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Association of 24-Hour Cortisol Production Rates, Cortisol-Binding Globulin, and Plasma-Free Cortisol Levels with Body Composition, Leptin Levels, and Aging in Adult Men and Women

Oregon Health and Science University, Portland, Oregon 97239

Address all correspondence and requests for reprints to: Jonathan Q. Purnell, M.D., Oregon Health and Science University, Division of Endocrinology, Diabetes, and Clinical Nutrition, L607, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239. E-mail: purnellj{at}ohsu.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present study was designed to examine the hypothesis that hypothalamic-pituitary-adrenal axis activity as measured by 24-h cortisol production rate (CPR) and plasma levels of free cortisol is linked to increased body fat in adults, and that increased cortisol levels with aging results from increased CPR. Fifty-four healthy men and women volunteers with a wide range of body mass indexes and ages underwent measurement of CPR by isotope dilution measured by gas chromatography-mass spectroscopy, cortisol-binding globulin, and free cortisol in pooled 24-h plasma, body composition, and leptin. Cortisol clearance rates were determined from the 10-h disappearance curves of hydrocortisone after steady-state infusion in a separate group of lean and obese subjects with adrenal insufficiency. Although CPR significantly increased with increasing body mass index and percentage body fat, free cortisol levels remained independent of body composition and leptin levels due to increased cortisol clearance rates. CPR and free cortisol levels were, however, significantly higher in men than women. In addition, 24-h plasma free cortisol levels were increased with age in association with increased CPR, independent of body size. This increase in hypothalamic-pituitary-adrenal axis activity may play a role in the alterations in body composition and central fat distribution in men vs. women and with aging.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GIVEN THE OCCURRENCE of obesity and central distribution of fat with hypercortisolemia (Cushing’s syndrome), many studies have sought to determine whether cortisol plays a role in the expression of obesity in the general population, in the expression of central fat patterning in men and women, and with changes in body composition with aging. Although some of these studies have shown an association of increased cortisol production rates (CPRs) with increased body weight or fat mass (FM) (1, 2, 3, 4, 5, 6), others have not (7, 8, 9). Likewise, some studies have found higher CPR in men than women (10), whereas others have found no sex difference (11). In studies of aging, which in humans is characterized by an increase in percentage body fat (12) and central obesity (13), cortisol levels have been found to be low to normal (14, 15, 16, 17, 18) or increased (15, 19, 20). In those studies that found increased cortisol levels in older compared with younger subjects, it was inferred that this resulted from increased cortisol secretion, although CPR, cortisol-binding globulin (CBG), and free cortisol levels were not directly measured.

These previous studies relied on measured cortisol in timed urine or serum samples to estimate CPR, which have a number of limitations that can affect their accuracy and may have led to the contrary findings in the studies noted above. These include failure to account for circadian variation of cortisol secretion, inadequate sample collection, failure to account for enterohepatic clearance of cortisol, and the effect of individual variation in CBG concentrations on serum cortisol levels (21, 22, 23). In addition, no studies to date have measured CPR and 24-h levels of free cortisol in the same patient population to determine the biological significance of differences in CPR.

The purpose of the present study is to measure CPR using the technique of steady-state, stable isotope tracer infusion of deuterated cortisol over 24 h with isotope dilution determined by mass spectrometry (22, 23, 24), along with measurement of free cortisol and CBG levels, in healthy volunteers representing a range of body weights and ages. The relationships between CPR and cortisol levels with age, gender, and body composition were then examined. We hypothesized that CPR would correlate positively with indices of FM and would increase as a function of aging. If this were true, this increased CPR could help explain the observed differences in body composition and fat distribution in healthy older subjects.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Twenty-four men and 30 women at least 18 yr old were recruited through campus advertisements, local newspapers, and clinics at the University of Washington. All subjects were healthy, at their usual weight, free of active medical diseases, and were chosen to provide a range of body weights and ages. Subjects were excluded if they had diabetes, cancer, depression or other psychiatric illness, or were taking medication for a psychiatric diagnosis, alcoholism (more than two drinks per day), substance abuse (including smoking), untreated hypothyroidism, or exercised more than 30 min, three times a week. Subjects with controlled hypertension on medication or taking thyroid hormone replacement therapy (HRT) were included if adequacy of the replacement dose was documented by blood pressures no greater than 140/90 or TSH levels between 0.5 and 5 µU/liter. Seventeen of the women were postmenopausal, including eight women who were receiving chronic HRT (estrogen or estrogen/progestin combination) and nine who were not. Admissions for CPR measurements for women were not timed to occur during specific times of their menstrual cycle or HRT.

To investigate the effect of obesity on cortisol clearance without interference from endogenous cortisol secretion, patients with primary adrenal insufficiency (AI) were recruited from clinics at Oregon Health and Science University (OHSU) and the surrounding community to measure cortisol clearance, including four obese [body mass index (BMI), 29–43 kg/m²] and three lean (BMI, 19–25 kg/m²) subjects. AI was defined as spontaneous serum cortisol of levels less than 5 µg/dl (138 nmol/liter) after 12 h without glucocorticoid replacement and a peak serum cortisol level of less than 5 µg/dl (138 nmol/liter) 60 min after a 250-µg ACTH stimulation test (25).

The Human Subjects Committees at the University of Washington and OHSU approved all procedures, and consent was obtained before study entry.

Procedures

CPR. Healthy subjects were admitted to the University of Washington General Clinical Research Center (GCRC) the evening before study. Two separate peripheral iv lines were started, one for infusion of the deuterium-labeled cortisol isotope (d₃-cortisol; 9,12,12-²H₃-cortisol, 99.8 atom %; Cambridge Isotope Labs, Andover, MA), and the other for drawing blood. Starting at 0200 h, a continuous infusion of deuterium-labeled cortisol (20 µg/h) was administered at a constant rate over the next 30 h (until 0800 h of the final study day). Five-milliliter blood samples were drawn every 30 min from the second iv line during the final 24 h of the deuterated cortisol infusion (0800–0800 h). The total amount of deuterated cortisol infused over 24 h (480 µg) was less than 5% of a normal endogenous 24-h CPR. Plasma was separated, and 50 µl from each sample were combined into a 24-h pooled sample for mass spectrophotometric analysis. Plasma from the pooled sample (0.5 ml) then underwent an extraction and derivatization process to form fluoroacyl derivatives of cortisol as previously described to enhance detection by gas chromatography-negative ion chemical ionization mass spectrometry (26). The area under the curves (AUCs) for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d₀, m/z 782) and d₃-cortisol (d₃, m/z 785) were used to determine the isotopic dilution ratio (d₃/d₀) in 24-h pooled plasma samples (PS). Retention times of the fluoroacyl derivatives were verified by authentic standards. The CPR was then calculated from the product infusion rate (IR) and the ratio of the isotopic enrichment to isotopic dilution in plasma (CPR = IR x isotopic enrichment/PS). Standard, control, and subject samples were run in triplicate. The intraassay variability [five pools taken from the same 24-h samples and measured five times in the same gas chromatography-mass spectrometry (GC/MS) run] was 9%. The interassay variability (five separate pools taken from the same 24-h samples and measured five separate times by GC/MS) was 5%. The biological variation in the same individual studied on five separate occasions over the span of 16 months was 12%.

Cortisol clearance rates. Subjects with AI were admitted to the inpatient unit of the OHSU GCRC in the evening and had their usual hydrocortisone replacement held. After placement of an iv in each arm, 50 µg of hydrocortisone was infused in one iv from 2000 h until 0800 h the next morning to achieve steady-state levels of cortisol. Beginning at 0400 h during the hydrocortisone infusion, 5 ml of blood was obtained through the opposite iv every 30 min for 10 h for measurement of cortisol levels and calculation of clearance rates.

Clearance rate for cortisol was calculated as follows: the steady-state concentration of cortisol in plasma was assumed to be the mean of the plasma cortisol levels between 0400 and 0800 h in AI subjects receiving a constant hydrocortisone infusion. Cortisol clearance was then calculated by dividing the IR (micrograms/hour of infused cortisol = 4.2 µg/h) by the steady-state concentration (micrograms/deciliter). The half-life of cortisol C(t1/2) was calculated using the formula C(t1/2) = C₀ (e^-t1/2). C₀ is the concentration of cortisol at time zero and was derived from logarithmic transformation of the decay curve (0800–1400 h) and was set as the y-intercept of the transformed curve, assuming monoexponential decay. "k" is the slope of the logarithmic data line fit. "t" represents time, and C(t1/2) was calculated as C(time zero) divided by 2. The equation was then solved for t1/2.

Total and free cortisol and CBG. Total cortisol levels were measured in individual plasma samples taken every 30 min during the 24-h sampling by two-site chemiluminescent immunometric assay (Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA) in the core laboratory of the OHSU GCRC. Intraassay coefficients of variation were 3–5%, and interassay coefficients of variation were 6–10%. Assay sensitivity was 0.8 µg/dl (22.1 nmol/liter). All samples from a single individual were run in duplicate in the same assay. In a second 24-h pooled sample (50 µl from each 30-min sample), free cortisol was measured by 18-h equilibrium dialysis and RIA of the dialyzate (27), and CBG (28) was quantified by RIA (Quest Diagnostics). The intraassay coefficient of variation, interassay coefficient of variation, and assay sensitivity of the free cortisol assay were 9.8%, 12.6%, and 0.03 µg/dl (0.828 nmol/liter), respectively. The intraassay coefficient of variation, interassay coefficient of variation, and assay sensitivity of the CBG assay were 5.8%, 9.2%, and 0.01 mg/liter (0.192 nmol/liter), respectively.

Body composition and leptin. Percentage body fat (% fat), total body FM, and fat-free mass (FFM) were determined in 44 (25 women and 19 men) of the normal, healthy subjects at the time of their inpatient GCRC stay by the method of underwater weighing (29). Leptin was measured in fasting PS using a commercial kit (Linco Research, Inc., St. Charles, MO).

Statistical analysis

AUC cortisol levels were calculated using the trapezoidal method for the entire 24 h and in blocks of 6 h beginning at 2400 h based on previous observations of increased levels occurring just past 2400 and 1200 h in older men and women (20). For comparisons between groups, paired t testing or its nonparametric equivalent, the signed rank test, was used if the data were normally or nonnormally distributed, respectively. Correlational relationships were tested using linear regression or multiple-linear regression analysis. When appropriate, nonnormally distributed variables were natural log-transformed before performing linear regression analysis. Comparison of values in multiple groups were first tested using one-way ANOVA; if significant, then the first and third tertiles were compared using t testing.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The mean age of the group was 46 yr (range, 19–70), and the mean BMI was 32 kg/m² (range, 19–64 kg/m²) (Table 1). Both CPR and CPR adjusted for body surface area, i.e. CPR/BSA, ranged 5-fold from lowest to highest values (Table 1); 24-h plasma free cortisol ranged 3-fold from lowest to highest values (Table 1). CPR and CPR/BSA were positively correlated with 24-h AUC cortisol levels (CPR vs. AUC cortisol, r = 0.30, P = 0.03; CPR/BSA vs. AUC cortisol, r = 0.36, P = 0.008) and 24-h plasma free cortisol levels (CPR vs. free cortisol, r = 0.65, P < 0.001; CPR/BSA vs. free cortisol, r = 0.71, P < 0.001; Fig. 1). CBG levels correlated with 24-h AUC cortisol (r = 0.37, P = 0.007) but not with CPR, CPR/BSA, nocturnal cortisol, or 24-h plasma free cortisol (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Correlation between CPR/BSA and 24-h plasma free cortisol levels (r = 0.71; P < 0.001). Men are shown in closed circles, women in open circles. To convert cortisol values from metric (milligrams per deciliter) to SI (nanomoles per liter), multiply by 27.59.

Men had significantly higher unadjusted CPR (although after adjustment for BSA this difference was no longer statistically significant, P = 0.17), lower CBG, and higher 24-h plasma free cortisol than women (Table 1). To assess the effect of menopausal status on the above variables, the women were divided into groups consisting of those who were premenopausal, postmenopausal but not taking HRT, and postmenopausal taking HRT. These groups did not differ with regard to BMI, percentage body fat, FM, FFM, or leptin levels (data not shown). CBG levels tended to be higher in the postmenopausal women taking HRT (mean ± SD, 27 ± 3.2 mg/liter or 518 ± 61.4 nmol/liter) than the premenopausal women (23 ± 4.6 mg/liter or 442 ± 88.3 nmol/liter) and postmenopausal women not taking HRT (24 ± 4.2 mg/liter or 461 ± 80.6 nmol/liter), but these differences between groups did not quite reach statistical significance by ANOVA (P = 0.06). Including a variable for postmenopausal status in multiple linear regression analysis did not significantly change the results for the associations of body composition, leptin, and age with CPR/BSA and free cortisol described below.

Body composition and leptin

Correlation analysis was used to test the relationships between CPR, cortisol levels, and CGB with body composition and leptin levels separately for men and women (Table 2). Unadjusted CPR was significantly positively correlated with all measures of body weight, body composition, and leptin in men. After adjustment for BSA, CPR/BSA remained significantly positively correlated with percentage body fat and leptin levels. Because leptin levels are closely associated with percentage body fat, multiple linear regression analysis was performed with CPR/BSA as the dependent variable and percentage body fat and leptin as independent variables. In this analysis, CPR/BSA was significantly associated with percentage body fat (standard coefficient, 0.70; P = 0.03) but not leptin (standard coefficient, -0.31; P = 0.31). Neither CBG nor free cortisol was associated with body composition or leptin levels. In women, unadjusted CPR was positively associated with BMI and percentage body fat only; but CPR/BSA, CBG, and free cortisol were not associated with any of the body composition parameters or leptin.

Obese and lean AI subjects exhibited first-order exponential decay curves, and cortisol clearance was significantly higher in the obese group than the lean group (mean ± SD, 19.7 ± 3.8 liters/h vs. 11.3 ± 1.3 liters/h; P = 0.02). C(t1/2) was lower in obese than lean subjects (mean ± SD, 1.5 ± 0.1 h vs. 2.2 ± 0.5 h; P = 0.03; Fig. 2).

View larger version (19K): [in this window] [in a new window]   FIG. 2. C(t1/2) in obese (•) and lean () subjects with AI. Time zero = 0800, when a constant infusion of hydrocortisone was discontinued (see Subjects and Methods for full details).

Increasing age correlated positively with CPR/BSA and 24-h plasma free cortisol in both men and women when examined separately and, therefore, were combined in subsequent analysis. In this combined group, increasing age correlated positively with CPR/BSA (r = 0.28; P < 0.05; Fig. 3), 24-h AUC cortisol (r = 0.37; P = 0.006), and 24-h plasma free cortisol (r = 0.37; P = 0.006; Fig. 3). CBG levels were not correlated with increasing age in the men, women, or the combined group (data not shown). Multiple linear regression was used to determine the independent effects of age and gender on these variables of cortisol metabolism. The addition of gender did not affect the significant relationships between age and CPR/BSA (standard coefficient, 0.29; P < 0.05), 24-h AUC cortisol (standard coefficient, 0.36; P = 0.008), or 24-h free cortisol (standard coefficient, 0.39; P = 0.002).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Correlation between age and 24-h plasma free cortisol (top) (r = 0.37, P = 0.006), and CPR/BSA (bottom) (r = 0.28; P = 0.04). Men are shown in closed circles, women in open circles. To convert cortisol values from metric (milligrams per deciliter) to SI (nanomoles per liter), multiply by 27.59.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Mean ± SE plasma cortisol levels every 30 min over 24 h in the oldest subjects [highest tertile for age, open triangle; n = 18] and youngest subjects [lowest tertile for age, closed triangle; n = 18]. Significance values represent comparison of AUC cortisol levels measured between 1200 and 1800 h, and between 2400 and 0600 h between the oldest and youngest tertiles. To convert cortisol values from metric (milligrams per deciliter) to SI (nanomoles per liter), multiply by 27.59.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

It is possible that enhanced CPR, when coupled with increased free cortisol levels, could adversely affect body composition by increasing FM and decreasing lean mass as seen in Cushing’s disease. This hypothesis is supported by several animal models of obesity (e.g. ob/ob and db/db mice, and fa/fa rats) in which the expression of obesity can be prevented or reversed by adrenalectomy or treatment with the glucocorticoid receptor antagonist RU-486 (33, 34, 35, 36). In the present study, CPR was significantly associated with increasing percentage body fat in both men and women; but CPR/BSA, 24-h plasma free cortisol, and 24-h cortisol binding globulin levels were not significantly associated with either FM or non-FM in either group. These findings, plus our finding of an elevated cortisol clearance rate in obese vs. lean subjects, support the conclusion that enhanced CPR in obese subjects is due to increased plasma volume, cortisol clearance, or both, resulting in normal levels of biologically available cortisol. Therefore, activation of the hypothalamic-pituitary- adrenal (HPA) axis in obese humans is unlikely to be a primary cause of total FM accumulation.

It remains of interest, however, to note the large normal range of both CPR (a >5-fold range from lowest to highest values) and 24-h plasma free cortisol levels (a >3-fold range from lowest to highest values) in the present study. The strong association of increasing CPR, even after adjustment for body size, with increasing 24-h plasma free cortisol (Fig. 1) implies that increased HPA axis activity does result in significantly higher levels of available cortisol to the tissues of the body. Therefore, although our data do not support a role for activation of the HPA axis as a cause of excess total body fat in human obesity, it is still possible that increased CPR and free cortisol may significantly affect systems involved in fat patterning (i.e. visceral fat accumulation) and glucose metabolism in humans. In the text that follows, data from the present study will be used to illustrate this concept with respect to determination of visceral fat accumulation in men vs. women and with aging.

Men have a greater amount of visceral fat than women even after matching for total FM (37). In the present study, we find that men have significantly higher CPR, higher 24-h plasma free cortisol levels, and lower 24-h CBG levels than women. Although previous studies have separately reported no difference in CPR (5), total or free cortisol levels (11, 38), or CBG levels (38, 39) between men and women, our data measured these values within the same subject group, used 24-h sampling to avoid discrepancies due to circadian cycling, and are in agreement with data from more recent studies (10, 31, 40). Therefore, our data support a role for an increase in HPA axis activity in the expression of greater central obesity in men than women.

Another population in which visceral fat accumulation has been reported is in aging humans (41), and activity of the HPA axis in aging has been the focus of a number of investigators. Our study reports for the first time that CPR, CPR/BSA, 24-h AUC cortisol, and 24-h plasma free cortisol are all positively correlated with age in the same study group. In a study by Van Cauter et al. (20) that combined published data from seven different groups, a significant relationship between increasing age and increasing average daily cortisol levels in men and women was demonstrated. It was assumed that increases in serum cortisol levels reflected an increase in the basal cortisol secretion rate. It is possible, however, that the observed increase in cortisol concentration represented impairment in cortisol clearance with aging, possibly due to an age-associated reduction in FFM (12). If a defect in metabolic clearance were the primary abnormality in aging leading to increased blood cortisol levels, then CPR would be expected to be normal or low. In the present study, however, CPR was found to increase with aging and to be associated with elevated plasma free cortisol levels. These data, therefore, support a role for dysregulation of HPA axis activity and elevated daily plasma free cortisol levels in the expression of central obesity with aging.

The increase in AUC cortisol with aging was found to be the result of an increase in cortisol levels between 1200 and 1800 h and between 2400 and 0600 h. Although a disruption of the normal nocturnal cortisol levels could be predicted by data from other studies (18, 20, 42), the clear rise in cortisol levels after the noontime meal with aging deserves further study. A similar increase in cortisol in the early afternoon after lunch has been reported in a group of centrally obese men with the BclI 4.5-kb variant of the glucocorticoid receptor by Rosmond et al. (43) compared with a group of less centrally obese men. The reason for this early afternoon rise in cortisol is not clear. A meal-stimulated rise in cortisol has been reported before, particularly after ingestion of a high-protein meal (44, 45). Gastric inhibitory peptide (GIP) secretion is increased by meals and can induce cortisol secretion by the adrenal gland in an ACTH-independent manner when GIP receptors are ectopically expressed in adrenal glands (46, 47); however, GIP receptors are not normally expressed or known to be functional in normal adrenal tissue. Understanding this apparently meal-associated abnormality in cortisol secretion could, therefore, involve interactions between other gut-dependent hormones and central systems involved in generating the circadian rhythm of cortisol.

It is important to point out, however, that the HPA axis is not the only pituitary axis whose secretion has been shown to become dysregulated with aging. GH secretion declines with age, and GH replacement has been shown to reduce FM, increase lean mass, and reduce abdominal fat in healthy, older men and women (48, 49, 50). It is possible, therefore, that abnormalities in both cortisol and GH secretion contribute to changes in fat distribution with aging and may reflect a primary abnormality in one axis affecting the other (i.e. hypercortisolism inhibiting GH secretion or vice versa) or abnormalities in secretion of both hormone axes may be due to a common upstream regulatory abnormality. Testing the independence or interrelatedness of these pituitary systems will require prospective studies that manipulate each axis independently and measure the effect on the other axis and body composition.

In summary, we have measured 24-h CPR and daily free cortisol, demonstrating a wide range of normal CPR and cortisol levels in humans of various body weights and ages. Increasing body weight is associated with increasing CPR, which is balanced by enhanced cortisol clearance, resulting in daily plasma free cortisol levels that are invariant to increasing body size. These data indicate that human obesity is not characterized by a dysregulation of the HPA axis. On the other hand, we demonstrate that men have higher CPR and daily free cortisol than women and that aging is associated with an increase in CPR, independent of weight, which accounts for the increased daily free cortisol of the oldest vs. youngest subjects. Given that CPR/BSA is strongly and positively associated with 24-h plasma free cortisol levels, activation of the HPA axis may, therefore, be a determinant of the increased central fat distribution and associated disturbances in glucose and lipid metabolism previously documented between men and women and with aging.

	Acknowledgments

 
We thank Holly Callahan for invaluable services as patient coordinator and Dr. John Brunzell for support of these studies.

	Footnotes

 
This work was supported by the General Clinical Research Centers of the University of Washington (M01-RR00037), Oregon Health and Science University (M01-RR00334), and National Institutes of Health Grant R03-DK61996 (to J.Q.P.).

Abbreviations: AI, Adrenal insufficiency; AUC, area under the curve; BMI, body mass index; CBG, cortisol-binding globulin; CPR, cortisol production rate(s); CPR/BSA, CPR adjusted for body surface area; C(t1/2), half-life of cortisol; FM, fat mass; FFM, fat-free mass; GC/MS, gas chromatography-mass spectrometry; GCRC, General Clinical Research Center; GIP, gastric inhibitory peptide; HPA, hypothalamic-pituitary-adrenal; HRT, hormone replacement therapy; IR, infusion rate; OHSU, Oregon Health and Science University; PS, plasma sample(s).

Received March 12, 2003.

Accepted September 24, 2003.

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