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High Fluid Intake Increases Urine Free Cortisol Excretion in Normal Subjects

Developmental Endocrinology Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 10892-1862

Address all correspondence and requests for reprints to: M. Veronica Mericq, National Institutes of Child Health and Human Development, Developmental Endocrinology Branch, SDE, Building 10, Room 10N262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: mericqV{at}cc1.NICHD.NIH.gov

	Abstract

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To test the hypothesis that increased fluid intake increases the urine free cortisol, we prescribed 5 liters of fluid intake per day or a normal fluid intake according to a randomized cross-over design in six normal volunteers. Each period lasted 5 days, with a 2-day washout period of normal fluid intake between the two periods. Urine free cortisol, 17-hydroxycorticosteroids, and creatinine were measured daily during each study period, and the average value over each 5-day period was calculated for each subject. High fluid intake caused a significant increase in the mean urine free cortisol [126 ± 33 (SD) vs. 77 ± 18 µg/day, P < 0.005]. The frequency of urine free cortisol results that exceeded the upper normal limit of 95 µg/day was also much higher during high fluid intake (23/30 vs. 6/30, P < 0.005). By contrast, urine 17-hydroxycorticosteroids (high fluid vs. normal fluid: 5.3 ± 1.5 vs. 5.0 ± 1.7 mg/day, respectively, P = not significant) and urine creatinine (1.51 ± 0.48 vs. 1.45 ± 0.37 g/day, P = not significant) did not differ between the two study periods. We conclude that high fluid intake (5 liters/day) increases free cortisol excretion without an increase in urine 17-hydroxycorticosteroids. Thus, mild to moderate increases in urine cortisol excretion may not indicate hypercortisolism in individuals who have a high fluid intake and urine volume.

	Introduction

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THE URINE free cortisol is one of the principal screening tests for hypercortisolism (1). Although urine free cortisol is sometimes conceived as representing all cortisol that is filtered at the glomerulus, the amount of filtered cortisol actually greatly exceeds the amount of free cortisol measured in the urine. For example, a glomerular filtration rate of 180 liters/1.73 m² per day (2) and a mean plasma free cortisol concentration of approximately 5 µg/L (3, 4) would produce a filtered cortisol load of 900 µg/1.73 m² per day. This amount is approximately 20 times the upper limit of the normal urine free cortisol, as determined by highly specific assays on chromatographed samples (5, 6). Thus, most of the filtered cortisol must either be metabolized or reabsorbed before it is excreted. Two recognized renal metabolic reactions are catalyzed by 11ß-hydroxysteroid dehydrogenase, the site of the defect in the syndrome of apparent mineralocorticoid excess (7, 8), and 20-ketosteroid reductase, an enzyme implicated in rare patients with Cushing syndrome and normal or low urine free cortisol (9, 10).

The fact that most filtered cortisol is metabolized or reabsorbed suggested the hypothesis that increased fluid intake and the resulting increase in urine volume might reduce the fraction of filtered cortisol that is metabolized or reabsorbed and thus increase the urine free cortisol. To test this hypothesis we measured urine cortisol excretion under conditions of normal and increased fluid intake in normal volunteers.

	Subjects and Methods

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Subjects

We studied six normal volunteers (three females, three males) with normal body mass index (mean 23.2 ± 3.6) and ages from 22–45 yr. The study protocol was approved by the institutional review board of the National Institute of Child Health and Human Development, and informed consent was obtained from each subject. Volunteers were assigned, according to a randomized, cross-over design to 5 days of normal fluid intake and 5 days of high fluid intake. A 2-day washout period of normal fluid intake separated the two arms. During the 5 days of high fluid intake, patients were asked to drink 5 liters of fluid per day, with water comprising most of the excess fluid above their normal fluid intake.

Urine assays

Creatinine in each 24-h urine specimen was measured at the National Institutes of Health Clinical Center. Urine free cortisol was measured at Hazleton Labs. (Vienna, VA) by a modification of a previously described RIA (11). The cortisol antibody was raised against cortisol-3-carboxymethyloxime conjugated to BSA. The antibody cross-reactivity (relative to 100% for cortisol) was 37% for 11-deoxycortisol; 25% for cortisone; 3% for corticosterone; 0.8% for 17-hydroxyprogesterone; and < 0.02% for progesterone, pregnenolone, dehydroepiandrosterone, and testosterone. After dichloromethane extraction, samples were chromatographed on celite columns (with elution of the cortisol fraction by 40% ethyl acetate) before assay. The intra - and interassay coefficients of variation were 9.5% and 13%, respectively.

An aliquot from each urine collection was also analyzed for 17-hydroxycorticosteroids by a modification of the Porter-Silber method (10). The intra - and interassay coefficients of variation were 5.9% and 7–14%, respectively. All 10 samples from each subject were measured in the same urine free cortisol or 17-hydroxycorticosteroid assay to avoid interassay variation. Additionally, during the period of high fluid intake, the volume of the urine aliquots was increased 3-fold to assay a similar fraction of the total 24-h collection for each study period.

Statistical analysis

Comparisons between the normal and high-fluid periods were made with the two-tailed paired Student’s t test. The frequency of elevated urine free cortisol results for each study period was compared by the -square test. Unless otherwise stated, data are presented as mean ± SD.

	Results

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Urine volume and creatinine

As expected, urine volume was significantly greater during the 5 days of high fluid intake than during the period of normal intake (3800 ± 1033 mL vs. 1070 ± 376 mL, P < 0.005, Fig. 1). By contrast, urine creatinine excretion did not change significantly [1.51 ± 0.48 vs. 1.45 ± 0.37 g/day, P = not significant (NS)].

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of high fluid intake on urine volume, urine free cortisol, urine 17-hydroxycorticosteroids (17OHCS), and urine creatinine excretion in healthy normal volunteers. Each symbol represents data from one of six subjects, and each data point shown is mean of five consecutive daily measurements for that subject during each phase of study. Shaded area, Normal range (9–95 µg/day) for urine cortisol excretion. Vertical bars, Mean ± SEM for data from all six subjects. * P < 0.005, high fluid intake vs. normal fluid intake.

High fluid intake caused a significant increase in urine free cortisol (126 ± 33 vs. 77 ± 18 µg/day, P < 0.005, Fig. 1) but not in urine 17-hydroxycorticosteroids (5.3 ± 1.5 vs. 5.0 ± 1.7 mg/day, P = NS). The urine 17-hydroxycorticosteroid excretion per gram of creatinine excretion was nearly identical between the two arms [3.49 ± 0.51 (high fluid ) vs. 3.45 ± 0.57 mg/g creatinine per day, P = NS). Additionally, the frequency of elevated urine free cortisol levels (normal range, 9–95 µg/day) was significantly greater during the period of high fluid intake (23/30 vs. 6/30, P < 0.005).

	Discussion

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We observed significantly higher urine cortisol excretion during high fluid intake than during normal fluid intake. This higher cortisol excretion occurred without an increase in urine 17-hydroxycorticosteroid excretion, indicating that the rise in urine cortisol did not appear to result from activation of the hypothalamic-pituitary-adrenal axis. Although this study may lack sufficient power to exclude a real increase in 17-hydroxycorticosteroid excretion, our observations seem more consistent with the hypothesis that increased fluid intake decreases the renal metabolism or reabsorption of filtered cortisol.

Like many urine free cortisol assays in current use, the RIA used in this study lacks absolute specificity for cortisol and thus yields values approximately twice as great as highly specific methods (5, 6). The cause of such higher values is cross-reaction of closely related cortisol precursors and metabolites (5, 6). Because these cross-reacting steroids (and hence their fractional renal reabsorption and metabolism) may differ among assays, it cannot be assumed that the effect of high fluid intake will be precisely the same for different assays. Indeed, the effect of fluid load could be on these metabolites rather than on cortisol itself, and it would be of interest to examine the effects of fluid loading using more specific assay methods for urine cortisol.

Despite the widespread clinical use of urine free cortisol measurements for the diagnosis of hypercortisolism, there have been few clinical studies of the effect of fluid intake on urine cortisol excretion. In 1959, Schedl and colleagues (12), measuring urine cortisol by fluorometry and by double isotope dilution, stated that there was no correlation between cortisol excretion and urine flow, but did not provide the data for this conclusion. In 1974, Baum and colleagues (13) published an abstract stating that water loading increased the urine free cortisol and that water deprivation did the reverse. To our knowledge, a full account of this study was never published. Bertrand et al. (14) analyzed the factors affecting the spontaneous excretion of free cortisol in 203 overnight urine collections from 7- to 18-yr-old school children. In these overnight urine collections (median volume 235 mL), total cortisol excretion was strongly related to urine volume (P < 0.0001), with an increase of 24% (95% confidence limits: 17–32%) with each 100-mL increase in urine volume. Thus, the limited previous data are consistent with our observation that increased fluid intake increases the excretion of free cortisol.

Our observations do not address the relative contribution of metabolism vs. reabsorption in explaining the discrepancy between the amount of filtered and excreted cortisol. Although earlier studies made the assumption that reabsorption accounted for essentially all of the discrepancy (12, 15, 16), more recent studies have demonstrated renal metabolism of cortisol both by 20-ketosteroid reductase (9) and by 11ß-hydroxysteroid dehydrogenase (17). Thus, the increase in urine cortisol excretion during high fluid intake may reflect decreased renal metabolism of cortisol as well as decreased cortisol reabsorption.

Our study did not formally address the effect of more modest urine volume increases such as would be encountered more commonly in clinical practice. However, the six individual 24-h urine collections with the lowest volume (3–3.5 liters) during high fluid intake had a mean ± SD urine free cortisol of 107 ± 27 µg/day, with five of the six values above the normal limit of 95 µg/day. Thus, it appears that more modest increases in urine volume also produce proportional increases in urine free cortisol.

The clinical significance of these observations is that 77% of the 24-h urine samples collected from normal volunteers during high fluid intake had urine free cortisol levels that were above the upper limit of the normal range. Thus, it seems likely that patients with high fluid intake, such as patients with psychogenic polydipsia or diabetes insipidus, may also be at greater risk of excreting increased levels of cortisol during periods of high fluid intake and urine volume. If this hypothesis is correct, mild to moderate increases in urine cortisol in such patients should be confirmed by an alternate method, such as the overnight dexamethasone suppression test, diurnal cortisol levels in plasma, or urine 17-hydroxycorticosteroid excretion before concluding that the increased urine cortisol level is in fact caused by increased cortisol secretion (18).

	Footnotes

 
¹ Commissioned officer in the United States Public Health Service.

Received April 7, 1997.

Revised August 12, 1997.

Accepted October 29, 1997.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mericq, M. V. Articles by Cutler, G. B. Search for Related Content PubMed PubMed Citation Articles by Mericq, M. V. Articles by Cutler, G. B., Jr. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB HYDROCORTISONE