Adrenal Gland Volume and Dexamethasone-Suppressed Cortisol ...

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The Journal of Clinical Endocrinology & Metabolism 92(4):1358 –1363 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2674

Adrenal Gland Volume and Dexamethasone-Suppressed Cortisol Correlate with Total Daily Salivary Cortisol in African-American Women Sherita Hill Golden, Saurabh Malhotra, Gary S. Wand, Frederick L. Brancati, Daniel Ford, and Karen Horton Departments of Medicine (S.H.G., G.S.W., F.L.B., D.F.), Psychiatry (G.S.W.), and Radiology (K.H.), Johns Hopkins University School of Medicine, and Department of Epidemiology (S.H.G., F.L.B., D.F.), Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205; and Department of Medicine (S.M.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213 Context: Population-based studies of associations between subclinical hypercortisolism and risk for disease states, such as type 2 diabetes mellitus, have been difficult to assess because of imprecise measures of glucocorticoid exposure. Alternative measures (salivary cortisol and adrenal gland volume) have not been systematically compared with 24-h urine free cortisol (UFC) in a healthy population. Objective: Our objectives were: 1) to determine whether 24-h UFC and total daily salivary cortisol correlated with each other, adrenal gland volume, and salivary cortisol after dexamethasone suppression and 2) to evaluate the association of adrenal gland volume with salivary cortisol after dexamethasone suppression. Design, Setting, and Participants: This was a cross-sectional study of 20 healthy, premenopausal African-American women aged 18 – 45 yr.

Adrenal gland volume was measured by computed tomography scan. Dexamethasone-suppressed salivary cortisol was measured at 0800 h after administration of 0.5 mg dexamethasone at 2300 h the prior evening. Results: Dexamethasone-suppressed salivary cortisol levels correlated strongly with individual, timed salivary cortisol measurements, total daily salivary cortisol (rs ⫽ 0.75; P ⫽ 0.0001; n ⫽ 20), and adrenal gland volume (rs ⫽ 0.66; P ⫽ 0.004; n ⫽ 17). Total daily salivary cortisol and adrenal gland volume also correlated (rs ⫽ 0.46; P ⫽ 0.04; n ⫽ 19). In contrast, 24-h UFC levels did not correlate with any of the other hypothalamic-pituitary-adrenal axis measures. Conclusion: A dexamethasone suppression test or adrenal gland volume may be alternative measures for characterizing subtle subclinical hypercortisolism in healthy adults. (J Clin Endocrinol Metab 92: 1358 –1363, 2007)

Main Outcome Measures: Salivary cortisol was assessed at six time points throughout the day simultaneous with 24-h UFC collection.

O

BESITY, TYPE 2 DIABETES, and cardiovascular disease continue to impose major public health burdens, and type 2 diabetes continues to rise in epidemic proportions. Identification of novel risk factors for these diseases is urgently required to guide the development of additional preventive measures. Chronic psychological stress, sometimes manifested as depression or anger, is a risk factor for both type 2 diabetes (1– 6) and cardiovascular disease (7–13), but the mechanism remains unclear. Neuroendocrine changes induced by these psychological states, specifically activation of the hypothalamic-pituitary-adrenal (HPA) axis, might provide a unifying explanation. Patients with major depression have evidence of hypercortisolism, including an exaggerated cortisol response to ACTH hormone, failure to suppress cortisol levels after administration of dexamethasone, and adrenal gland hypertrophy (14, 15). Whereas adrenal gland hypertrophy suggests chronic HPA axis activation, two studies found no relation between plasma cortisol levels

First Published Online February 6, 2007 Abbreviations: HCG, Human chorionic gonadotropin; HPA, hypothalamic-pituitary-adrenal; UFC, urine free cortisol. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

and adrenal volume (16, 17). The correlation between 24-h urine free cortisol (UFC) and adrenal volume was not assessed in these studies. With the exception of the Heart and Soul Study (18), a prospective cohort study of psychosocial factors and health outcomes in individuals with coronary heart disease, most population-based studies of the association between depression and metabolic outcomes have not assessed HPA axis activity as a potential link. Epidemiological investigations are limited by imprecise measures of glucocorticoid exposure. The presumed gold standard, a 24-h UFC, is cumbersome to perform in large-scale studies. Moreover, it is uncertain whether the 24 h assessment of UFC is the best way to assess the relationship between cortisol dynamics and metabolic consequences in apparently healthy adults. One reasonable alternative to measurement of HPA axis activity is collection of salivary cortisol, which has several advantages: 1) salivary cortisol can be collected in the freeliving state without venipuncture; 2) it is stable for several days before processing (19) and stable under frozen and mailed conditions (20); and 3) it reflects free, not total, cortisol. Recently, 2300 h salivary cortisol has been added to the initial screening algorithm for Cushing’s syndrome as a method for assessing the diurnal pattern of cortisol and may

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Golden et al. • Adrenal Volume and Dex-Suppressed Salivary Cortisol

provide a means of evaluating subclinical hypercortisolism (21). Another alternative is the 1-mg overnight dexamethasone suppression test, which is also used in the clinical assessment of Cushing’s syndrome (22) and is abnormal in affective illness (14). An abnormal cortisol response to dexamethasone also indicates a state of hypercortisolism, presumably related to impaired glucocorticoid-negative feedback. A third alternative is adrenal volume, an integrated, noninvasive measure of HPA axis activity, which can be assessed during abdominal computed tomography scans. None of these alternative measures, however, have been systematically compared with the 24-h urine cortisol collection in a healthy population. The objectives of our study were 2-fold. First, we sought to determine whether 24-h urine free cortisol (the presumptive gold standard) and total daily salivary cortisol correlated with each other, adrenal gland volume, and salivary cortisol after dexamethasone suppression in a group of healthy subjects without pathological hypercortisolism. Second, we sought to evaluate the association of adrenal gland volume with salivary cortisol after dexamethasone suppression. We hypothesized that larger adrenal glands would indicate more chronically elevated levels of glucocorticoids. These studies were conducted in African-American women to reduce heterogeneity of findings and because this population is particularly at risk for metabolic disorders. Subjects and Methods Study population We recruited 20 healthy African-American women between the ages of 18 and 45 yr. The mean age of study participants was 32 ⫾ 8 yr and the mean body mass index and waist circumference were 24.3 ⫾ 1.19 kg/m2 and 75.2 ⫾ 1.43 cm, respectively. The body mass index range was 16.0 –32.6 kg/m2. Women with the following conditions were excluded from the study because these conditions are known to alter the normal function of the HPA axis or interfere with measurement of cortisol: 1) diabetes or clinical cardiovascular disease (including a history of myocardial infarction, coronary artery bypass surgery, stroke, peripheral vascular disease, angina, claudication, or transient ischemic attacks); 2) body mass index greater than 35 kg/m2; 3) use of oral or inhaled steroids within the past year, a diagnosis of adrenal insufficiency, or a diagnosis of Cushing’s syndrome; 4) treatment with ketoconazole, tegretol, dilantin, or other medications known to perturb the HPA axis; 5) chronic night shift work; 6) pregnancy or use of oral contraceptives [a urinary human chorionic gonadotropin (HCG) test was performed on all participants to exclude pregnancy]; 7) diagnosed and/or treated depression or anxiety disorder (self-reported diagnosis and/or treatment with medications over the past 2 wk); 8) chronic diseases such as renal disease (defined as a serum creatinine ⬎ 1.4 mg/dl), liver disease (defined as transaminases ⬎ 2 times the upper limit of normal and/or a known diagnosis of hepatitis B or C), or HIV; 9) allergy or hypersensitivity reactions to dexamethasone or other glucocorticoids; or 10) any current use of illicit drug or heavy use of alcohol (defined as five or more drinks/episode for men and four or more drinks/episode for women per month). Nicotine addiction in smokers was assessed using the Fagerstrom instrument and individuals with a score above 7 were considered nicotine dependent and were not eligible for further participation (23). Written informed consent was obtained from each participant. This study was approved by the Institutional Review Board at the Johns Hopkins University School of Medicine.

J Clin Endocrinol Metab, April 2007, 92(4):1358 –1363

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Study protocol During the screening visit, the following assessments were performed: history and physical examination; comprehensive metabolic panel and serum HCG test; serum and urine toxicology screen; and assessment of nicotine addiction in smokers using the Fagerstrom instrument. Participants were then admitted to our inpatient General Clinical Research Center. On the evening of admission (d 1), a repeat serum HCG was performed. Measurement of weight and height were performed to calculate body mass index. Participants wore light-weight, nonconstricting underwear and scrub suits. Height (without shoes) was measured using a wall-mounted ruler. Weight was measured using a balance scale. Waist circumference was measured at the midpoint between the iliac crests and the lower rib cage. Twenty-four hour urine collection for UFC began the following morning at 0800 h (d 2). Urine was collected in a jug containing boric acid. UFC was measured using liquid chromatography and tandem mass spectroscopy using a validated assay (Nichols Institute, San Juan Capistrano, CA). The interassay coefficient of variation ranges between 9.0 and 13.2% and the intraassay coefficient of variation ranges between 11.5 and 13.3%. Salivary cortisol was collected in salivettes simultaneously with UFC at six time points during the day before meals, according to the MacArthur protocol (http://macses.ucsf.edu/research/allostatic/ notebook/salivarycort.html): on awakening (0800 h); 45 min after awakening (0845 h); 2.5 h after awakening (1030 h); 8 h after awakening (1600 h); 12 h after awakening (2000 h); and bedtime (2300 h). Salivary cortisol measurements were performed in our laboratory by RIA (Diagnostics Product Co., Los Angeles, CA) using a model 1470 ␥-counter (PerkinElmer, Shelton, CT). The inter- and intraassay coefficients of variation for the assay are both less than 10%. Participants underwent abdominal computed tomography scan in the afternoon to calculate adrenal gland volume. Adrenal volume was determined using a Volume Zoom, 4-slice multidetector-row scanner (Siemens, Melvern, PA) or Sensation 16, 16-slice multidetector-row scanner (Siemens). An AP localizer scout was used to determine the level, and then a noncontrast scan was performed through the adrenal glands. The following parameters were used: window width 410, window center 10, and slice thickness 1 mm, reconstructed every 1 mm. The adrenal contour was manually traced on each slice with a console cursor. Care was taken to try to exclude adjacent fat. However, because the shape of the adrenals can be somewhat irregular, thresholds values were established to exclude adjacent fat from the calculated volume. For example, only tissue with attenuation values between ⫺25 and 200 houndsfield units within the region of interest would be included. The scanner software then automatically calculated the adrenal volume by summing the area on each slice (17). The following morning (d 3), participants were discharged with 0.5 mg dexamethasone to be taken at 2300 h the night of discharge for an overnight low-dose dexamethasone suppression test. Dexamethasone doses less than 1 mg have been shown to allow detection of subtle degrees of HPA axis hyperactivity (24). Participants were given a salivette to perform an 0800 h salivary cortisol on the next day at home and were instructed to return the samples to our laboratory. All salivettes were returned by participants.

Analysis One individual (G.S.W.) performed salivary cortisol assays, a second person (Quest Diagnostics, Baltimore, MD) performed the urinary cortisol assays, and a third person (K.H.) calculated adrenal gland volume. Two individuals (S.M., S.H.G.) performed the data analysis. All individuals were blind to the others’ results. Because salivary cortisol, UFC, and adrenal gland volume were not normally distributed, they were log transformed for analyses. Spearman’s correlation coefficients were calculated to determine the cross-sectional association between 24-h UFC and each of the following: 1) individual, timed salivary cortisol measurements, 2) total daily salivary cortisol, 3) adrenal gland volume, and 4) dexamethasone suppressed cortisol. Total daily salivary cortisol was calculated as the sum of the six individual timed salivary cortisol measurements. Correlation coefficients were also calculated to determine the correlation between dexamethasone suppressed cortisol, a more dynamic measure of HPA axis activity and each of the following: 1) in-

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dividual, timed salivary cortisol measurements, 2) total daily salivary cortisol, and 3) adrenal gland volume. Spearman’s correlation coefficient was determined because it does not assume normality of the distributions being compared and is a more conservative than the Pearson’s estimate (25). P ⬍ 0.05 was used to determine statistical significance. Statistical analyses were performed using Stata version 8.2 (College Station, TX).

Results Association of cortisol parameters

Table 1 summarizes the hormonal characteristics of the 20 healthy African-American women in this study. Dexamethasone-suppressed salivary cortisol levels correlated strongly with individual, timed salivary cortisol measurements (Table 2), total daily salivary cortisol (rs ⫽ 0.75; P ⫽ 0.0001; n ⫽ 20), and adrenal gland volume (rs ⫽ 0.66; P ⫽ 0.004; n ⫽ 17). Adrenal gland volume correlated strongly with total daily salivary cortisol (rs ⫽ 0.46; P ⫽ 0.04; n ⫽ 19) as well as timed salivary cortisol measurements at 0845 (rs ⫽ 0.55; P ⫽ 0.01; n ⫽ 19), 1030 (rs ⫽ 0.48; P ⫽ 0.04; n ⫽ 19), and 1600 (rs ⫽ 0.53; P ⫽ 0.02; n ⫽ 19) (Table 2). In contrast, 24-h UFC levels did not correlate with any of the other HPA axis measures: individual, timed salivary cortisol measurements (rs ⫽ 0.07 to 0.40; n ⫽ 20), total daily salivary cortisol (rs ⫽ 0.26; P ⫽ 0.26; n ⫽ 20), adrenal gland volume (rs ⫽ 0.18; P ⫽ 0.48; n ⫽ 18), or dexamethasone-suppressed cortisol (rs ⫽ 0.16; P ⫽ 0.54; n ⫽ 18) (Table 2). Figure 1 shows that adrenal gland volume was more strongly correlated with dexamethasonesuppressed salivary cortisol than 24-h UFC. Similarly, total daily salivary cortisol was more strongly correlated with dexamethasone-suppressed cortisol than 24-h UFC (Fig. 2). There were two individuals with adrenal gland volumes that were significantly lower than the remainder of the group. When analyses were repeated with these two individuals excluded, the correlations between adrenal gland volume and dexamethasone-suppressed cortisol (rs ⫽ 0.60; P ⫽ 0.014) and 24-h UFC (rs ⫽ 0.08; P ⫽ 0.76) were similar. Discussion

We found that among healthy, premenopausal AfricanAmerican women, dexamethasone-suppressed salivary cortisol was positively correlated with total daily salivary cortisol as well as adrenal gland volume and that adrenal gland volume also correlated with total daily salivary cortisol. In contrast, 24-h UFC showed poor correlations with total daily


salivary cortisol, adrenal gland volume, and dexamethasonesuppressed cortisol. Cushing’s syndrome is the prototypical clinical example of extreme hypercortisolism with metabolic consequences including hypertension, glucose intolerance and/or type 2 diabetes mellitus, hyperlipidemia, enhanced cardiovascular disease risk, osteoporosis, and neuropsychological disturbances (22). Studies of patients with incidentally discovered adrenal gland tumors suggest that even subclinical hypercortisolism (defined as having two of three abnormalities of HPA axis function: elevated 24-h UFC, failure to suppress cortisol in response to dexamethasone, and decreased ACTH levels) is associated with adverse metabolic consequences. Compared with patients with incidentally discovered adrenal gland tumors who do not have subclinical hypercortisolism, those with subclinical hypercortisolism have reduced insulin sensitivity and glucose intolerance (26 –28), reduced bone mineral density (29), greater central adiposity (27, 28), more lipid abnormalities (27, 28, 30), and are more likely to have hypertension (27, 28, 30) and type 2 diabetes (27, 30). Thus, we hypothesize that subclinical hypercortisolism induced by depression and anxiety might also result in adverse metabolic consequences. However, we also suspect that other individuals without clear-cut depression or anxiety disorders may also have mild persistent or intermittent hypercortisolism and that even individuals within the upper limit of the normal range for cortisol production may be at risk for metabolic disorders. To test this hypothesis, however, the need to identify easy-to-measure and reliable markers of mild hypercortisolism in the research setting is paramount. The 24-h UFC has traditionally been considered the gold standard for assessing pathological hypercortisolism; however, studies in nondepressed individuals with subclinical hypercortisolism have not found consistent associations of 24-h UFC with metabolic abnormalities. One study found no correlation between 24-h UFC and visceral abdominal fat or insulin sensitivity (31), and in another study, 24-h UFC was similar in Pima Indians and Caucasians despite higher percent body fat in the Pima Indians (32). In two studies, Duclos et al. (33, 34) found that 24-h UFC was similar in women with peripheral and abdominal fat distributions, although women with abdominal obesity had a higher ratio of cortisol to cortisone. Whereas some studies have found elevated 24-h

TABLE 1. Median (interquartile range) 24-h UFC, adrenal gland volume, and individual timed, total daily, and dexamethasonesuppressed salivary cortisol for 20 healthy African-American women

a

Hormonal parameters

Median (interquartile range)

24-h UFC (␮g/24 h) Adrenal gland volume (cm3) Timed salivary cortisol (␮g/dl) 0800 h 0845 h 1030 h 1600 h 2000 h 2300 h Total daily salivary cortisol (␮g/dl)a Dexamethasone-suppressed salivary cortisol (␮g/dl)

19.4 (11.2, 31.6) 4.0 (3.34, 5.37)

Total daily salivary cortisol equals the sum of the six timed salivary cortisol samples.

0.34 0.29 0.24 0.13 0.07 0.06 1.08 0.05

(0.20, (0.18, (0.17, (0.08, (0.04, (0.02, (0.77, (0.03,

0.53) 0.52) 0.41) 0.14) 0.08) 0.1) 1.65) 0.16)



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TABLE 2. Spearman’s correlation coefficients between natural log of 24-h UFC and dexamethasone-suppressed salivary cortisol and the natural log of timed salivary cortisol, total daily salivary cortisol, and adrenal gland volume Timed salivary cortisol measurements 0800 h

0845 h

1030 h

1600 h

2000 h h

2300

Total daily cortisol

24-h UFC 0.16 (0.51) 0.07 (0.78) 0.08 (0.72) 0.06 (0.80) 0.40 (0.08) 0.27 (0.25) 0.26 (0.26) Dexamethasone suppres- 0.45 (0.05) 0.66 (0.002) 0.77 (0.0001) 0.70 (0.0007) 0.62 (0.004) 0.44 (0.05) 0.75 (0.0001) sion of salivary cortisol Adrenal gland volume 0.20 (0.40) 0.55 (0.01) 0.48 (0.04) 0.53 (0.02) 0.31 (0.20) ⫺0.02 (0.95) 0.46 (0.04)

Adrenal gland volume

0.18 (0.48) 0.66 (0.004) 1.0

Values are correlation coefficients, rs (P values).

UFC in abdominal obesity (35) and type 1 diabetes (36, 37), other studies have found reduced 24-h UFC levels in morbid (38) and abdominal obesity (39). Most of these studies, like ours, used one 24-h urine collection period. In contrast, other measures of HPA axis activity have shown more consistently positive correlations with metabolic abnormalities. For example, several studies have found abnormal responses to the dexamethasone suppression test in abdominal obesity and diabetes. Duclos et al. (33) and Ljung et al. (24) found a positive correlation between waist to hip ratio and cortisol after dexamethasone suppression.

Other studies found that the response to the dexamethasone suppression test and adrenal gland volume are related to diabetes mellitus. In one study, nondepressed individuals with diabetes mellitus had a significantly greater prevalence of nonsuppressed responses to dexamethasone at 1600 h, compared with normal controls (40), and in another study, 55% of nondepressed patients with diabetes mellitus (types 1 and 2) had an abnormal 1-mg dexamethasone suppression test (41). Chiodini et al. (42) found an increased prevalence of subclinical hypercortisolism in individuals with type 2 diabetes, compared with controls, and Godoy-Matos et al. (43) found that individuals with diabetes had significantly higher

FIG. 1. A, Correlation of log adrenal gland volume and log dexamethasone-suppressed salivary cortisol. B, Correlation of log adrenal gland volume to log 24-h UFC.

FIG. 2. A, Correlation of log total daily salivary cortisol to log dexamethasone-suppressed salivary cortisol. B, Correlation of log total daily salivary cortisol to log 24-h UFC.

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adrenal gland volume than controls. Their study also demonstrated a significant correlation between visceral fat and adrenal gland volume in the whole group of diabetic cases and controls (43). Tsagarakis et al. (44) found that the dexamethasone suppression test was better than 24-h UFC and other modalities at unmasking subclinical hypercortisolism in patients with incidentally discovered adrenal gland tumors. However, Findling et al. (45) found that some patients with Cushing’s syndrome, particularly those with mild hypercortisolism, have a normal dexamethasone suppression test, suggesting that it should not be used as the sole criterion to confirm hypercortisolism. Castro et al. (46) found that the combination of 2300 h salivary cortisol and an overnight dexamethasone suppression test helped to identify individuals with hypercortisolism. We found that cortisol after dexamethasone correlates with 2300 h salivary cortisol, total daily salivary cortisol, and adrenal gland volume; however, as in other studies, we found that 24-h UFC did not correlate with adrenal gland volume (16, 17). In general, these studies suggest that subclinical HPA axis hyperactivity is related to metabolic abnormalities in nondepressed individuals. Similar to our findings, this literature also suggests that dexamethasone suppression or adrenal gland volume may be superior to 24-h UFC for characterizing cortisol dynamics that fall within the normal range or are modestly above the normal range as found in individuals with subclinical hypercortisolism. These findings may reflect the fact that increases in adrenal gland volume and changes in the dexamethasone suppression test occurs over weeks or months and thus are better able to capture mild but chronic hypercortisolism. In contrast, the 24-h UFC reflects cortisol secretion during a much more restricted time interval, making it less able to be a marker for mild, intermittent HPA axis activation. Indeed it is well known how variable 24-h UFC levels can be even in patients with endogenous Cushing’s syndrome (47, 48). In essence 24-h UFC determination reflects cortisol exposure at only one point in time, whereas adrenal gland volume and the dexamethasone suppression test are more reflective of integrated cortisol exposure over a longer time period. Future studies in nondepressed individuals without adrenal disease are warranted to determine whether the dexamethasone suppression test and/or adrenal volume are better methods for assessing subclinical hypercortisolism related to stress and depression in populationbased studies. Our study has several strengths. It is one of the few studies to examine correlations between multiple measures of HPA axis activity, particularly the correlation between 24-h UFC and salivary cortisol and adrenal volume, in healthy, nondepressed individuals. We examined HPA axis measures in African-American women, a group that has not been evaluated as often. Because there are race and gender differences in the HPA axis activity (49, 50), our results are not confounded by heterogeneity. Some limitations should be kept in mind in interpreting our data. Our first salivary cortisol was a timed collection at 0800 h instead of being collected within 30 min of awakening, and there are data to suggest that the salivary cortisol response to awakening correlates best with total cortisol secretion throughout the day (51). However, we are unaware


of studies that have compared awakening morning cortisol with 24-h UFC and our 0800 h salivary cortisol was still strongly correlated with total daily cortisol secretion (rs ⫽ 0.73; P ⬍ 0.0001) and dexamethasone suppressed salivary cortisol (rs ⫽ 0.45; P ⫽ 0.05). Because our population only included African-American women, our results may not be generalizable to other ethnicities/races and men; however, using one race and gender helped to reduce confounding because our study population was small. Finally, our sample size was small, limited to 20 women; however, many of our correlations were very strong and remained so, even when outliers were excluded. Identifying reliable, efficient measures of HPA axis activity and incorporating them into an existing population-based study of cardiovascular disease and diabetes that also has data on measures of psychological stress will allow for future research to determine whether subclinical HPA axis hyperactivity is a risk factor for adverse metabolic outcomes. Future studies should focus on determining how additional measures, such as the dexamethasone suppression test, cumulative salivary cortisol levels, and adrenal gland volume, are related to psychological stress and risk factors for metabolic diseases. Acknowledgments We thank our research coordinator, Ms. Bennette DrummondFitzgerald, for her excellent recruitment and study coordination skills. Received December 5, 2006. Accepted January 25, 2007. Address all correspondence and requests for reprints to: Dr. Sherita Hill Golden, Johns Hopkins University School of Medicine, Division of Endocrinology and Metabolism, 2024 East Monument Street, Suite 2-616, Baltimore, Maryland 21205. E-mail: [email protected]. This work was supported by the Johns Hopkins University School of Medicine General Clinical Research Grant M01-RR00052 from the National Center for Research Resources/National Institutes of Health and a Patient-Oriented Mentored Scientist Award (to S.H.G.) through the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland (5 K23 DK071565). Disclosure Summary: The authors have nothing to disclose.

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