ORIGINAL
ARTICLE
Cortisol Measures Across the Weight Spectrum Melanie Schorr,* Elizabeth A. Lawson,* Laura E. Dichtel, Anne Klibanski, and Karen K. Miller Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Context: There are conflicting reports of increased vs decreased hypothalamic-pituitary-adrenal (HPA) activation in obesity; the most consistent finding is an inverse relationship between body mass index (BMI) and morning cortisol. In anorexia nervosa (AN), a low-BMI state, cortisol measures are elevated. Objective: This study aimed to investigate cortisol measures across the weight spectrum. Design and Setting: This was a cross-sectional study at a clinical research center. Participants: This study included 60 women, 18 – 45 years of age: overweight/obese (OB; N ⫽ 21); AN (N ⫽ 18); and normal-weight controls (HC; N ⫽ 21). Measures: HPA dynamics were assessed by urinary free cortisol, mean overnight serum cortisol obtained by pooled frequent sampling every 20 minutes from 2000 – 0800 h, 0800 h serum cortisol and cortisol-binding globulin, morning and late-night salivary cortisol, and dexamethasone-CRH testing. Body composition and bone mineral density (BMD) were assessed by dual-energy x-ray absorptiometry. Results: Cortisol measures demonstrated a U-shaped relationship with BMI, nadiring in the overweight-class I obese range, and were similarly associated with visceral adipose tissue and total fat mass. Mean cortisol levels were higher in AN than OB. There were weak negative linear relationships between lean mass and some cortisol measures. Most cortisol measures were negatively associated with postero-anterior spine and total hip BMD. Conclusions: Cortisol measures are lowest in overweight-class I obese women—lower than in lean women. With more significant obesity, cortisol levels increase, although not to as high as in AN. Therefore, extreme underweight and overweight states may activate the HPA axis, and hypercortisolemia may contribute to increased adiposity in the setting of caloric excess. Hypercortisolemia may also contribute to decreased BMD and muscle wasting in the setting of both caloric restriction and excess. (J Clin Endocrinol Metab 100: 3313–3321, 2015)
ctivation or suppression of the hypothalamic-pituitary-adrenal (HPA) axis is associated with alterations in body composition. Pathologic hypercortisolemia, as in Cushing’s syndrome, results in weight gain, increased intra-abdominal fat, decreased lean body mass, and bone loss (1, 2). In contrast, patients with hypocortisolemia due to adrenal insufficiency lose
A
weight (3). More subtle changes in HPA function may be present in women at the extremes of the weight spectrum, but such abnormalities are not well characterized, particularly in severely obese individuals, and their association with body composition and bone mineral density (BMD) in both underweight and overweight states is unclear.
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received April 24, 2015. Accepted July 8, 2015. First Published Online July 14, 2015
* M.S. and E.A.L. are co-first authors. Abbreviations: AN, anorexia nervosa; BMD, bone mineral density; BMI, body mass index; CBG, cortisol-binding globulin; CrCl, creatinine clearance; dex-CRH, dexamethasone suppression-CRH stimulation; FMI, fat mass index; HC, healthy control; HPA, hypothalamicpituitary-adrenal; IBW, ideal body weight; LC/MS/equilibrium dialysis, liquid chromatography tandem mass spectrometry, equilibrium dialysis; LNSC, late-night salivary cortisol; OB, overweight/obese; PA, postero-anterior; UFC, urinary free cortisol; WHO, World Health Organization.
doi: 10.1210/JC.2015-2078
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
press.endocrine.org/journal/jcem
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There are conflicting reports of HPA dynamics in overweight and obese subjects. The most consistent finding in some, but not all, studies is a negative linear relationship between body mass index (BMI) and morning serum or salivary cortisol (4 – 8). Moreover, previous studies have used waist circumference or waist-to-hip ratio to evaluate the relationship between abdominal adiposity and cortisol measures with inconsistent results (9 –11). Only one prior study radiographically measured adipose tissue, and showed no significant association between visceral adipose tissue, as measured by computed tomography scan, and 24-hour urinary free cortisol (UFC) (12). Whether abnormalities in HPA function contribute to weight gain, adiposity, or their metabolic consequences is therefore unknown. Anorexia nervosa (AN), a psychiatric disorder characterized by food restriction despite extremely low body weight and fat mass, is associated with elevated UFC (13, 14) and late-night salivary cortisol (LNSC) (15), as well as lack of cortisol suppression on 1 mg overnight dexamethasone suppression testing (13, 16) and dexamethasone suppression-CRH stimulation (dex-CRH) testing (14). Despite hypercortisolemia, these women do not appear Cushingoid, presumably due to lack of substrate. With weight gain, however, there is a positive correlation between baseline UFC and percent increase in truncal fat (17). Some studies have shown normalization of cortisol measures with weight gain (13, 18), but no study has evaluated the association of BMI or body composition with comprehensive cortisol measures in AN. Pathologic hypercortisolemia is a known risk factor for metabolic bone disease. Cushing’s syndrome is associated with increased bone resorption, decreased BMD, and increased risk of fractures (2, 19). AN is also associated with increased bone resorption, decreased BMD, and increased fracture risk (20, 21). Of note, UFC and mean overnight serum cortisol are negatively correlated with BMD in AN (22, 23). Although there is increasing recognition that obesity is associated with increased fracture risk (24), the relationship between cortisol measures and BMD in overweight/obese individuals remains unknown. We hypothesized that although mean cortisol levels would be higher in women with AN compared with normal-weight controls, they would be comparable between overweight/obese women and normal-weight controls. However, we predicted that higher cortisol levels would be associated with visceral adiposity in overweight/obese women, similar to patients with Cushing’s syndrome, and that cortisol levels would be negatively associated with BMD across the weight spectrum.
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
Materials and Methods This study was approved by the Institutional Review Board of Partners Health Care, Inc. Written informed consent was obtained from all subjects prior to procedures. We studied 60 premenopausal women, 18 – 45 years old; 21 overweight or obese (OB), 18 AN, and 21 normal-weight healthy controls (HC). Patient characteristics and mean overnight serum cortisol every 20 minutes between 2000 and 0800 h and its relationship to BMD in AN, but not OB, were previously reported (23, 25–27). Morning serum cortisol, cortisol-binding globulin (CBG), UFC, morning and LNSC, and dex-CRH testing results have not been previously published, and the relationship between these HPA measures and body composition or BMD has not been reported. All women in the OB group had BMIs between 25 and 40 kg/m2. Overweight was defined as BMI 25–29.9 kg/m2; class I obesity, BMI 30 –34.9 kg/m2; and class II obesity, BMI 35–39.9 kg/m2, as categorized by the World Health Organization (WHO) (28). Subjects with AN met Diagnostic and Statistical Manual of Mental Disorders IV criteria, including intense fear of gaining weight, body image disturbance, weight ⬍ 85% of ideal body weight (IBW), and amenorrhea for at least 3 consecutive months. Percent IBW was calculated using 1983 Met Life tables taking into account frame size. HC were at least 90% of IBW with a BMI ⬍ 25 kg/m2. Exclusion criteria for all subjects included diabetes mellitus, drug or alcohol abuse, abnormal thyroid function, use of medications known to affect cortisol levels (including estrogen or depot medroxyprogesterone), and pregnancy or breastfeeding. Additional exclusion criteria for the OB and HC groups included significant medical problems, amenorrhea (current or past), disordered eating, or significant anxiety or depression. BMD and body composition were assessed by dual-energy x-ray absorptiometry (Hologic 4500, Hologic, Inc), with a precision of 0.01 g/cm2 at the lumbar spine and a precision of 3% for fat mass (29). Fat mass index (FMI) was calculated by dividing fat mass in kilograms by height in meters2 (30). Kelly et al (30) developed sex-specific FMI categories using the population prevalence of each of the WHO BMI categories described above to generate FMI categories with similar prevalence. The classification scheme for FMI in women is as follows: normal FMI, 5–9 kg/m2; excess fat, 9 –13 kg/m2; class I obese, 13–17 kg/m2; class II obese, 17–21 kg/m2; and class III obese, at least 21 kg/m2 (30). Participants were asked to collect 24-hour urine samples, as well as 2300 and 0700 h salivary samples within a week of an inpatient, overnight visit. Creatinine clearance (CrCl) was measured for each sample, and UFC/CrCl was calculated for each sample to correct for the decreased creatinine and filtered cortisol associated with AN (31). At the inpatient visit, an IV catheter was placed and subjects were allowed to acclimate to their rooms for at least 2 hours, followed by frequent sampling of blood every 20 minutes from 2000 to 0800 h. Subjects fasted starting at 2000 h and were allowed to sleep through the night. Fasting cortisol and CBG levels were obtained at 0800 h. Serum samples were pooled for mean overnight serum cortisol levels. OB and HC presented for overnight visits during the follicular phase of the menstrual cycle. Subjects also presented at a morning outpatient visit for a dex-CRH test as previously described (32). Participants were instructed to take oral dexamethasone (0.5 mg every 6 h) for 48 hours to reduce endogenous variability in cortisol levels. At 0800 h, 2 hours after the final dexamethasone dose, corticorelin
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doi: 10.1210/JC.2015-2078
Table 1.
press.endocrine.org/journal/jcem
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Subject Characteristics and Cortisol Measures
Subject Characteristics Age, y BMI, kg/m2 Visceral adipose tissue, g Total fat mass, kg Lean body mass, kg Cortisol measures UFC, g/d (13.5– 88.7)a UFC/CrCl, g 䡠 kg/mg 䡠 d (0.15–1.23)a Overnight serum cortisol, g/dL (4.0 –11.2)a Overnight serum unbound cortisol, ng/mL (3.7–12.7)a Morning serum cortisol, g/dL (4.1–39.7)a Morning serum unbound cortisol, ng/mL (0 –98.0)a LNSC, g/dL (0.01– 0.09)a Morning salivary cortisol, g/dL (0 –1.01)a CBG, g/mL (11.7–31.7)a Dex-CRH 0-min cortisol, g/dL (0.05– 0.73)a Dex-CRH 15-min cortisol, g/dL (0 –1.4)a Dex level prior to CRH stimulation, ng/dL (237– 857)a
AN (n ⴝ 18)
HC (n ⴝ 21)
OB (n ⴝ 21)
Combined (n ⴝ 60)
ANOVA P Value
AN v HC P Value
AN v OB P Value
HC v OB P Value
26 ⫾ 6 18.2 ⫾ 1.0 90 ⫾ 39 9.3 ⫾ 2.3 40.7 ⫾ 3.6
27 ⫾ 7 22.3 ⫾ 1.4 215 ⫾ 85 16.9 ⫾ 3.6 43.4 ⫾ 4.8
30 ⫾ 8 31.2 ⫾ 4.3 481 ⫾ 185 32.5 ⫾ 9.1 51.7 ⫾ 7.3
28 ⫾ 7 24.1 ⫾ 6.0 255 ⫾ 196 19.4 ⫾ 11.0 45.1 ⫾ 7.0
NS ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
⬍.0001 .002 .0001 NS
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
82.0 ⫾ 42.3 1.16 ⫾ 0.52 11.6 ⫾ 3.3 14.2 ⫾ 6.7 26.9 ⫾ 8.1 51.0 ⫾ 21.1 0.06 ⫾ 0.03 0.71 ⫾ 0.43 24.4 ⫾ 3.9 1.86 ⫾ 3.85 2.05 ⫾ 4.22 386 ⫾ 99
51.1 ⫾ 18.8 0.69 ⫾ 0.27 7.6 ⫾ 1.8 8.2 ⫾ 2.3 21.9 ⫾ 8.9 44.7 ⫾ 26.7 0.05 ⫾ 0.02 0.43 ⫾ 0.29 21.7 ⫾ 5.0 0.39 ⫾ 0.17 0.61 ⫾ 0.41 547 ⫾ 155
67.0 ⫾ 35.2 0.61 ⫾ 0.25 6.2 ⫾ 1.7 6.8 ⫾ 2.3 18.3 ⫾ 6.8 34.9 ⫾ 21.6 0.04 ⫾ 0.01 0.29 ⫾ 0.15 20.2 ⫾ 3.0 0.34 ⫾ 0.22 0.46 ⫾ 0.21 554 ⫾ 204
63.7 ⫾ 33.0 0.76 ⫾ 0.39 8.1 ⫾ 3.1 9.2 ⫾ 4.8 22.0 ⫾ 8.6 42.9 ⫾ 24.1 0.05 ⫾ 0.02 0.48 ⫾ 0.35 21.9 ⫾ 4.4 0.81 ⫾ 2.17 0.61 ⫾ 0.51 499 ⫾ 174
.04 .0004 ⬍.0001 ⬍.0001 .01 NS .08 .005 .02 .07 .09 .004
.01 .0007 ⬍.0001 ⬍.0001 NS
NS .0002 ⬍.0001 ⬍.0001 .003
NS NS .05 NS NS
.02 NS
.001 .006
NS NS
.004
.003
NS
Abbreviations: dex, dexamethasone; NS, not significant. a
Values are mean ⫾ 2 SD of the HC group.
(1 g/kg Acthrel; max dose, 100 g; Ferring Pharmaceuticals) was administered iv. Serum cortisol levels were measured 5 minutes before and 15 minutes after corticorelin administration. Baseline dexamethasone levels were measured. The iv was placed at least 15 minutes prior to drawing blood.
Biochemical analysis Urinary free cortisol was measured by immunoassay (Immulite, Diagnostics Product Corp), with a sensitivity of 0.20 g/dL and reference range of 20 –70 g/day. Serum cortisol was measured by chemiluminescent microparticle immunoassay (Architect System, Abbot Diagnostics), with a sensitivity of 0.8 g/dL. Serum-free cortisol was measured by liquid chromatography tandem mass spectrometry, equilibrium dialysis (LC/MS/equilibrium dialysis) (Quest Diagnostics), with a sensitivity of 0.1 g/dL. Salivary cortisol was measured using EIA (Salimetrics), with a sensitivity of 0.003 mg/dL and reference range of 0.007– 0.115 g/dL. In Table 1 and Figure 1, a normal range was calculated and reported as mean ⫾ 2 SD of the HC group for all cortisol measures. CBG was measured by ELISA (BioVendor), with a sensitivity of 0.01 ng/mL. Unbound serum cortisol was estimated using Coolens equation (33). Dexamethasone was measured using HPLC-tandem mass spectrometry (Esoterix, Inc), with a sensitivity of 5 ng/dL. Coefficients of variation were ⬍ 10% for all assays.
.05. Data are reported as mean ⫾ SD or as a correlation coefficient (R) with an associated P value.
Results Baseline characteristics The groups did not differ in mean age (Table 1). Per study design, BMI was highest in the OB group, lowest in the AN group, and intermediate in the HC group (Table 1). BMI ranged from 16.0 to 39.8 kg/m2. As expected,
Data analysis JMP Statistical Discoveries (SAS Institute, Inc) was used for statistical analyses. Three outliers for morning salivary cortisol and LNSC were excluded (1 AN, 2 OB) using a quantile analysis. Clinical characteristics and hormone levels were compared using Fisher’s Least Significant Difference Test. Additional correction for multiple comparisons is not indicated when this method is used for three-group comparisons (34). Data modeling was performed to determine the best fit between cortisol measures and BMI or body composition measurements. Multivariate leastsquare analyses were constructed to control for potential confounders. Statistical significance was defined as a two-tailed P ⱕ
Figure 1. Percentage of subjects in the AN and OB groups with elevated cortisol measures as defined as greater than the mean ⫹ 2 SDs of the HC group. Cortisol measures were elevated in 15–45% of AN subjects. UFC was the only cortisol measure elevated in any OB subjects.
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visceral adipose tissue and total fat mass were also highest in the OB group, lowest in the AN group, and intermediate in the HC group (Table 1). Lean body mass was significantly greater in the OB group compared with the AN or HC groups (Table 1). UFC Mean UFC was significantly higher in the AN group compared with the HC group (P ⫽ .01); there was no difference in mean UFC between the OB and HC groups (Table 1). Mean UFC/CrCl was significantly higher in the AN group compared with the OB (P ⫽ .0002) and HC (P ⫽ .0007) groups; there was no difference in mean UFC/ CrCl between the OB and HC groups (Table 1). Overnight serum cortisol Mean overnight serum cortisol was also significantly higher in the AN group compared with the OB (P ⬍ .0001) and HC (P ⬍ .0001) groups (Table 1). These differences remained significant when estimating mean overnight serum unbound cortisol (P ⬍ .0001), which was calculated given a significantly higher CBG level in the AN group compared with the OB group (Table 1). Although the OB group had significantly higher mean overnight serum cortisol than the HC group (P ⫽ .05), there was no significant difference between the two groups in mean overnight serum unbound cortisol (Table 1). LNSC The differences in LNSC between the three groups did not reach significance (P ⫽ .08) (Table 1). Morning cortisol In addition to having higher UFC and overnight serum cortisol measures, the AN group also had significantly higher morning serum and salivary cortisol measures compared with the OB group (P ⫽ .003 and .001, respectively)
Table 2.
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
(Table 1). Morning serum unbound cortisol was not significantly different between the three groups (Table 1). dex-CRH stimulation testing results Morning serum cortisol after dexamethasone suppression and after subsequent CRH administration trended toward being significantly different between the three groups (Table 1). The AN group had significantly lower baseline dexamethasone levels compared with the OB (P ⫽ .003) and HC (P ⫽ .004) groups (Table 1). After controlling for differences in CBG levels and baseline dexamethasone levels, morning serum cortisol after dexamethasone suppression and after subsequent CRH administration was not significantly different between the three groups. Elevated cortisol measures in AN and OB groups The percentage of subjects in the AN and OB groups with elevated cortisol measures, as defined as greater than the mean ⫹ 2 SD in the HC group, was determined. The mean ⫹ 2 SD of UFC in the HC group was 89 g/day. The mean ⫹ 2 SD of LNSC in the HC group was 0.09 g/dL. The mean ⫹ 2 SD of dex-CRH 15-minute serum cortisol in the HC group was 1.4 g/dL, which is the same as the published cutoff for the dex-CRH test (32). Cortisol measures were elevated in 15– 45% of AN subjects (Figure 1). Three AN subjects “failed” the dex-CRH test with a 15-minute serum cortisol ⬎ 1.4 g/dL (Figure 1). The dexamethasone levels in these three cases were 282, 332, and 386 ng/dL, and none of these subjects were receiving medications that are inducers of cytochrome P450 variants 3A4, 5, and 7, of which dexamethasone is a substrate. UFC was the only elevated cortisol measure in OB subjects; it was elevated in 25% of the OB group (Figure 1). These OB subjects had no specific symptoms or
U-Shaped Relationships Between Most Cortisol Measures and BMI or Adiposity BMI
UFC UFC/CrCl Mean overnight serum cortisol Mean overnight serum unbound cortisol Morning serum cortisol Morning serum unbound cortisol LNSC Morning salivary cortisol
Visceral Adipose Tissue
Total Fat
R
P
R
P
R
P
Nadir BMI, kg/m2
0.47 0.55 0.66 0.58
.004 .0004
ARTICLE
Cortisol Measures Across the Weight Spectrum Melanie Schorr,* Elizabeth A. Lawson,* Laura E. Dichtel, Anne Klibanski, and Karen K. Miller Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Context: There are conflicting reports of increased vs decreased hypothalamic-pituitary-adrenal (HPA) activation in obesity; the most consistent finding is an inverse relationship between body mass index (BMI) and morning cortisol. In anorexia nervosa (AN), a low-BMI state, cortisol measures are elevated. Objective: This study aimed to investigate cortisol measures across the weight spectrum. Design and Setting: This was a cross-sectional study at a clinical research center. Participants: This study included 60 women, 18 – 45 years of age: overweight/obese (OB; N ⫽ 21); AN (N ⫽ 18); and normal-weight controls (HC; N ⫽ 21). Measures: HPA dynamics were assessed by urinary free cortisol, mean overnight serum cortisol obtained by pooled frequent sampling every 20 minutes from 2000 – 0800 h, 0800 h serum cortisol and cortisol-binding globulin, morning and late-night salivary cortisol, and dexamethasone-CRH testing. Body composition and bone mineral density (BMD) were assessed by dual-energy x-ray absorptiometry. Results: Cortisol measures demonstrated a U-shaped relationship with BMI, nadiring in the overweight-class I obese range, and were similarly associated with visceral adipose tissue and total fat mass. Mean cortisol levels were higher in AN than OB. There were weak negative linear relationships between lean mass and some cortisol measures. Most cortisol measures were negatively associated with postero-anterior spine and total hip BMD. Conclusions: Cortisol measures are lowest in overweight-class I obese women—lower than in lean women. With more significant obesity, cortisol levels increase, although not to as high as in AN. Therefore, extreme underweight and overweight states may activate the HPA axis, and hypercortisolemia may contribute to increased adiposity in the setting of caloric excess. Hypercortisolemia may also contribute to decreased BMD and muscle wasting in the setting of both caloric restriction and excess. (J Clin Endocrinol Metab 100: 3313–3321, 2015)
ctivation or suppression of the hypothalamic-pituitary-adrenal (HPA) axis is associated with alterations in body composition. Pathologic hypercortisolemia, as in Cushing’s syndrome, results in weight gain, increased intra-abdominal fat, decreased lean body mass, and bone loss (1, 2). In contrast, patients with hypocortisolemia due to adrenal insufficiency lose
A
weight (3). More subtle changes in HPA function may be present in women at the extremes of the weight spectrum, but such abnormalities are not well characterized, particularly in severely obese individuals, and their association with body composition and bone mineral density (BMD) in both underweight and overweight states is unclear.
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received April 24, 2015. Accepted July 8, 2015. First Published Online July 14, 2015
* M.S. and E.A.L. are co-first authors. Abbreviations: AN, anorexia nervosa; BMD, bone mineral density; BMI, body mass index; CBG, cortisol-binding globulin; CrCl, creatinine clearance; dex-CRH, dexamethasone suppression-CRH stimulation; FMI, fat mass index; HC, healthy control; HPA, hypothalamicpituitary-adrenal; IBW, ideal body weight; LC/MS/equilibrium dialysis, liquid chromatography tandem mass spectrometry, equilibrium dialysis; LNSC, late-night salivary cortisol; OB, overweight/obese; PA, postero-anterior; UFC, urinary free cortisol; WHO, World Health Organization.
doi: 10.1210/JC.2015-2078
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
press.endocrine.org/journal/jcem
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There are conflicting reports of HPA dynamics in overweight and obese subjects. The most consistent finding in some, but not all, studies is a negative linear relationship between body mass index (BMI) and morning serum or salivary cortisol (4 – 8). Moreover, previous studies have used waist circumference or waist-to-hip ratio to evaluate the relationship between abdominal adiposity and cortisol measures with inconsistent results (9 –11). Only one prior study radiographically measured adipose tissue, and showed no significant association between visceral adipose tissue, as measured by computed tomography scan, and 24-hour urinary free cortisol (UFC) (12). Whether abnormalities in HPA function contribute to weight gain, adiposity, or their metabolic consequences is therefore unknown. Anorexia nervosa (AN), a psychiatric disorder characterized by food restriction despite extremely low body weight and fat mass, is associated with elevated UFC (13, 14) and late-night salivary cortisol (LNSC) (15), as well as lack of cortisol suppression on 1 mg overnight dexamethasone suppression testing (13, 16) and dexamethasone suppression-CRH stimulation (dex-CRH) testing (14). Despite hypercortisolemia, these women do not appear Cushingoid, presumably due to lack of substrate. With weight gain, however, there is a positive correlation between baseline UFC and percent increase in truncal fat (17). Some studies have shown normalization of cortisol measures with weight gain (13, 18), but no study has evaluated the association of BMI or body composition with comprehensive cortisol measures in AN. Pathologic hypercortisolemia is a known risk factor for metabolic bone disease. Cushing’s syndrome is associated with increased bone resorption, decreased BMD, and increased risk of fractures (2, 19). AN is also associated with increased bone resorption, decreased BMD, and increased fracture risk (20, 21). Of note, UFC and mean overnight serum cortisol are negatively correlated with BMD in AN (22, 23). Although there is increasing recognition that obesity is associated with increased fracture risk (24), the relationship between cortisol measures and BMD in overweight/obese individuals remains unknown. We hypothesized that although mean cortisol levels would be higher in women with AN compared with normal-weight controls, they would be comparable between overweight/obese women and normal-weight controls. However, we predicted that higher cortisol levels would be associated with visceral adiposity in overweight/obese women, similar to patients with Cushing’s syndrome, and that cortisol levels would be negatively associated with BMD across the weight spectrum.
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
Materials and Methods This study was approved by the Institutional Review Board of Partners Health Care, Inc. Written informed consent was obtained from all subjects prior to procedures. We studied 60 premenopausal women, 18 – 45 years old; 21 overweight or obese (OB), 18 AN, and 21 normal-weight healthy controls (HC). Patient characteristics and mean overnight serum cortisol every 20 minutes between 2000 and 0800 h and its relationship to BMD in AN, but not OB, were previously reported (23, 25–27). Morning serum cortisol, cortisol-binding globulin (CBG), UFC, morning and LNSC, and dex-CRH testing results have not been previously published, and the relationship between these HPA measures and body composition or BMD has not been reported. All women in the OB group had BMIs between 25 and 40 kg/m2. Overweight was defined as BMI 25–29.9 kg/m2; class I obesity, BMI 30 –34.9 kg/m2; and class II obesity, BMI 35–39.9 kg/m2, as categorized by the World Health Organization (WHO) (28). Subjects with AN met Diagnostic and Statistical Manual of Mental Disorders IV criteria, including intense fear of gaining weight, body image disturbance, weight ⬍ 85% of ideal body weight (IBW), and amenorrhea for at least 3 consecutive months. Percent IBW was calculated using 1983 Met Life tables taking into account frame size. HC were at least 90% of IBW with a BMI ⬍ 25 kg/m2. Exclusion criteria for all subjects included diabetes mellitus, drug or alcohol abuse, abnormal thyroid function, use of medications known to affect cortisol levels (including estrogen or depot medroxyprogesterone), and pregnancy or breastfeeding. Additional exclusion criteria for the OB and HC groups included significant medical problems, amenorrhea (current or past), disordered eating, or significant anxiety or depression. BMD and body composition were assessed by dual-energy x-ray absorptiometry (Hologic 4500, Hologic, Inc), with a precision of 0.01 g/cm2 at the lumbar spine and a precision of 3% for fat mass (29). Fat mass index (FMI) was calculated by dividing fat mass in kilograms by height in meters2 (30). Kelly et al (30) developed sex-specific FMI categories using the population prevalence of each of the WHO BMI categories described above to generate FMI categories with similar prevalence. The classification scheme for FMI in women is as follows: normal FMI, 5–9 kg/m2; excess fat, 9 –13 kg/m2; class I obese, 13–17 kg/m2; class II obese, 17–21 kg/m2; and class III obese, at least 21 kg/m2 (30). Participants were asked to collect 24-hour urine samples, as well as 2300 and 0700 h salivary samples within a week of an inpatient, overnight visit. Creatinine clearance (CrCl) was measured for each sample, and UFC/CrCl was calculated for each sample to correct for the decreased creatinine and filtered cortisol associated with AN (31). At the inpatient visit, an IV catheter was placed and subjects were allowed to acclimate to their rooms for at least 2 hours, followed by frequent sampling of blood every 20 minutes from 2000 to 0800 h. Subjects fasted starting at 2000 h and were allowed to sleep through the night. Fasting cortisol and CBG levels were obtained at 0800 h. Serum samples were pooled for mean overnight serum cortisol levels. OB and HC presented for overnight visits during the follicular phase of the menstrual cycle. Subjects also presented at a morning outpatient visit for a dex-CRH test as previously described (32). Participants were instructed to take oral dexamethasone (0.5 mg every 6 h) for 48 hours to reduce endogenous variability in cortisol levels. At 0800 h, 2 hours after the final dexamethasone dose, corticorelin
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doi: 10.1210/JC.2015-2078
Table 1.
press.endocrine.org/journal/jcem
3315
Subject Characteristics and Cortisol Measures
Subject Characteristics Age, y BMI, kg/m2 Visceral adipose tissue, g Total fat mass, kg Lean body mass, kg Cortisol measures UFC, g/d (13.5– 88.7)a UFC/CrCl, g 䡠 kg/mg 䡠 d (0.15–1.23)a Overnight serum cortisol, g/dL (4.0 –11.2)a Overnight serum unbound cortisol, ng/mL (3.7–12.7)a Morning serum cortisol, g/dL (4.1–39.7)a Morning serum unbound cortisol, ng/mL (0 –98.0)a LNSC, g/dL (0.01– 0.09)a Morning salivary cortisol, g/dL (0 –1.01)a CBG, g/mL (11.7–31.7)a Dex-CRH 0-min cortisol, g/dL (0.05– 0.73)a Dex-CRH 15-min cortisol, g/dL (0 –1.4)a Dex level prior to CRH stimulation, ng/dL (237– 857)a
AN (n ⴝ 18)
HC (n ⴝ 21)
OB (n ⴝ 21)
Combined (n ⴝ 60)
ANOVA P Value
AN v HC P Value
AN v OB P Value
HC v OB P Value
26 ⫾ 6 18.2 ⫾ 1.0 90 ⫾ 39 9.3 ⫾ 2.3 40.7 ⫾ 3.6
27 ⫾ 7 22.3 ⫾ 1.4 215 ⫾ 85 16.9 ⫾ 3.6 43.4 ⫾ 4.8
30 ⫾ 8 31.2 ⫾ 4.3 481 ⫾ 185 32.5 ⫾ 9.1 51.7 ⫾ 7.3
28 ⫾ 7 24.1 ⫾ 6.0 255 ⫾ 196 19.4 ⫾ 11.0 45.1 ⫾ 7.0
NS ⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
⬍.0001 .002 .0001 NS
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
82.0 ⫾ 42.3 1.16 ⫾ 0.52 11.6 ⫾ 3.3 14.2 ⫾ 6.7 26.9 ⫾ 8.1 51.0 ⫾ 21.1 0.06 ⫾ 0.03 0.71 ⫾ 0.43 24.4 ⫾ 3.9 1.86 ⫾ 3.85 2.05 ⫾ 4.22 386 ⫾ 99
51.1 ⫾ 18.8 0.69 ⫾ 0.27 7.6 ⫾ 1.8 8.2 ⫾ 2.3 21.9 ⫾ 8.9 44.7 ⫾ 26.7 0.05 ⫾ 0.02 0.43 ⫾ 0.29 21.7 ⫾ 5.0 0.39 ⫾ 0.17 0.61 ⫾ 0.41 547 ⫾ 155
67.0 ⫾ 35.2 0.61 ⫾ 0.25 6.2 ⫾ 1.7 6.8 ⫾ 2.3 18.3 ⫾ 6.8 34.9 ⫾ 21.6 0.04 ⫾ 0.01 0.29 ⫾ 0.15 20.2 ⫾ 3.0 0.34 ⫾ 0.22 0.46 ⫾ 0.21 554 ⫾ 204
63.7 ⫾ 33.0 0.76 ⫾ 0.39 8.1 ⫾ 3.1 9.2 ⫾ 4.8 22.0 ⫾ 8.6 42.9 ⫾ 24.1 0.05 ⫾ 0.02 0.48 ⫾ 0.35 21.9 ⫾ 4.4 0.81 ⫾ 2.17 0.61 ⫾ 0.51 499 ⫾ 174
.04 .0004 ⬍.0001 ⬍.0001 .01 NS .08 .005 .02 .07 .09 .004
.01 .0007 ⬍.0001 ⬍.0001 NS
NS .0002 ⬍.0001 ⬍.0001 .003
NS NS .05 NS NS
.02 NS
.001 .006
NS NS
.004
.003
NS
Abbreviations: dex, dexamethasone; NS, not significant. a
Values are mean ⫾ 2 SD of the HC group.
(1 g/kg Acthrel; max dose, 100 g; Ferring Pharmaceuticals) was administered iv. Serum cortisol levels were measured 5 minutes before and 15 minutes after corticorelin administration. Baseline dexamethasone levels were measured. The iv was placed at least 15 minutes prior to drawing blood.
Biochemical analysis Urinary free cortisol was measured by immunoassay (Immulite, Diagnostics Product Corp), with a sensitivity of 0.20 g/dL and reference range of 20 –70 g/day. Serum cortisol was measured by chemiluminescent microparticle immunoassay (Architect System, Abbot Diagnostics), with a sensitivity of 0.8 g/dL. Serum-free cortisol was measured by liquid chromatography tandem mass spectrometry, equilibrium dialysis (LC/MS/equilibrium dialysis) (Quest Diagnostics), with a sensitivity of 0.1 g/dL. Salivary cortisol was measured using EIA (Salimetrics), with a sensitivity of 0.003 mg/dL and reference range of 0.007– 0.115 g/dL. In Table 1 and Figure 1, a normal range was calculated and reported as mean ⫾ 2 SD of the HC group for all cortisol measures. CBG was measured by ELISA (BioVendor), with a sensitivity of 0.01 ng/mL. Unbound serum cortisol was estimated using Coolens equation (33). Dexamethasone was measured using HPLC-tandem mass spectrometry (Esoterix, Inc), with a sensitivity of 5 ng/dL. Coefficients of variation were ⬍ 10% for all assays.
.05. Data are reported as mean ⫾ SD or as a correlation coefficient (R) with an associated P value.
Results Baseline characteristics The groups did not differ in mean age (Table 1). Per study design, BMI was highest in the OB group, lowest in the AN group, and intermediate in the HC group (Table 1). BMI ranged from 16.0 to 39.8 kg/m2. As expected,
Data analysis JMP Statistical Discoveries (SAS Institute, Inc) was used for statistical analyses. Three outliers for morning salivary cortisol and LNSC were excluded (1 AN, 2 OB) using a quantile analysis. Clinical characteristics and hormone levels were compared using Fisher’s Least Significant Difference Test. Additional correction for multiple comparisons is not indicated when this method is used for three-group comparisons (34). Data modeling was performed to determine the best fit between cortisol measures and BMI or body composition measurements. Multivariate leastsquare analyses were constructed to control for potential confounders. Statistical significance was defined as a two-tailed P ⱕ
Figure 1. Percentage of subjects in the AN and OB groups with elevated cortisol measures as defined as greater than the mean ⫹ 2 SDs of the HC group. Cortisol measures were elevated in 15–45% of AN subjects. UFC was the only cortisol measure elevated in any OB subjects.
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Schorr et al
Cortisol Measures Across the Weight Spectrum
visceral adipose tissue and total fat mass were also highest in the OB group, lowest in the AN group, and intermediate in the HC group (Table 1). Lean body mass was significantly greater in the OB group compared with the AN or HC groups (Table 1). UFC Mean UFC was significantly higher in the AN group compared with the HC group (P ⫽ .01); there was no difference in mean UFC between the OB and HC groups (Table 1). Mean UFC/CrCl was significantly higher in the AN group compared with the OB (P ⫽ .0002) and HC (P ⫽ .0007) groups; there was no difference in mean UFC/ CrCl between the OB and HC groups (Table 1). Overnight serum cortisol Mean overnight serum cortisol was also significantly higher in the AN group compared with the OB (P ⬍ .0001) and HC (P ⬍ .0001) groups (Table 1). These differences remained significant when estimating mean overnight serum unbound cortisol (P ⬍ .0001), which was calculated given a significantly higher CBG level in the AN group compared with the OB group (Table 1). Although the OB group had significantly higher mean overnight serum cortisol than the HC group (P ⫽ .05), there was no significant difference between the two groups in mean overnight serum unbound cortisol (Table 1). LNSC The differences in LNSC between the three groups did not reach significance (P ⫽ .08) (Table 1). Morning cortisol In addition to having higher UFC and overnight serum cortisol measures, the AN group also had significantly higher morning serum and salivary cortisol measures compared with the OB group (P ⫽ .003 and .001, respectively)
Table 2.
J Clin Endocrinol Metab, September 2015, 100(9):3313–3321
(Table 1). Morning serum unbound cortisol was not significantly different between the three groups (Table 1). dex-CRH stimulation testing results Morning serum cortisol after dexamethasone suppression and after subsequent CRH administration trended toward being significantly different between the three groups (Table 1). The AN group had significantly lower baseline dexamethasone levels compared with the OB (P ⫽ .003) and HC (P ⫽ .004) groups (Table 1). After controlling for differences in CBG levels and baseline dexamethasone levels, morning serum cortisol after dexamethasone suppression and after subsequent CRH administration was not significantly different between the three groups. Elevated cortisol measures in AN and OB groups The percentage of subjects in the AN and OB groups with elevated cortisol measures, as defined as greater than the mean ⫹ 2 SD in the HC group, was determined. The mean ⫹ 2 SD of UFC in the HC group was 89 g/day. The mean ⫹ 2 SD of LNSC in the HC group was 0.09 g/dL. The mean ⫹ 2 SD of dex-CRH 15-minute serum cortisol in the HC group was 1.4 g/dL, which is the same as the published cutoff for the dex-CRH test (32). Cortisol measures were elevated in 15– 45% of AN subjects (Figure 1). Three AN subjects “failed” the dex-CRH test with a 15-minute serum cortisol ⬎ 1.4 g/dL (Figure 1). The dexamethasone levels in these three cases were 282, 332, and 386 ng/dL, and none of these subjects were receiving medications that are inducers of cytochrome P450 variants 3A4, 5, and 7, of which dexamethasone is a substrate. UFC was the only elevated cortisol measure in OB subjects; it was elevated in 25% of the OB group (Figure 1). These OB subjects had no specific symptoms or
U-Shaped Relationships Between Most Cortisol Measures and BMI or Adiposity BMI
UFC UFC/CrCl Mean overnight serum cortisol Mean overnight serum unbound cortisol Morning serum cortisol Morning serum unbound cortisol LNSC Morning salivary cortisol
Visceral Adipose Tissue
Total Fat
R
P
R
P
R
P
Nadir BMI, kg/m2
0.47 0.55 0.66 0.58
.004 .0004
