Original Article

Circadian rhythm of urinary steroid metabolites

Walid K Jerjes1, Anthony J Cleare2, Timothy J Peters1 and Norman F Taylor1

Abstract
Addresses 1 Department of Clinical Biochemistry, Guys, Kings College London School of Medicine, Bessemer Road, London SE5 9RS and 2 Section of Neurobiology of Mood Disorders, Division of Psychological Medicine, The Institute of Psychiatry and Guys, Kings College London School of Medicine, De Crespigny Park, London SE5 8AF, UK Correspondence: Mr Walid K Jerjes Email: w_jerjes@yahoo.co.uk

Background Samples submitted for urinary steroid prole analysis are often untimed, but inuence of collection time on interpretation is unknown. We report circadian rhythms of the major steroid metabolites and derived ratios in urine collected at 3-h intervals over 24 h, after rst establishing that disturbance of sleep associated with collection does not alter rhythms on the succeeding day. Methods Assay of steroid metabolites (gas chromatography) and creatinine in urine collections made by 10 men and 10 women every 3 h starting at 2100 for 24 h. Data were subjected to cosinor analysis. Results Summed cortisol and androgen metabolites exhibited signicant circadian rhythms, as expected, but with a surprisingly long time-lag (maxima at around 1400). Amplitudes were different, so that the ratio cortisol/androgen metabolites also showed a signicant rhythm. The ratios 5a/5b tetrahydrocortisol and 20-hydroxy/20-oxo cortisol metabolites showed signicant rhythms which were not in phase with total cortisol metabolites, while 11-hydroxy/11-oxo cortisol metabolites showed no rhythm. There were no gender differences in time of maxima. Previously established gender differences in metabolite levels were conrmed. Creatinine levels showed no circadian rhythm. Conclusion Circadian variation should be considered when interpreting results from urine steroid analysis. Calculation of steroid/steroid or steroid/creatinine ratios is not informative in untimed collections.
Ann Clin Biochem 2006; 43: 287294

Introduction
Urinary steroid proling by gas chromatography (GC) or gas chromatography--mass spectrometry is useful for investigation of disordered steroid production and metabolism and, when 24-h collections are made, for determination of steroid production rates. During the provision of a referral service, we have noted a substantial clinical demand for analysis of untimed collections, most commonly taken from children with possible precocious adrenarche and a general belief that relating steroid levels to creatinine would oset any disadvantage of a short collection. The circadian rhythm of these metabolites remains unreported. This study set out to examine the variability of steroid metabolites and creatinine obtained in sequential 3-h urine collections over one day in adult volunteers under carefully controlled conditions of collection, since the circadian rhythm of cortisol secretion is entrained by the lightr 2006 The Association for Clinical Biochemistry

dark1,2 and sleep-wake cycles3, while age, weight and the timing of meals may also be inuential. Since volunteers had to wake up during the night during 24 h sampling we checked whether this disturbed the rhythm the following day by comparing results with those of 3 h sampling during the daytime only. Urine steroid proling in sequential collections may have a role in the investigation of disorders in which circadian variation of cortisol is altered, such as Cushings syndrome4 and depression.5 Single serum cortisol determinations are accepted as unreliable for evaluation of adrenocortical function6 and stress associated with sequential blood sampling may also make it dicult to establish the cortisol rhythm unless an indwelling cannula is used; even then, diculties often arise with blockages, pain and the need for resiting cannulae. Urinary free cortisol shows a similar rhythm to plasma cortisol, but with a delay of about 3 h.7 However, less than 5% of secreted cortisol is cleared as free corti287

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sol; the rest is excreted as cortisol metabolites.8 Levels of total cortisol metabolites (TCM) provide a sensitive means of detecting changes in rates of cortisol secretion, as in asthmatics treated with inhaled glucocorticoids.9 Quantication of individual metabolites permits examination of changes in cortisol metabolism such as cortisol--cortisone interconversion. Changes in this equilibrium are associated with some disorders such as obesity10 and hirsutism,11,12 but whether there are circadian changes in normal or pathological situations has never been explored. We also examined possible relationships of age and body mass index with circadian rhythm.

Depression scale (HADS) were 2.871.9, 3.473.3 and 3.373.1, 3.773.1, respectively, for anxiety and 3.272.3; 2.773.3 and 2.172.4; 2.572.8, respectively, for depression. Signicant levels of depression and anxiety are usually dened as 10 or more.15

Steroid metabolite assay

Urinary steroid prole analysis used high-resolution gas chromatography of methyloxime-trimethylsilyl ether (MO-TMS) derivatives, as previously described.16 The intra- and interassay coecients of variation were between 7.1--21.1% and 11.2--21.9%, respectively, for dierent metabolites. Each metabolite was calculated as mg/3 h period. Derived sums were as previously reported.17 Urinary androgen metabolites (AM) were determined from the sum of androsterone and aetiocholanolone. TCM were the sum of tetrahydrocortisone (THE), tetrahydrocortisol (THF), allo -tetrahydrocortisol (5aTHF), a-cortolone, b-cortolone, a-cortol, and b-cortol. 20-hydroxy metabolites of cortisol (20OH) were the sum of a-cortolone, b-cortolone, a-cortol, and b-cortol, and 20-oxo metabolites of cortisol (20OXO) were the sum of THE, THF, and 5aTHF. The ratio of THF 5aTHF/THE (11OH/11OXO) was calculated as an index of total net 11b-hydroxysteroid dehydrogenase (11bHSD) activity. The ratio 5a/5b THF was calculated as an index of 5a versus 5b reduction and 20OH/20OXO as an index of net 20-HSD activity.

Materials and methods

Volunteer subjects
All volunteers were healthy and recruited from among hospital sta and students. One group (10 m, 10f) made sequential 3-h urine collections from 0600 to 2100 (DT group). Men and women were matched for age (32712 and 347 11 years) and body mass index (BMI) (2474 and 2475 kg/m2), respectively (all mean7SD). A second group (10 m, 10f, 17 were also in the day time [DT] group) made sequential 3-h urine collections, starting at 2100, for 24 h (DT night time [NT] group). They were also matched for age (31712 and 33712 years) and BMI (2474, and 2375 kg/m2), respectively. Subjects were all studied during winter, between October 2001 and March 2002. No female subjects were taking the oral contraceptive. Menstrual cycle was not recorded, since there are no changes of the studied steroid metabolites over the menstrual cycle (NFT unpublished observation) and no relationship of menstrual cycle and cortisol circadian rhythm has been found using saliva sampling.13 All subjects had been medication-free for at least two weeks, did not smoke, were asked to limit their intake of caeine and alcohol during the collection period and had no history of hypersensitivity to corticosteroids. All had normal dietary habits, taking breakfast, lunch and dinner at about the same time. All subjects habitually went to bed between 2300 and 0100 and got up between 0600 and 0800. All subjects provided written informed consent before participation. Kings Research Ethics Committee Approval for this study was obtained.

Creatinine assay
Urinary creatinine was measured by a Bayer Advia 1650. Values were calculated as nmol/3 h in order to examine steroid/creatinine ratios.

Circadian rhythm analysis

Individual and population mean cosinor analysis to determine circadian rhythm parameters of each variable was performed using TSA-Seriel Cosinor software (Expert Soft Technologie, Laboratoire dInformatique BioMe dicale, France) for analysis of biological time series by least-squares estimation. Population mean cosinor analysis is based on the means of parameter estimates obtained from individuals in the study sample to derive the following parameters: (1) the goodness of t of a cosinor curve tted to the data; (2) midline estimate statistic of rhythm (MESOR), dened as the rhythm-adjusted mean; (3) amplitude, dened as half the extent of rhythmic change in a cycle approximated by a tted curve (dierence between nadir and peak); (4) acrophase, dened as the time of peak in the cosinor curve tted to the data. The acrophase is expressed as a phase angle in degrees, so the formula (Value in degrees/3601) 24 h) can be used to establish the clock time of the peak.

Questionnaires
All groups had a good sleep quality. Total Pittsburgh Sleep Quality Index (PSQI) scores were (men, DT) 2.471.3; (women, DT) 2.071.3 and (men, DT NT) 3.372.3; (women, DT NT) 2.872.0, respectively (all mean7SD). Good is dened as a score of 7 or less.14 They did not have clinically signicant levels of anxiety or depression. Scores for the Hospital Anxiety and
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Statistical analysis
Comparisons between results for the entire period in men and women were made by the repeated measures analyses of variance (ANOVA). Additionally, we performed post hoc t-tests on values for each of the 3-h blocks over 24 h to determine if there were any dierences in cortisol levels at a particular time of the day. The coecient of correlation between circadian rhythm parameters, age and BMI was calculated by the general linear regression method.

(a) 2000 1800 1600 1400 1200 1000 800 600 400 200 0

Cortisol metabolites (TCM) Women Men * *

** * * * * *

g/3 h

2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time (b) 600 500 g/3 h 400 300 200 100 0 2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time (c) 5.0 4.0 Ratio 3.0 2.0 1.0 0.0 2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time TCM/AM * * * ** Androgen metabolites (AM) ** ** Women Men

Results
Comparison of TCM excretion between 0600 and 2100 (DT) and the excretion for the same period within the DT NT series (Table 1) showed there was a signicant circadian rhythm in both men and women which did not dier between the DT and DT NT conditions. Thus, the following analyses were based on DT NT collections only.

Gender differences
Levels of TCM were signicantly higher in men (P 0.008) and at all time points: Po0.005 for 1200--1500, and Po0.05 for the rest of the periods (Figure 1a). Levels of AM were also signicantly higher in men (Po0.0005) and at all time points except for 0600--0900 and 1800--2100: Po0.005 for 0900--1800, and Po0.05 for 2100--0600 (Figure 1b). The ratio of 11OH/11OXO was signicantly higher in men (Po0.05), but no dierences were observed in post hoc t-tests between men and women at any 3-h period (Figure 2a). The ratio of 5a/5b THF was signicantly higher in men (F (1,18) 9.8, P 0.006) and at all time points (2100--1200, Po0.05; 1200--2100, Po0.005) (Figure 2b). The ratio of 20OH/20OXO was signicantly lower in men (Po0.05), but no dierences were observed for any 3-h period over 24 h (Figure 2c).

Women Men

Figure 1 Mean values and standard errors of (a) total urinary cortisol metabolite excretion; (b) urinary androgen metabolites and (c) cortisol metabolites/androgen metabolites at 3-h intervals over 24 h. *Po0.05, **Po0.005

Table 1 Effect of prior night-time collections on circadian rhythm parameters of TCM between 0600 and 2100: comparison of collections made only in this period (DT) with collections made as part of a 24 h series (DT+NT) Males DT DT+NT Females t-test DT N/A 71% Po0.0001 0.6 0.7 0.8 DT+NT 72% Po0.001 t-test N/A

% Rhythm 77% Po0.0001 72% po0.001 (goodness of t) MESOR (mg/3 h) 1025 (6571325) 1031 (6701330) Amplitude (mg/3 h) 513 (429635) 542 (337627) Acrophase 206 (235 to 177) 210 (240 to 189)

467 (291561) 470 (284570) 0.8 222 (220270) 235 (214296) 0.6 201 (221 to 184) 212 (256 to 169) 0.9

Values are expressed as means (95% condence intervals), Acrophase is presented as phase angle in degrees where 3601=24 h DT, day time; NT, night time
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(a) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Jerjes et al.
11OH/11OXO Men Women

2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time (b) 2.0 1.5 Ratio 1.0 0.5 0.0 2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time (c) 20OH/20OXO Men Women * * * * * ** 5 /5 THF Men Women ** **

There were signicant rhythms for 5a/5b THF and 20OH/20OXO, but no signicant rhythm of the 11OH/ 11OXO ratio in either men or women. The MESORs for TCM, 11OH/11OXO ratio and 5a/5b THF ratio were higher in men than in women, while the 20OH/ 20OXO ratio MESOR was lower. For amplitude, only TCM diered between groups, being signicantly higher in men than women. The acrophase of TCM occurred signicantly earlier than those for the 5a/5b THF and 20OH/20OXO ratios in both men and women. Calculated acrophases are also shown for the 11OH/11OXO ratio in Table 2, but since the circadian rhythm was not signicant these would not be valid. There were no signicant dierences between men and women in age, BMI and acrophase of TCM. When the data were combined, there was a signicant negative correlation between age and acrophase of TCM (r 0.93, Po0.001), whereas a non-signicant negative correlation was seen between BMI and acrophase of TCM (r 0.64, P 0.07).

Ratio

Discussion
0.50 0.40 Ratio 0.30 0.20 0.10 0.00 2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time

Effect of sleep disruption

This study established that waking every 3 h to pass urine does not have a signicant eect on subsequent steroid levels, so our 24-h data are likely to provide a valid indicator of the normal rhythm of steroid metabolite excretion. This accords with studies of the eects of sleep disruption on HPA axis activity, reviewed by Steiger,18 which indicate that the cortisol circadian rhythm in blood is relatively robust. Night shift work does not result in any change in the acrophase in the rst days, although there is a decrease of rhythm amplitude.19 After East--West air travel, adjustment of the cortisol rhythm requires several days.20 Experimental regimes of sleep deprivation have shown relatively small shortterm changes.21 Anticipation of awakening induces a marked increase of ACTH,22 so that, paradoxically, a study in which an experimenter wakes the subject may be less disruptive than our protocol, in which the volunteers planned their own awakenings. However, no eect on this of our collection regime was evident.

Figure 2 Mean values and standard errors of (a) 11OH/ 11OXO ratio; (b) 5a/5b THF ratio and (c) 20OH/20OXO ratio at 3-h intervals over 24 h. *Po0.05, **Po0.005

Circadian rhythm
Cosinor analysis derived population mean circadian parameter estimates by gender are detailed in Table 2. Both males and females showed a signicant rhythm over 24 h of TCM and AM. The amplitude of the TCM rhythm was lower than that of the AM rhythm, so that TCM/AM also showed a circadian rhythm (Figure 1c). Creatinine level showed no circadian rhythm, so that AM/creatinine and TCM/creatinine showed circadian rhythms with similar characteristics to AM and TCM alone (Figure 3 a and b). The amplitude and MESOR of AM were higher in men compared to women, with no changes in the acrophase. The acrophase of TCM was not statistically dierent from AM, although TCM lagged behind AM by 56 and 44 min for men and women, respectively.
Ann Clin Biochem 2006; 43: 287294

Circadian rhythm of TCM and AM

To our knowledge, this is the rst study to examine the circadian rhythm of urinary steroid metabolites in healthy volunteers. Men and women show a similar phase of the rhythm. The circadian rhythm of AM shows a lower amplitude than TCM. This is to be expected because androgens arise from the gonads as well as from the adrenals.23 Since the ratio TCM/AM showed a circadian rhythm, we consider that it would be unwise to assess androgen production by calculation of this ratio in untimed samples. Although

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Table 2 Comparison of circadian rhythm parameters of steroids in healthy men and women over 24 h Parameters Men TCM 11-OH/11-Oxo 5a/5b THF 20OH/20OXO AM TCM/AM AM/creatinine TCM/creatinine Women TCM 11-OH/11-Oxo 5a/5b THF 20OH/20OXO AM TCM/AM AM/creatinine TCM/creatinine % Rhythm (goodness of t) 81%, 22%, 68%, 51%, 51%, 63%, 50%, 62%, Po0.0001 P=0.1 Po0.005 Po0.05 P=0.04 P=0.02 P=0.03 P=0.001 Po0.0001 P=0.2 Po0.001 Po0.05 P=0.04 P=0.001 P=0.04 P=0.006 MESOR Amplitude Acrophase Equivalent time (h) to to to to to to to to 190) 253) 302)a 360)b 176) 191) 135) 180) 1412 1804 2216 2240 1316 1536 1148 1404

884 (5901180)** 0.92 (0.791.05)* 1.31 (1.061.55)** 0.25 (0.200.30)* 420 (356484)*** 3.08 (2.1740) 191 (132209)* 490 (362602)*

406 (437465)*** 0.086 (0.050.16) 0.29 (0.180.41) 0.059 (0.0370.087) 89 (40166)** 1.06 (1.011.18) 38 (2551)* 214 (192255)***

213 271 334 340 199 234 177 211

(255 (288 (336 (240 (212 (277 (219 (242

85%, 30%, 78%, 51%, 52%, 66%, 49%, 65%,

393 0.75 0.73 0.33 191 2.78 145 327

(316470) (0.610.90) (0.520.94) (0.270.39) (139243) (1.713.81) (126196) (233422)

183 (173241) 0.063 (0.080.19) 0.17 (0.0680.27) 0.046 (0.0120.079) 40 (2682) 0.7 (0.651.02) 21 (1339) 135 (139176)

214 265 340 360 203 236 201 217

(253 (355 (380 (389 (205 (271 (172 (255

to to to to to to to to

175) 240) 305)c 330)c 186) 202) 224) 180)

1416 1740 2240 2400 1332 1544 1324 1428

Values are expressed as means (95% condence intervals), Acrophase is presented as phase angle in degrees where 3601=24 h. For men versus women: *Po0.05, **P o0.005, ***Po0.0005. Within the group: acrophase TCM versus metabolite ratios: aPo0.05, bPo0.005 and cPo0.0005. Equivalent times are given for convenience. MESOR and amplitude are expressed as mg/3 h for steroids and their ratios, and mg/nmol for AM and TCM over creatinine ratios

(a)

TCM/creatinine Men 1000 Women 900 800 700 600 500 400 300 200 100 0 2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time AM/creatinine Men Women

(b) 400 350 300 250 200 150 100 50 0 g/nmol

2100 2400 0300 0600 0900 1200 1500 1800 2400 0300 0600 0900 1200 1500 1800 2100 Clock time

our data were obtained in adults, this conclusion can probably be applied to the assessment of androgen production in children with signs of early puberty or adrenarche. It should be noted, however, that untimed collection may otherwise be very informative in children. Higher levels of TCM and AM excretion in men, shown by ANOVA and comparison of MESOR and Amplitude, agree with our previous ndings based on single 24-h urine sample collections.8,24 Comparing the phase of the cortisol rhythm by various measures, serum cortisol shows a peak between 0700 and 1100,6 while urinary free cortisol peaks at around 1200 midday7 and we have similar ndings in our volunteers (Jerjes et al., unpublished data). Thus, there is a lag time between serum cortisol and urine free cortisol of 2--3 h and a further delay of about 2 h for the appearance of cortisol metabolites. This delay between serum change and the appearance of urinary metabolites should be taken into account in dynamic studies where urine analysis would be informative, such as ACTH stimulation tests in investigation of congenital adrenal hyperplasia.

g/nmol

Figure 3 Mean values and standard errors of (a) total urinary cortisol metabolite excretion/creatinine and (b) urinary androgen metabolites/creatinine at 3-h intervals over 24 h

Cortisol metabolite ratios

Values for cortisol metabolite ratios over 24 h are in agreement with our previous reports based on single
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24 h urine sample collections.8,25 The 11OH/11OXO ratio represents an index of 11b-HSD enzyme activity: type 1 is predominant in the liver and acts as a reductase (cortisone to cortisol),26 while type 2 is predominantly renal and acts as a dehydrogenase (cortisol to cortisone).27 Decrease of this ratio is associated with increase of cortisol metabolic clearance rate, as in apparent cortisone reductase deciency,11 polycystic ovary syndrome,12 growth hormone excess28 and obesity.10,29 These states are probably all due to decreased 11b-HSD type 1 activity.30 Increase in this ratio is seen in patients with apparent mineralocorticoid excess,31 in which 11b-HSD type 2 is decient, and in Cushings syndrome.32 The latter may be due to an acute increase of cortisol exceeding the capacity of 11b-HSD type 2, since it is also seen after ACTH stimulation.33 It might thus have been expected that the circadian peak in cortisol secretion would be associated with signicant daily increase in the 11OH/11OXO ratio due to a mass eect of cortisol, but this is clearly not the case. Indeed, values were highest at 2100--0300, when urinary cortisol metabolite secretion was at a nadir. Thus, untimed urine samples should permit an accurate assessment of 11b-HSD status. In contrast, the ratio 5a/5b THF shows a striking daily rhythm, with an acrophase that lags behind that of TCM by 8.1h (males) and 8.4 h (females). This ratio reects relative activities of the 5a- and 5b-reductases in the liver and probably other tissues, including fat. It is increased in obese subjects, suggesting an enhancement of 5a-reductase activity, since fat contains 5areductase activity, but not 5b-reductase activity.34 It is likewise decreased in anorexia nervosa.35 Comparison of a variety of clinical disorders indicates that 5a/b reduction and 11-HSD activity are independently modulated,32 but changes do occur secondarily to marked changes in cortisol 11-oxoreduction. The ratio is increased in deciency of 11b-HSD type 2 (apparent mineralocorticoid excess,36) and decreased in apparent deciency of11b-HSD type1 (apparent cortisone reductase deciency,37). Acute cortisol increase after ACTH stimulation does not result in changes,33 although Cushings syndrome is associated with decrease of this ratio.38 Again, there is no evidence for a mass eect within our data. The acrophase for the ratio 20OH/20OXO lags behind that of TCM by 8.5 h (males) and 9.7 h (females). This ratio does not change in many clinical states, but it is elevated in alcoholic liver disease,39 which may be due to increased liver activity of 20-reductases or decreased A-ring reduction. The signicant changes of 5a/5b THF and 20OH/ 20OXO ratios over 24 h are not co-dependent nor do they appear to be due to changed mass eect of cortisol over the day. They are unlikely to result from changes in enzyme level, since the duration is short. Alternative
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causes include changes in substrate, inhibitor, cofactor or hormone concentrations, perhaps related to meal intake, but no candidate compounds can currently be suggested. Whether they represent passive eects or are evidence for active modulation of steroid hormone action, perhaps related to circadian entrainment of physiological functions, is currently unclear. Our ndings do indicate that interpretation of cortisol metabolite ratios in untimed urine collections would be misleading if reference limits did not take the circadian uctuation into account.

Correlates of circadian rhythm parameters

The nding of a signicant negative correlation between age and acrophase is in line with previous reports of an earlier acrophase of plasma cortisol in older subjects.40 This might be due to a changed sleepwake cycle, since older subjects tend to show a forward shift in sleeping times.41 The negative trend between BMI and acrophase of urinary cortisol metabolites might suggest that obese subjects have an earlier peak of cortisol, although none of our volunteers were clinically obese. This could reect a faster cortisol metabolic clearance rate or it might be due to the confounding eects of age, since subjects tend to get fatter with age. Food intake stimulates cortisol release. Careful studies by Follenius et al.,42 using frequent plasma sampling, showed a marked eect of a meal at noon and less consistent eects at other times, relatively independent of customary eating patterns. In our study, no eect is apparent. This may be because the urine collections were made at relatively wide intervals, and timing of meals was not controlled experimentally. Investigation of the serum cortisol rhythm in disorders such as depression and Cushings syndrome is not only of interest in the circadian physiology in those diseases but also for their diagnostic utility. Approximately 50% of depressed patients fail to suppress cortisol in response to dexamethasone administration43 and this attenuated feedback probably related to elevated circulating cortisol levels and hypersecretion of corticotrophin-releasing hormone and ACTH.44,45 Dahl et al.5 reported that patients with depression showed a preserved plasma cortisol circadian rhythm but with lower amplitude and an earlier acrophase. The disappearance of the cortisol circadian rhythm in Cushings disease is well known.46 Patients show deterioration of serum cortisol rhythmicity as the disease progresses.4 Abnormal circadian rhythms may be part of a cascade of dysfunction in the HPA axis, in which excessive cortisol production damages glucocorticoid receptors, which impairs feedback inhibition of CRH, leading to further excessive secretion of ACTH and cortisol.47 We conclude that 3-h urinary cortisol collections over 24 h are a valid way to measure circadian uctua-

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tions in the excretion of androgen and cortisol and its metabolites. We suggest that such uctuations need to be taken into account when interpreting results of urinary steroid analyses. Calculation of steroid/steroid or steroid/creatinine ratios is not informative in untimed collections. With improved understanding of the circadian rhythm of steroid metabolites in normal subjects, such alterations in excretion patterns may have a future place in diagnosing and monitoring disease.

14

15 16

Acknowledgements
We thank the volunteers who took part in this study and Mr J Keating and department sta for carrying out the creatinine analyses.

17

18 19

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Accepted for publication 31 March 2006

Ann Clin Biochem 2006; 43: 287294