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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 1 215-219
Copyright © 2003 by The Endocrine Society

Original Article

Urinary Free Cortisol Increases in Adolescent Caucasian Females during Perimenarche

Richard S. Legro, Hung Mo Lin, Laurence M. Demers and Tom Lloyd

Departments of Obstetrics and Gynecology (R.S.L.), Health Evaluation Sciences (H.M.L., T.L.), and Pathology (L.M.D.), College of Medicine and University Hospitals, Penn State College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Richard S. Legro, M.D., Department of Obstetrics and Gynecology, Room C3608, 500 University Drive, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033. E-mail: rsl1{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Urinary free cortisol (UFC) excretion has been thought to be constant during female reproductive maturation when normalized for body surface area. We sought to determine whether there are longitudinal changes in urinary free cortisol excretion during perimenarche in adolescent females. We performed a longitudinal study of 24-h UFC excretion obtained at 6-month intervals over a 4-yr period in a cohort of 112 adolescent non-Hispanic white perimenarchal females from south central Pennsylvania. The overall mean values (mean ± SD) for UFC/24 h for all measurements between ages 12 and 17 yr was 67.4 ± 43.8 µg/24 h (to convert to nanomoles per day, multiply by 2.759). In our model, we found a significant positive association between UFC excretion with both gynecological age (P = 0.002) and chronological age (P = 0.0001). For every incremental increase in Tanner stage, the UFC/BSA increased by 3.0 µg/24 h per square meter. Correcting the UFC values by both creatinine and BSA creates a fairly constant number (6.3 ± 3.1 µg/mg per square meter per 24 h) over the age range 12–17 yr represented in this study. An increase in cortisol excretion may be part of normal reproductive maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PERIMENARCHE IS AN understudied area with sparse prospective longitudinal data to edify. Cortisol is thought to have a minor role, if any, in reproductive maturation. The secretory rate of cortisol is thought to be constant during puberty (1). Published reports on normal urinary free cortisol (UFC) values in adolescent women have been limited by small numbers, incomplete timed urinary collections (<24 h), and limited longitudinal follow-up (2, 3, 4, 5). The largest study of this age group that included 57 girls was cross-sectional but reported constant values with age when UFC was normalized for body surface area, consistent with the finding of a constant secretory rate (6). Smaller longitudinal studies have also shown no increase in UFC when normalized for body surface area (7).

We examined age-related trends, both in terms of chronological and gynecological age, in UFC in a sample of 112 premenarchal girls who participated in the Penn State Young Women’s Health Study over a 4-yr period surrounding menarche to analyze the progression of the individual over time in relation to developmental milestones. Our prior study of sex steroids and gonadotropins had suggested clear developmental increases (8), and we theorized there would be similar trends in cortisol. There is evidence of this in mammals (9), and a recent study of salivary cortisol in adolescent humans had also shown increased levels with age (10).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The mean age at initiation into the study, begun in 1990, was 11.9 ± 0.5 yr (mean ± SD). All procedures involving human subjects were reviewed and approved by the Institutional Review Board for clinical research studies at the Pennsylvania State University College of Medicine. All subjects gave written informed consent. Details of the recruitment and retention strategies and baseline anthropometric, endocrine, and bone measurements have been reported (8). The study originally started as a randomized trial of calcium supplementation to examine the effects of calcium on bone mass accretion. The initial positive effect of calcium supplementation on bone accretion in this cohort disappeared over time as hormonal factors became dominant (11). At the baseline visit, 48% were randomized to calcium and remained around 50% for each subsequent visit because calcium supplementation continued throughout the study.

The study group represents a Caucasian female adolescent population of European ancestry attending public school in central Pennsylvania. All study participants were seen and examined at 6-month intervals throughout the study. The average relative difference between actual and target visit date was 1.5%. The physical characteristics of the study groups were similar to appropriate age-matched national normative values (12). All women were examined for height, weight, and Tanner breast staging by the same research nurse at each visit. body surface area was calculated according to the formula: exp[-3.751 + 0.422 log(height) + 0.515 log(weight)]. During the 4 yr of this study, 88 (79%) of the original 112 participants remained in the study. No significant differences were noted between those who dropped out and those who remained in the study in terms of baseline age, height, weight, or baseline bone measurements.

The percentage body fat was determined at 6-month intervals by dual-energy x-ray absorptiometry (DXA) scan, and skinfold measurements were obtained with calipers at five sites: triceps, subcapsular, umbilicus, suprailiac, and midthigh (13). Body composition analyses, including percentage body fat, was obtained from the DXA scans performed with a QDR-2000 instrument (Hologic, Inc., Waltham, MA). As has been reported by others (14), our observed coefficient of variation was less than 0.7% for the day-to-day quality control scans using the manufacturer’s spine phantom. All body composition scans were obtained using the pencil beam mode in the presence of the three-step acrylic/acrylic-aluminum wedge standard that simulates lean and soft tissue (Hologic, Inc.; Ref. 15). Body composition analysis was performed using software version 5.71A (Hologic, Inc.).

The date of menarche was based on interviews and questionnaire data. The mean age of menarche for the cohort was 13.3 ± 1.0 yr. Gynecological age was based on the year of menarche (0 yr). Twenty-four-hour urine specimens were obtained from each subject every 6 months during the 4 yr of the study for measurement of UFC; 99.7% of scheduled visits were completed. The urines were collected independent of menstrual bleeding and cycle day. Data obtained during birth control pill use were excluded. UFC assays were performed throughout the study period using established RIA methods that employ methanol extraction before assay. The day-to-day reproducibility of the urinary cortisol measurements during this study averaged 8% at a mean concentration of 35 µg/24 h. Assay sensitivity was 5 µg/24 h. Cross-reactivity for the cortisol assay with other steroids is minimal except for 11-deoxycortisol and prednisolone in which the cross-reactivity is 11% and 76%, respectively. Steroids that had less than 1% cross-reactivity with our cortisol assay included aldosterone, cortisone, progesterone, estrone, and estradiol.

Statistical procedures were accomplished using a range of procedures in SAS (SAS Institute, Inc., Cary, NC) and Splus (StatSci, Seattle, WA). For the various endocrine and body composition parameters obtained every 6 months, starting up to 2 yr before menarche and up to 3.5 yr post menarche, the multivariate analyses of variance was performed using PROC MIXED (SAS Institute, Inc.) and accounting for the time effect. Unlike the usual ANOVA and regression models, this model accommodates the within-subject and between-subject variability that are inherent in a longitudinal study. Linearity of the various parameters over time was examined. We also stratified the analysis according to calcium supplementation, and there was no effect of this on any of the measured outcomes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The overall mean values (mean ± SD) for UFC/24 h for all measurements between ages 12 and 17 yr was 67.4 ± 43.8 µg/24 h (to convert to nanomoles per day, multiply by 2.759). We examined the changes in relation to gynecological age and chronological age in the cohort (Figs. 1Go and 2Go). In our model, we found a significant positive association between UFC excretion with both gynecological age (P = 0.035, test of third degree of polynomial term of the curve) and chronological age (P = 0.028). The mean values (mean ± SD) for UFC/creatinine between ages 12 and 17 yr in our cohort was 9.4 ± 4.6 µg/mg per 24 h. We also found significant time-related increases in UFC/creatinine excretion by chronological age (P = 0.002, test of slope) and gynecological age (P < 0.001, test of slope). The mean values (mean ± SD) for UFC/BSA between ages 12 and 17 yr in our cohort was 44.70 ± 27.4 µg/m2 per 24 h. The UFC/BSA patterns during ages 12–17 yr resemble the patterns of UFC excretion both with chronological and gynecological ages. When both gynecological and chronological ages are included in our multivariate model, neither is an independent predictor of UFC.



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  		Figure 1. Changes (mean ± SE) in daily UFC, UFC/creatinine ratio, UFC/body surface area, and UFC/creatinine/BSA by chronological age. n, Number of subjects at each time point (to convert to nanomoles, multiply by 2.759).

 


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  		Figure 2. Changes (mean ± SE) in daily UFC, UFC/creatinine ratio, and UFC/body surface area by gynecological age (age at menarche = 0). n, Number of subjects at each time point (to convert to nanomoles, multiply by 2.759).

 
For every incremental increase in Tanner stage, the UFC/BSA increased by 3.0 µg/24 h per square meter (Fig. 3Go). This was significant both by the trend test (P = 0.003) as well as ANOVA [Tanner stage II vs. 5 (P = 0.011), Tanner stage III vs. 5 (P = 0.003)]. Alternatively, correcting the UFC values by both creatinine and BSA created a fairly constant number (6.3 ± 3.1 µg/mg per square meter per 24 h) over the age range 12–17 yr represented in this study [P = 0.66, test of slope for chronological age, and P = 0.17, test of slope for gynecological age]. There were no significant changes in body composition either as determined by DXA or using skinfold thickness over the course of this study (Table 1Go).



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  		Figure 3. Changes (mean ± SE) in daily UFC/body surface area excretion Tanner stage of the breast. n, Number of subjects at each time point (to convert to nanomoles, multiply by 2.759).

 

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  		Table 1. Subject characteristics [mean ± SD (n)]

 
We examined the relationship between growth rate and cortisol excretion in our cohort with both chronological and gynecological ages. For girls with the same age at menarche or the same chronological age, there was a nonsignificant relationship between the increase in height and values for cortisol, cortisol/BSA, and cortisol/creatinine.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings of increased UFC, both when corrected for creatinine excretion and BSA with aging during the perimenarchal years, were novel. These findings may be related to the large size of our cohort, its exclusively female focus, its ethnic and socioeconomic homogeneity, and the longitudinal study design with a high retention rate. As such they may not be applicable to other ethnic groups or cultures. These findings suggest that normative values for UFC excretion must also be corrected for chronological or reproductive age as well as for body surface area. There appears to be as much as an approximately 40% difference between baseline and peak UFC/BSA values in a healthy population of young women over the perimenarchal period. Alternatively, correcting the UFC values by both creatinine and BSA creates a fairly constant number over the age range 12–17 yr represented in this study.

Twenty-four-hour urinary collections provide a better time-integrated view of adrenocortical activity than isolated fluctuating levels of serum cortisol (16) and for this reason are often used as a screening test for Cushing’s syndrome. Our study was unable to assess whether increased excretion reflects changes in production rate, circulating levels of binding proteins, or renal function over this period of development. We have previously demonstrated both rapid and significant changes in reproductive hormones, primarily related to the hypothalamic-pituitary-ovarian axis during perimenarche (8). Our collections were obtained by design independent of cycle day (the study was initiated among premenarchal women) and thus reflect quantitative changes over the whole menstrual cycle as these developed in the individual women. Our sample size was large enough to allow for random distribution of visits throughout the menstrual cycle and allowed for meaningful inferences about cortisol excretion. Additionally we found no change in body fat composition over this period that may, especially in females, confound UFC (17). We found no association between growth rate and cortisol excretion, but this may be because our cohort was began around age 12 yr, right at the time that the growth rate peaks (~ 1 yr before menarche) (18). Therefore, a younger cohort may be more appropriate to examine this relationship.

Our findings pose as many questions as they provide answers. Our study design does not allow us to answer questions about production rates during puberty, and we have no abnormal or other comparison groups. Differences have been noted in the sensitivity of the hypothalamic-pituitary-adrenal axis in African-American girls, compared with Caucasian girls (19). Thus, our findings remain applicable only to females of Caucasian descent. A recent finding in girls with congenital adrenal hyperplasia showed an increased clearance of serum cortisol and corresponding decreased half-life, compared with age-matched boys (20). Thus, our findings may be gender specific. The primary mechanism of increased cortisol clearance in this group of endocrinologically abnormal girls was projected to be a pubertal-related inhibition of 11-ß hydroxysteroid dehydrogenase, type I, which converts cortisone to the more potent glucocorticoid cortisol (21). This, combined with other mechanisms triggered by the endocrinology of puberty, such as increased circulating IGF-1 levels, contributes to increased renal excretion of cortisol. IGF-1 increases the glomerular filtration rate by a direct effect on the glomerular vessels (22).

The authors theorized this may contribute to a relative hypocortisolism during puberty in girls with congenital adrenal hyperplasia and thus increased difficulty in suppression of the hypothalamic-pituitary-adrenal axis (21). We could speculate a similar mechanism at work here in our endocrinologically normal cohort. Increased cortisol clearance would favor the growth and maturation of the reproductive system by avoiding the peripheral catabolic effects of potent glucocorticoids. Protein breakdown and glucose shunting away from the reproductive tract would be a few of the undesirable glucocorticoid effects during perimenarche. Such a relative hypocortisolism would also favor the anabolic effects of insulin and insulinlike growth factors. Hyperinsulinemia and insulin resistance have been well documented during puberty and theorized to favor growth (23, 24). But without further data regarding the production rates and kinetics of cortisol clearance during perimenarche, this remains speculation. We limit our conclusions therefore to our collected evidence, that reproductive maturity is associated with increased urinary cortisol excretion even after correction for body surface area or creatinine excretion.


   Acknowledgments
 


   Footnotes
 
This work was supported by Public Health Service Grant RO1-HD25973 (to T.L.), Grant K24 HD01476 (to R.S.L.), and General Clinical Research Grant MO1-RR10732 (to Pennsylvania State University).

Abbreviations: DXA, Dual-energy x-ray absorptiometry; UFC, urinary free cortisol.

Received February 19, 2002.

Accepted September 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  7. Honour JW, Kelnar CJ, Brook CG 1991 Urine steroid excretion rates in childhood reflect growth and activity of the adrenal cortex. Acta Endocrinol (Copenh) 124:219–224
  8. Legro RS, Lin HM, Demers LM, Lloyd T 2000 Rapid reproductive maturation during perimenarche independent of body composition. J Clin Endocrinol Metab 85:1021–1026[Abstract/Free Full Text]
  9. Van Kampen M, Fuchs E 1998 Age-related levels of urinary free cortisol in the tree shrew. Neurobiol Aging 19:363–366[CrossRef][Medline]
  10. Kiess W, Meidert A, Dressendorfer RA, Schriever K, Kessler U, Konig A, Schwarz HP, Strasburger CJ 1995 Salivary cortisol levels throughout childhood and adolescence: relation with age, pubertal stage, and weight. Pediatr Res 37(4 Pt 1):502–506
  11. Lloyd T, Johnson-Rollings N, Chinchilli V 1997 The effect of enhanced bone gain achieved with calcium supplementation during ages 12–16 does not persist in late adolescence. In: Burckhardt P, Dawson-Hughes B, Heaney RP, eds. Proc Third International Symposium of Nutritional Aspects of Osteoporosis, Boston, MA, Serono Symposium Series, Springer Verlag, pp 11–25
  12. Schlicker SA, Borra ST, Regan C 1994 The weight and fitness status of United States children. Nutr Rev 52:11–17[Medline]
  13. Katch FI, Behnke AR, Katch VL 1979 Estimation of body fat from skinfolds and surface area. Hum Biol 51:411–424[Medline]
  14. Johnston Jr CC, Slemenda CW, Melton 3rd LJ 1991 Clinical use of bone densitometry. N Engl J Med 324:1105–1109[Medline]
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  19. Yanovski JA, Yanovski SZ, Cutler Jr GB, Chrousos GP, Filmer KM 1996 Differences in the hypothalamic-pituitary-adrenal axis of black girls and white girls. J Pediatr 129:130–135[CrossRef][Medline]
  20. Charmandari E, Hindmarsh PC, Johnston A, Brook CG 2001 Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: alterations in cortisol pharmacokinetics at puberty. J Clin Endocrinol Metab 86:2701–2708[Abstract/Free Full Text]
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