups (31.5 ± 11.1 µg/d in controls vs. 38.0 ± 16.9 µg/d in AN; P not significant).

Cluster analysis of cortisol concentration. Table 2Go describes data derived from Cluster analysis of the cortisol concentrations. Girls with AN had significantly higher mean cortisol, nadir of cortisol concentration, valley mean cortisol concentration, amplitude of and area under concentration peaks, and total AUC than healthy controls. Figure 1AGo demonstrates Cluster data from two girls with AN and two controls. Fifty-seven percent (13 of 23) girls with AN had mean cortisol values that were more than 2 SD above the mean for controls.


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  		TABLE 2. Cluster and deconvolutional analysis of cortisol in adolescent girls with AN and healthy adolescents

 


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  		FIG. 1. Cluster analysis of cortisol concentration and deconvolutional analysis of cortisol secretion AN and healthy adolescents. A, Cluster analysis in two girls with AN (two left panels) and two healthy adolescent girls (two right panels). Mean, nadir, valley mean, peak mass, peak amplitude of cortisol concentration, and total AUC were greater in girls with AN than in controls. B, Deconvolutional analysis in the two girls with AN and the two healthy controls analyzed by Cluster in A. The upper panels show cortisol concentrations over the sampling period, whereas the lower panels show the individual secretory bursts. Girls with AN had a greater number of secretory episodes than healthy adolescents of comparable CA and BA and higher pulsatile and total cortisol secretion.

 
Deconvolutional analysis of cortisol secretion. Data derived from deconvolutional analysis of cortisol secretion are shown in Table 2Go. The number of secretory bursts over the 12-h sampling period was higher in the group with AN, resulting in significantly higher pulsatile and total cortisol secretion in this group. The half-life of cortisol trended higher in the group with AN. Basal cortisol secretion, burst mass, and approximate entropy did not differ between the groups. Figure 1BGo shows deconvolutional analysis of cortisol secretion in two girls with AN and two controls.

Predictors of cortisol concentration and secretion

Nutritional status. Inverse correlations were observed between measures of cortisol concentration (mean, nadir, valley mean, and total AUC) and markers of nutritional status (BMI, percent fat mass, leptin, insulin, and IGF-I) in the combined group of girls with AN and controls (Table 3Go). These markers of nutritional status predicted pulsatile and total secretion and also levels of UFC/cr.SA, but not basal cortisol secretion. In addition, the number of nocturnal secretory bursts correlated inversely with these measures of nutritional status (r = –0.37, P = 0.01 with % body fat; r = –0.45, P = 0.004 with glucose; r = –0.46, P = 0.003 with insulin; r = –0.41, P = 0.006 with IGF-I).


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  		TABLE 3. Relationship between cortisol concentration and secretion parameters and markers of nutritional status

 
On stepwise regression when BMI and percent body fat were entered into the model, percent body fat emerged as the sole predictor of all measures of cortisol concentration and secretion, contributing to 32%, 33%, 21%, and 19% of the variability of cortisol total AUC, mean cortisol, nadir cortisol, and valley mean, respectively, and 16% of the variability of both pulsatile and total cortisol secretion. On further stepwise regression when percent body fat, glucose, insulin, and IGF-I were entered into the model, insulin, and percent body fat predicted cortisol AUC and mean cortisol, contributing to 38% and 8% of the variability of cortisol AUC, and 37% and 8% of the variability of mean cortisol. Insulin was the sole predictor of nadir cortisol, valley mean, and cortisol half-life, contributing to 23%, 28%, and 16% of the variability, respectively. Percent body fat was the sole predictor of pulsatile secretion, total secretion, and UFC/cr.SA, contributing to 17%, 18%, and 11% of the variability of these measures. On stepwise regression, glucose was the sole significant predictor of the number of secretory bursts, contributing to 20% of its variability.

Thyroid hormones. Total T4 did not predict any measure of cortisol secretion or concentration. Free T4 predicted the frequency of secretory bursts (r = –0.35; P = 0.02), but not half-life or any other measure of secretion or concentration. Conversely, strong inverse correlations were observed between total T3 and measures of cortisol concentration (r = –0.52. P = 0.0003 for cortisol AUC; r = –0.53, P = 0.0002 for mean cortisol; r = –0.43, P = 0.005 for valley mean cortisol; r = –0.44, P = 0.004 for nadir cortisol). Total T3 also correlated inversely with the frequency of secretory bursts (r = –0.37; P = 0.01), total pulsatile secretion (r = –0.41; P = 0.006), and total secretion (r = –0.41; P = 0.006). However, total T3 did not correlate with cortisol half-life, and on stepwise regression analysis when measures of nutritional status were entered into the model with total T3, total T3 did not emerge as an independent predictor of cortisol concentration or secretory bursts. Inverse correlations were observed between total T3 and measures of nutritional status. including BMI (r = –0.32; P = 0.03), percent body fat (r = –0.52; P = 0.0003), fasting glucose (r = –0.41; P = 0.009), fasting insulin (r = –0.45; P = 0.004), leptin (r = –0.48; P = 0.001), and IGF-I (r = –0.60; P < 0.0001).

Changes in cortisol concentration after an oral glucose load. Girls with AN had higher cortisol concentration at 0 min of the 100-g oral glucose load than controls, but this did not reach statistical significance (20.5 ± 7.8 vs. 17.9 ± 5.2 µg/dl; P not significant). Values of cortisol were higher in girls with AN than in controls at 30 min (19.7 ± 7.8 vs. 15.3 ± 4.9 µg/dl; P = 0.04) and at 60 min after oral glucose (18.6 ± 7.6 vs. 13.2 ± 4.7 µg/dl; P = 0.01; Fig. 2Go). Nadir cortisol was significantly higher in AN than in healthy adolescent girls (16.6 ± 6.5 vs. 12.6 ± 4.4 µg/dl; P = 0.03). The mean decrease in cortisol during the oral glucose tolerance test (OGTT) (baseline-nadir values) was not different in the two groups (3.8 ± 4.5 µg/dl in AN vs. 5.3 vs. 3.5 µg/dl in controls; P not significant). The percent decrease in cortisol, however, was lower in girls with AN than in controls (16.6 ± 16.6% vs. 28.7 ± 18.0%; P = 0.04). Cortisol levels suppressed adequately with oral glucose in healthy controls, with nadir cortisol values being significantly lower than 0 min cortisol levels (12.6 ± 4.4 vs. 17.9 ± 5.2 µg/dl; P = 0.001). However, in AN girls, cortisol values did not suppress after oral glucose, as suggested by only a minimal decrease in nadir cortisol values from baseline 0 min values (16.6 ± 6.5 vs. 20.5 ± 7.8 µg/dl; P = 0.1).



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  		FIG. 2. Cortisol, glucose, and insulin levels at baseline (0 min) and 30 and 60 min after a 100-g oral glucose load in girls with AN (gray lines) and healthy adolescents of comparable maturity (black lines). Cortisol values were suppressed after oral glucose in healthy adolescents, but not in girls with AN, and were significantly higher in girls with AN than in healthy adolescents at 30 and 60 min. Glucose levels were significantly lower in AN than in controls at 0 min, but did not differ from controls at 30 and 60 min. Insulin levels were significantly lower in AN at 0 and 30 min, but did not differ from controls at 60 min. *, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001.

 
Nadir cortisol values during the OGTT correlated positively with various measures of cortisol concentration (Cluster analysis), such as total area under the curve (r = 0.36; P = 0.02), mean cortisol concentration overnight (r = 0.38; P = 0.02), and nadir cortisol concentration overnight (r = 0.36; P = 0.03), and also with measures of cortisol secretion (by deconvolutional analysis), such as cortisol half life (r = 0.54; P = 0.0004) and frequency of secretory bursts (r = 0.42; P = 0.008), such that girls with the highest overnight cortisol secretion and concentration had the highest nadir cortisol after the glucose load.

Effects of weight recovery. Ten girls with AN recovered weight (defined as a 10% increase in BMI) over the study period and underwent frequent sampling again at weight recovery. Paired analysis of these 10 girls with AN demonstrated lower cortisol concentration and secretion measures at weight recovery, but the differences from baseline were statistically significant only for the frequency of secretory bursts (7.1 ± 1.2 at baseline vs. 6.2 ± 0.6 after weight recovery; P = 0.02). Small nonsignificant decreases were noted in cortisol at different time points of the OGTT after weight recovery (data not reported). Eight of the 23 girls with AN recovered weight and also resumed menses over the study period. Similar results were noted on paired analysis of these eight girls at weight and menses recovery vs. at baseline, with a significant decrease in the frequency of secretory bursts (7.1 ± 0.8 vs. 6.0 ± 0.0; P = 0.007) and a nonsignificant decrease noted in other measures of pulsatile and total cortisol secretion.

Relationship between bone metabolism and measures of cortisol concentration. Table 4Go demonstrates the relationship between markers of cortisol concentration and bone turnover in girls with AN and controls. Strong inverse correlations were observed between PICP (a marker of bone formation) and mean cortisol, nadir cortisol, valley mean cortisol, and total AUC for cortisol in girls with AN. Thus, high cortisol concentration in AN was associated with a decrease in PICP. Conversely, the cortisol concentration did not correlate with PICP in controls. Similarly, inverse correlations were noted between two measures of cortisol concentration (mean and total AUC) and OC (another marker of bone formation) in AN, but not in controls. Cortisol concentration correlated weakly and inversely with NTX (a marker of bone resorption) in AN and controls. In addition, in girls with AN, cortisol half-life correlated negatively with PICP (r = –0.53; P = 0.01), OC (r = –0.50; P = 0.02), and NTX (r = –0.67; P = 0.0005). No relationship was observed between cortisol half-life and bone turnover markers in healthy controls.


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  		TABLE 4. Relationship between bone markers and cortisol concentration parameters

 
Cortisol secretory parameters by maturity. CA and BA correlated positively with half life (r = 0.41; P = 0.006, and r = 0.46; P = 0.002) and frequency of secretory bursts (r = 0.35; P = 0.02, and r = 0.37; P = 0.01), and inversely with mean area of secretory bursts (r = –0.42; P = 0.004, and r = –0.53; P = 0.0002) and mean burst amplitude (r = –0.43, P = 0.004, and r = –0.54; P = 0.002).

Relationship between UFC and measures of cortisol concentration and secretion. Both UFC/cr and UFC/cr.SA correlated positively with mean cortisol concentration (r = 0.44; P = 0.003, and r = 0.45; P = 0.002), total AUC for cortisol (r = 0.45; P = 0.002 for both), nadir cortisol (r = 0.36 and r = 0.37; P = 0.02), and valley mean cortisol concentration (r = 0.38 and r = 0.39; P = 0.01). UFC/cr and UFC/cr.SA also correlated with pulsatile (r = 0.43; P = 0.004, and r = 0.44; P = 0.003) and total cortisol secretion (r = 0.38; P = 0.02, and r = 0.41; P = 0.006), but not with basal cortisol secretion. Conversely, no correlation was observed between UFC and measures of cortisol concentration or secretion.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We report higher serum cortisol levels in girls with AN compared with healthy adolescent girls of comparable maturity and demonstrate that this increase is a consequence of an increased frequency of secretory bursts. Cortisol levels are predicted by markers of nutritional status and by glucose and insulin levels. Moreover, an oral glucose load fails to suppress cortisol levels in girls with AN, whereas suppression does occur in controls. High cortisol levels in girls with AN correlate inversely with markers of bone turnover and may contribute to low bone mineral density (BMD) in this condition by suppressing bone formation.

Although elevations in plasma and urinary cortisol levels have been reported in adults with AN (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23), levels of UFC have not been previously reported to be elevated in adolescent girls with this disorder (24, 25). Legro et al. (26) recently demonstrated that UFC levels increase with increasing age through puberty, and by standardizing UFC for creatinine and surface area (UFC/cr.SA), a fairly constant value is achieved for children in the age range of 12–17 yr. When UFC was standardized for cr and SA, we noted higher values in AN than in healthy adolescents (4), suggesting that cortisol values are indeed elevated in adolescents with AN as they are in adults.

We now report elevated serum cortisol in girls with AN compared with healthy adolescents and demonstrate through deconvolutional analysis that this is a function of an increased frequency of secretory bursts. To our knowledge, deconvolutional and Cluster analyses of cortisol secretion and concentration characteristics have not been previously reported in AN, a condition that serves as a model in which to study the effects of chronic undernutrition on hormonal secretion. We found no differences in secretory burst mass or amplitude between the groups. However, the increased frequency of secretory bursts resulted in increased pulsatile and total cortisol secretion in AN. Half-life trended higher in AN and together with increased pulsatile secretion resulted in higher mean cortisol concentration and total AUC in girls with AN, as demonstrated by Cluster analysis. Weight recovery resulted in a decrease in burst frequency. Our results differ from those obtained in adult men after acute fasting for a 3-d period, in whom increased cortisol concentration was a function of increased burst mass, whereas burst frequency and half life did not change (36). It is uncertain whether these differences are a consequence of differences in the duration of undernutrition or differences in age and gender of the subjects studied. We did not measure ACTH levels in our subjects during frequent sampling. It would be useful to examine ACTH and cortisol concentrations simultaneously at the different time points of frequent sampling to determine whether bursts of ACTH secretion correspond with secretory bursts determined from deconvolutional analysis of cortisol data.

Two small contradictory radioisotope studies have examined cortisol secretion in adults with AN. Boyar et al. (11) demonstrated higher 24-h mean plasma cortisol values with decreased cortisol clearance, but unchanged cortisol production in 10 adult women with AN, whereas Walsh et al. (23) reported higher cortisol production rates in adult women with this disorder. In our study increased pulsatile secretion suggests increased cortisol production in adolescents with AN, whereas the trend toward a longer half-life suggests decreased cortisol clearance. Thyroid hormones may affect cortisol clearance. Free T4 values trended lower and total T3 values were markedly lower in girls with AN compared with controls, the latter due to undernutrition. However, free T4 and total T3 levels did not correlate with cortisol half-life and did not emerge as independent predictors of cortisol concentration or secretory bursts. Therefore, it is unlikely that the alternations seen in thyroid hormones in AN caused the observed changes in cortisol levels or secretory patterns observed.

Although cortisol levels are elevated in AN, subjects do not demonstrate Cushingoid features. One proposed explanation for this observation is that it is a consequence of elevations in or increased binding affinity for corticosteroid-binding globulin (CBG) in AN with normal free cortisol values, or an acquired glucocorticoid resistance. However, no differences in CBG levels were found in adult women with AN vs. healthy matched controls (14) or in adult men after acute fasting (36). In addition, given that AN is associated with undernutrition and hypogonadism, one would expect low, rather than high, CBG levels. The binding capacity of CBG for cortisol does not differ in adults with AN compared with controls (12), and elevations have been reported not only in total, but also in free plasma cortisol in adult AN women (20, 37). To determine whether an acquired partial glucocorticoid resistance is the cause of hypercortisolemia in AN, investigators have examined expression of glucocorticoid receptors in adults with this disorder. The number of glucocorticoid receptors per cell does not differ in AN vs. controls in acute disease (37, 38). Moreover, hypercortisolemia in AN is associated with a marked reduction in the plasma ACTH response to CRH (13), suggestive of an appropriate pituitary response to hypercortisolism, which argues against glucocorticoid resistance.

Lack of substrate could account for the absence of Cushingoid features in AN. Hypercortisolism should cause increased lipogenesis, gluconeogenesis, increased protein breakdown, and redistribution of fat deposition. We have previously reported an increase in truncal fat during weight recovery in adults with AN, consistent with a steroid effect (39). Patients with active AN, however, do not have the substrate necessary for lipogenesis or gluconeogenesis. In fact, one may argue that lack of substrate is a cause of hypercortisolism in AN, with low glucose levels stimulating a release of counterregulatory hormones, including GH and cortisol. An example is the increase in cortisol and GH after insulin-induced hypoglycemia, which is the basis for assessing adequacy of GH and cortisol secretion using the insulin tolerance test (40, 41). In addition, endogenous and iatrogenic hypercortisolism can cause hyperglycemia or impaired glycemic responses to a glucose load (42, 43, 44).

We have demonstrated high GH levels in girls with AN (4) and have shown that the extent of elevation in GH levels correlates inversely with nutritional status. Similarly, in this study we demonstrate an inverse relationship between cortisol and measures of nutritional status, including glucose and insulin. Fat mass, glucose, and insulin emerge as independent predictors of cortisol secretion and concentration. Glucose levels were found to be the most significant predictor of the frequency of secretory bursts, the major contributor to increased cortisol secretion in AN. This suggests that low glucose and insulin levels in AN may indeed stimulate cortisol secretion. This would explain why plasma glucose levels are not in the hypoglycemic range in AN, although significantly lower than in fasting controls. Elevations in counterregulatory hormones may prevent hypoglycemia in this condition of chronic undernutrition. The link between nutritional status and cortisol secretion is additionally corroborated by cross-correlational analyses between cortisol and leptin (15, 45) and between insulin and leptin secretion (15), where changes in cortisol levels follow changes in leptin, and changes in leptin follow changes in insulin levels. In AN, an inverse correlation between plasma cortisol and insulin levels has been reported (46).

If undernutrition and low glucose levels contribute to hypercortisolism in AN, administration of glucose should decrease cortisol levels. To test this hypothesis, we examined cortisol levels 30 and 60 min after an oral glucose load in our subjects. Plasma cortisol levels fall during an OGTT in healthy adults (47, 48). In our healthy adolescents, an appropriate decrease occurred in cortisol after glucose administration. In girls with AN, however, cortisol levels trended downward with oral glucose, but the decrease did not reach statistical significance, despite equivalent glucose levels in girls with AN and controls at 30 and 60 min. A lack of suppression of cortisol levels after iv glucose loading has been reported in adults with AN (48). However, at least one report suggests differences in mean cortisol values during an oral vs. iv glucose tolerance test in a state of cortisol excess (49). It is uncertain whether a longer period of testing would have demonstrated a further lowering of cortisol levels. It is also uncertain whether prolonged undernutrition resets the hypothalamo-pituitary-adrenal axis, which cannot return to normal after a single glucose bolus, but will do so gradually after sustained weight recovery. Although decreases occurred in cortisol burst frequency with weight recovery, sustained weight recovery for a longer period of time may be necessary to see changes in other secretory characteristics and in cortisol response to an oral glucose load.

AN in adolescents is associated with low BMD (24, 25, 50, 51, 52). We have demonstrated that undernutrition with resultant decreases in BMI and IGF-I levels (1), and an acquired resistance to GH may contribute to low BMD in this condition (4). Given that cortisol inhibits proliferation of osteoblast precursors and differentiation to mature osteoblasts, simulates proliferation of osteoclasts, and has inhibitory effects on GH, which is anabolic to bone, one would expect hypercortisolism in AN to also contribute to low bone density in this condition. However, few studies have examined the effect of high cortisol levels on bone metabolism in AN. In this study we demonstrate that higher cortisol levels in AN correlate inversely with PICP and OC, markers of bone formation, but not with NTX, a marker of bone resorption, suggesting that hypercortisolemia in AN is associated with decreased bone formation, whereas lower cortisol levels in healthy controls do not affect bone turnover markers. Thus, hypercortisolemia in AN may contribute to low BMD by suppressing bone formation. Our results are corroborated by inverse correlations reported between UFC and bone mass in adults with AN (10) and a decrease in OC associated with high cortisol values in Cushing disease and AN (53).

We observed a direct relationship between maturity (CA and BA) and half-life and burst frequency of cortisol, and an inverse relationship with cortisol burst mass and amplitude. Data from adult studies similarly suggest an increase in cortisol half-life with increasing age (36, 54). Strong correlations were observed in our study between UFC/cr.SA, and measures of serum cortisol concentration as well as pulsatile and total cortisol secretion. These correlations were not observed between uncorrected UFC and measures of serum cortisol. Our data suggest that UFC/cr.SA may indeed be a better representation of cortisol status than urinary free cortisol values alone, in agreement with the findings of Legro et al. (26).

We thus report elevations in serum cortisol concentration (mean, nadir, valley mean, amplitude and area of concentration peaks, and total AUC) in adolescent girls with AN and demonstrate that this is subsequent to increased secretory burst frequency resulting in increased pulsatile secretion. UFC standardized for cr and SA is also elevated in AN. Cortisol secretion and concentration can be predicted by nutritional status, specifically fat mass, insulin, and glucose levels. However, a single oral glucose load does not suppress cortisol levels in AN, although it does so in controls. High cortisol levels in AN are inversely associated with bone formation markers, suggesting that hypercortisolism may contribute to low bone density in AN.


   Acknowledgments
 
We thank the skilled nurses of the GCRC for their role in carrying out the procedures required for this study, and Ellen Anderson and the GCRC bionutrition team for analysis of nutritional intake. We also thank our volunteer study staff for their roles in data collection and entry, and our subjects, without whom this study would not have been possible.


   Footnotes
 
This work was supported in part by NIH Grants M01-RR-01066, DK-062249, and K23-RR-018851.

Abbreviations: AN, Anorexia nervosa; AUC, area under the curve; BA, bone age; BMD, bone mineral density; BMI, body mass index; CA, chronological age; CBG, corticosteroid-binding globulin; cr, creatinine; NTX, N-telopeptide; OC, osteocalcin; OGTT, oral glucose tolerance test; PICP, type 1 procollagen; SA, surface area; UFC, urinary free cortisol.

Received April 16, 2004.

Accepted June 30, 3004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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