356 Article
Authors
J. Chan1, B. Sauvé2, S. Tokmakejian3, G. Koren2, 4, 5, 6, S. Van Uum4, 7
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ cortisol ● ▶ testosterone ● ▶ ratio ● ▶ hair ● ▶ obesity ●
Summary
received 13.12.2013 first decision 25.02.2014 accepted 03.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1374609 Exp Clin Endocrinol Diabetes 2014; 122: 356–362 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence S. Van Uum, MD, PhD, FRCPC Associate Professor Endocrinology and Metabolism Department of Medicine, Western University St. Joseph’s Health Care, Room B5-120 268 Grosvenor St. London Ontario N6A 4V2 Canada Tel.: + 1/519/646 6170 Fax: + 1/519/646 6058 [email protected]. on.ca
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Objective: Hair analysis has been demonstrated to accurately reflect exposure to drug abuse, environmental toxins and exogenous hormones. We tested the feasibility of measuring cortisol and testosterone in hair of healthy and obese subjects. Measurements: A modified immunoassay (ELISA) originally developed for saliva was used. Hair, urine and blood samples were collected from young nonobese and obese patients. Perceived stress (PSS) was measured using a validated questionnaire. Results: There was no difference in PSS between non-obese and obese subjects. Hair cortisol levels were significantly correlated with weight (r = 0.27, p < 0.05) and systolic blood pressure (r = 0.28, p < 0.05), while the correlation with BMI did not reach statistical significance (p = 0.063). Hair cortisol levels did not correlate with age or urinary cortisol. There was a negative correlation between hair testosterone and age
Introduction
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Proper assessment of endogenous production of cortisol and testosterone is essential for diagnosis of insufficient or overproduction of these hormones, and for monitoring of therapy. Measurement of total hormone concentrations in serum includes the large fractions of cortisol and testosterone that are bound to cortisol-binding globulin (90 %) (Dunn et al., 1981), and sex-hormone binding globulin (25–50 %), respectively. To better assess biologically active hormone fractions, methods have been developed to measure free cortisol, free testosterone and bioavailable testosterone (consisting of 1–2 % free and 50–75 % albumin bound). These methods are often technically demanding or have limited reliability. Saliva samples do more closely reflect free hormone levels (Walker et al., 1978). However, both saliva and
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
(r = − 0.47, p < 0.05) and BMI (r = − 0.40, p < 0.05). The correlation between hair testosterone and free androgen index (FAI) did not reach statistical significance (p = 0.098). The ratio of hair cortisol over hair testosterone (C/T) was higher in the obese group than in the young non-obese group. The C/T ratio correlated positively with age (r = 0.56, p < 0.01), waist circumference (r = 0.63, p < 0.01) and BMI (r = 0.62, p < 0.01), while the correlation between C/T ratio and FAI did not reach statistical significance. Conclusion: Hair cortisol levels increase, while hair testosterone levels decrease with obesity. The hair C/T ratio was significantly correlated with age, BMI and waist circumference better than hair cortisol or testosterone alone. As hair collection is non-invasive and is not influenced by moment-to-moment variations, the measurement of hormones in hair is a useful tool in research and possibly clinical practice.
blood samples often need to be taken at a specific time of the day, and repeat sampling may be required due to significant diurnal variations. Another limitation of these methods is that they do not assess bioactive hormone levels over a prolonged period of time. Hair analysis has been demonstrated to accurately reflect exposure to drug abuse, environmental toxins and exogenous hormones (Raul et al., 2004; Villain et al., 2004). Over the last few years, we and other groups have investigated hair as a matrix to measure cortisol and testosterone for assessment of systemic exposure to these hormones in chronic stress, endocrine and neuropsychiatric disorders, and in assessing cardiovascular health risks (Russell et al., 2012). Davenport et al. demonstrated in macaque monkeys that prolonged stress significantly increased cortisol levels in hair in parallel to the increase in
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Measurement of Cortisol and Testosterone in Hair of Obese and Non-Obese Human Subjects
salivary cortisol levels (Davenport et al., 2006). In humans we have demonstrated a significant correlation between hair cortisol levels and cortisol exposure in patients with Cushing syndrome and adrenal insufficiency (Thomson et al., 2010; Gow et al., 2011). In 2009, our group demonstrated that hair testosterone levels of hypogonadal men receiving testosterone injections were significantly higher than those not receiving treatment (Thomson et al., 2009). Importantly, hair testosterone of men treated with testosterone was not significantly different from eugonadal men. These findings suggest that testosterone can be measured in the hair as a matrix useful for monitoring testosterone therapy. We hypothesized that hair levels of cortisol and testosterone reflect free or bioactive hormone levels over prolonged periods of time, and may provide further insight into tissue exposure to these hormones in relation to clinical status. In the present explorative study, we aimed to determine hair levels of cortisol and testosterone in obese and non-obese individuals and to compare them to hormonal levels in serum and urine.
Methods
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Participants Non-obese (BMI < 30 mg/kg2) and obese (BMI ≥ 30 mg/kg2) subjects were recruited via advertising in local media. Exclusion criteria included use of any glucocorticosteroid treatment (either systemic or topical) within the last 6 months, dyed hair, and diagnosis of Cushing’s or Addison’s disease, use of testosterone medications or other drugs or medical conditions that affect testosterone secretion. All participants gave written informed consent before enrollment in the study. The study was approved by the local Research Ethics Board.
Procedures All participants were seen by a research nurse between 0730 h and 1000 h. Data on medical and surgical history, current intake of drugs, and use of alcohol and nicotine were obtained. All subjects filled out the Perceived Stress Scale questionnaire, a psychological instrument to quantify perceived stress for the period of 4–8 weeks before the date of administration (Cohen et al., 1983). All male participants also filled out the Saint Louis University Androgen Deficiency in the Aging Male (ADAM) questionnaire which has been developed and validated for screening of hypogonadism and low bioavailable testosterone levels (Morley et al., 2000). Blood pressure, weight, height and waist circumference were measured. A scalp hair sample was obtained as per guidelines established previously by the Motherisk Laboratory (Sauve et al., 2007; Yamada et al., 2007). Briefly, at least 20 mg (20–30 pieces) of hair is obtained from the vertex posterior using fine-tipped surgical scissors, as close to the scalp as possible. All volunteers collected 24-h urine for cortisol and creatinine, and a venous blood sample was taken for cortisol in all participants, and for total testosterone and SHBG in all male participants. A saliva sample was taken for cortisol measurement. All samples for steroid measurements in serum and urine were frozen at − 79 °C until analyzed.
and cortisol exposure was accurately weighed. 2 ml of methanol was added and the hair was minced finely with scissors and incubated for 16 h at 50 °C at 100 RPM. The next day, the methanol was transferred into a clean tube and evaporated to dryness using nitrogen gas. For measurement of cortisol and testosterone testing in hair we used a modified immunoassay (ELISA) from Alpco Diagnostics (Salem, NH, USA) originally developed for assaying these hormones in saliva. The reagents are commercially available (Alpco, Inc), and for quantitation we used calibrators prepared in house by spiking hair extract with the pure hormones to yield concentrations between 5–100 pg/mg of hair. The cortisol recovery was 94 % at 60 ng/ml, and 88 % at 6 ng/ml. The CV for cortisol measurement was 7 % at a normal concentration (64 ng/g) and 6 % at a high concentration (609 ng/g), the CV for between day measurements was 14 %. According to the manufacturer, the lowest level of detection for cortisol is 1.14 ng/mL, and we found intra-assay CVs of 7.2 % and 6.0 % for hair cortisol concentrations of 20 and 600 pg/mg, respectively13. For the saliva assay of testosterone, the lower limit of detection was 1.0 pg/mL, and the coefficient of variation was reported as 9 % (information manufacturer). Based on our method using a minimum of 10 mg of hair, 1 mL methanol, and concentrating the extract, the minimum detectable testosterone concentration in hair was approximately 0.05 pg/g (Thomson et al., 2009). Salivary cortisol was measured using the same salivary ELISA kit (Alpco, Inc.) as per manufacturer’s instructions as previously described. Serum cortisol was measured by Chemiluminescent Immunoassay (Siemens Centaur Analyzer) using trilevel serum quality control material for monitoring performance. Urinary cortisol was first extracted by Methylene Chloride, then constituted and tested on the same analyzer with the trilevel serum control. A urine extraction control was included in the extraction and testing. Testosterone was measured on the Centaur analyzer and SHBG was measured on the Immulite 2000 chemiluminescent analyzer (Diagnostic Products Corporation).
Statistical analysis All data are presented as median (range) unless indicated otherwise. A BMI of 30 kg/m2 was used as cut-off between non-obese and obese participants. Comparisons between groups were analyzed by the Mann-Whitney U test. Correlation coefficients were calculated using Spearman’s rank correlation. Descriptive statistics were calculated in both the arithmetic (natural) and logtransformed (geometric) scales, with the distribution analyzed for normality using the Shapiro-Wilkes test. Statistical significance was accepted at P < 0.05.
Results
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Participants In total 57 subjects were recruited, including 39 non-obese (20 women and 19 men) and 18 obese (11 women and 7 men) indi▶ Table viduals. Their baseline characteristics are presented in ● 1. Most participants were Caucasian. Blood pressure was higher in the obese group as compared to the non-obese group.
Cortisol Measurement of cortisol and testosterone Between 20–50 mg of hair from the 3 cm closest to the scalp, representing approximately 3 months of systemic testosterone
Cortisol levels in hair, urine, saliva or serum did not differ between ▶ Table 1). Therefore, for corthe non-obese and obese groups (● ▶ Fig. 1 relation studies, we used data for all subjects combined. ●
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
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Table 1 Baseline characteristics and results of cortisol measurements in non-obese and obese subjects. Obese
subjects n = 39
subjects n = 18
19/20 38 (20–76) 170 (127–187) 71 (45–92) 82 (60–106) 24 (18–29) 110 (92–138) 70 (56–82) 12 (3–31) 37 (2–213) 222 (25–545) 7.6 (2.3–12.8)# 46 (27–200)
7/11 48 (28–67)a 164 (158–185) 93 (76–30)a 106 (85–125)a 32 (30–50)a 119 (102–138)a 76 (64–88)a 18 (1–40) 37 (6–64) 196 (74–438) 4.6 (6.8–26.3)* 57 (33–424)
BMI = Body Mass Index; BP = blood pressure; PSS = Perceived Stress Scale. Data are presented as median (range). #n = 27; * n = 11 due to insufficient quantity. a P < 0.05 compared to non-obese subjects (Mann-Whitney U test)
r = 0.09 p = NS
400 300 200 100 0
0
20
40 60 Age (years)
Cortisol in Hair and Urine
Hair Cortisol and Weight
Cortisol in hair (pg/mg)
Cortisol in hair (pg/mg)
Hair Cortisol and Age
To assess the relation between testosterone levels in hair and age and obesity, we divided the 26 male participants in 3 groups: young non-obese participants (18–50 years), non-obese participants over 50 years of age, and obese subjects (BMI ≥ 30 mg/kg2). The baseline characteristics and results of hormonal measure▶ Table 3. Hair testosterone levels in ments are presented in ● non-obese subjects over 50 years of age were significantly lower than the levels in the 18–50 year old group. A similar difference was found for the Free Androgen Index, but not for total serum testosterone levels. The Free Androgen Index is a ratio used to determine abnormal androgen levels. It is calculated by measuring total testosterone concentration divided by sex hormonebinding globulin concentration multiplied by 100. In the obese group, total serum testosterone concentration and SHBG were lower than in both other groups, but neither the Free Androgen Index nor the hair testosterone levels were different compared to the non-obese groups. The ratio of hair cortisol over hair testosterone was higher in the obese group than in the young non-obese group, and a statistically non-significant trend toward a higher ratio in the nonobese subjects over 50 years as compared to the 18–50 year non-obese group (P = 0.1). The correlations between clinical and laboratory parameters were analyzed for the non-obese and obese male participants ▶ Table 4). Hair testosterone levels were negatively combined (● correlated with age, BMI and waist circumference, while the correlation between hair testosterone levels and the Free Androgen ▶ Fig. 2, Index did not reach statistical significance (P = 0.098) (●
80
r = 0.27 P< 0.05
400
Cortisol in hair (pg/mg)
male/female age (years) height (cm) weight (kg) waist (cm) BMI (kg/m2) systolic BP (mmHg) diastolic BP (mmHg) PSS score urinary cortisol (μmol/24 h) plasma cortisol (nmol/L) saliva cortisol (pmol/L) hair cortisol (pg/mg)
Non-obese
Testosterone
300 200 100 0
40
60
80 100 Weight (kg)
120
r = 0.11 p= NS
400 300 200 100 0
0
50 100 150 200 250 Urinary Cortisol (µmol/day)
Fig. 1 The relation between hair cortisol levels and age, weight and urinary cortisol. There was a significant positive correlation between hair cortisol levels and weight (n = 57 subjects; Spearman correlation test).
Table 2 Correlation coefficients for cortisol studies in all subjects (n = 57).
urine cortisol serum cortisol saliva cortisol age weight waist BMI SBP DBP PSS score
Hair Cortisol
Urine Cortisol
Serum Cortisol
0.11 0.16 0.27 0.04 0.27b 0.19 0.25 0.28b 0.20 0.01
− 0.08 0.21 0.14 0.15 0.06 0.03 0.01 − 0.04 − 0.08
0.54a − 0.23 − 0.13 − 0.15 − 0.09 − 0.15 − 0.23 − 0.21
Saliva Cortisol
0.03 0.17 0.07 0.15 0.01 0.04 − 0.41
Age
Weight
Waist
BMI
SBP
DBP
0.29b 0.37a 0.3b 0.24 0.32b − 0.09
0.94a 0.90a 0.59a 0.48a 0.17
0.88a 0.55a 0.48a 0.22
0.55a 0.47a 0.27b
0.74a − 0.04
0.12
BMI = Body Mass Index, SBP = Systolic Blood Pressure, DBP = Diastolic Blood Pressure. a P < 0.01, b P < 0.05 (Spearman’s rank correlation)
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
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shows the relation between cortisol levels in hair, for all subjects combined, and age, weight and 24-h urinary cortisol excretion. The correlations between cortisol measurements and clinical ▶ Table 2. Hair cortisol levels were parameters are presented in ● significantly correlated with weight and systolic blood pressure, while the correlation with BMI did not reach statistical significance (P = 0.063), possibly due to limited power.
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Table 3 Clinical characteristics and laboratory results for testosterone studies. Non-obese males ≤ 50 yr
Non-obese males > 50 yr
14 27 (20–50) 75 (55–92) 84 (70–99) 24 (19–29) 112 (92–122) 70 (60–80) 12 (7–31) 17 (11–33 ) 29 (14–56) 58 (39–109) 49 (27–199) 8.0 (4.2–11) 6.5 (4.2–58.8)
Obese males (BMI ≥ 30)
5 68 (54–76)a 84 (71–88) 99 (86–106)a 26 (22–29) 110 (92–138) 70 (60–82) 12 (6–22) 14 (12–20) 39 (35–48)a 36 (25–56)a 58 (37–91) 5.5 (2.7–6.7)a 10.6 (6.0–23.3)
7 51 (32–67)a,b 100 (93–117)b 106 (100–117)a,b 33 (31–38)a,b 120 (108–132)a 72 (68–82) 11 (1–27) 9 (4–13)a,b 16 (10–36)a,b 48 (24–75) 66 (39–424) 5.6 (2.4–17) 14.0 (8.0–46.0)a
BP = blood pressure; PSS = Perceived Stress Scale; SHBG = Sex Hormone Binding Globulin. Data are presented as median (range). a P < 0.05 vs. non-obese young males, b P < 0.05 vs. non-obese males > 50 years (Mann-Whitney U-test)
Table 4 Correlations coefficients for testosterone studies (all 26 males).
BMI waist serum test SHBG FAI PSS score hair cort hair test cort/test hair
Age
BMI
Waist
0.51a 0.66a − 0.53a 0.27 − 0.74a − 0.09 0.28 − 0.47b 0.56a
0.93a − 0.72a − 0.42b − 0.31 − 0.01 0.28 − 0.40b 0.62a
− 0.75a − 0.31 − 0.41b − 0.04 0.20 − 0.56a 0.63a
Serum Test
0.49b 0.46b − 0.02 − 0.40b 0.14 − 0.47b
SHBG
− 0.46b − 0.08 − 0.20 − 0.19 − 0.11
FAI
− 0.07 − 0.08 0.33 − 0.31
PSS score
Hair Cort
Hair Test
0.01 0.08 0.02
0.11 0.71a
− 0.54a
Cort = cortisol, Test = testosterone, FAI = Free Androgen Index a
P < 0.01 bP < 0.05 Spearman’s rank correlation
top panels). The ratio of cortisol over testosterone in hair was ▶ Fig. 2, bottom panels), positively correlated with age and BMI (● ▶ Table 4). and negatively correlated with serum testosterone (● To assess the relation between subjective androgen deficiency and testosterone levels in hair, we compared subjects who scored positive on the ADAM questionnaire (n = 11) with those who did not (n = 15). ADAM positive subjects differed from ADAM negative subjects with respect to age (51;22–76 vs. 32;20–67 years, P < 0.01), diastolic blood pressure (76;60–82 vs. 68;60–82 mmHg, P < 0.05) and increased subjective stress (PSS score (21;6–31 vs. 10;1–16, P < 0.05)), while no difference was found regarding weight, waist circumference, BMI, systolic blood pressure or saliva cortisol and urinary cortisol. ADAM positive subjects had lower serum testosterone (12;4–17 vs. 16;7– 33 nmol/L P < 0.05) and lower Free Androgen Index (40;25–69 vs. 58;24–109, P < 0.01). When we compared hair concentrations between ADAM positive and ADAM negative subjects, we found no difference in hair cortisol (63;39–424 vs. 51;27–114 pg/mg, P = NS) or hair testosterone (5.5;2.4–16.9 vs. 6.4;4.7–11.0 pg/mg, P < 0.05), but the ratio of cortisol over testosterone was significantly higher in the ADAM positive group then in the ADAM negative group (16.4;6.8–58.8 vs. 6.2;4.2–17.7, P = 0.002).
Discussion
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In this pilot study we find that hair cortisol levels are positively correlated to weight and systolic blood pressure, and that hair
testosterone levels are negatively correlated with age, BMI and ▶ Fig. 1, 2). The ratio of cortisol over testowaist circumference (● sterone in hair was positively correlated with age, BMI and waist circumference, while a negative correlation was found with ▶ Fig. 2). serum testosterone levels (● We recognize that this study included only a small number of participants, and that patient groups were not matched for comparison analyses. As most participants were Caucasian, the results can not necessarily be extrapolated to non-Caucasian subjects. However, as this is the first study measuring cortisol and testosterone in hair in relation to obesity, the results provide important data to be used for design and power calculations for future studies. In this light, it is important to determine if these results corroborate established relationships shown in serum, saliva and urine. In the present study, we found a positive correlation between hair cortisol levels and body weight, and a trend (P = 0.063) for a positive correlation with BMI, while there was no correlation between cortisol in hair and urine. Our trend for a positive correlation between hair cortisol levels and BMI is in line with 2 recent studies showing a significant positive correlation between these 2 parameters (Manenschijn et al., 2011; Stalder et al., 2012). Previous studies, using cortisol measurement in serum or urine to assess cortisol production, have found increased (Zelissen et al., 1991), similar (Andrew et al., 1998) or decreased (Vila et al., 2001) cortisol production in obesity. However, a study using stable isotope techniques demonstrated an increase of cortisol production in proportion to body weight and fat mass (Purnell et al., 2004). The increase in cortisol may origi-
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Parameter no. age (years) weight (kg) waist (cm) BMI (kg/m2) systolic BP (mmHg) diastolic BP (mmHg) PSS score serum testosterone (nmol/L) SHBG (nmol/L) free androgen index hair cortisol (pg/mg) hair testosterone (pg/mg) cortisol/testosterone (hair)
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12 8 4
20
40
60
20
0
20
30
40
8 4 0
0
50
100
Free Androgen Index
Hair C/T and Age
Hair C/T and BMI
Hair C/T and FAI
40
60
80
r = 0.62 P< 0.01
60
Cortisol / Testosterone (hair)
20
0
4
12
BMI (kg/m2)
40
0
8
r = 0.33 p=NS
16
Age (years)
r = 0.56 p< 0.01
60
12
0
80
r = –0.40 P< 0.05
16
Hair T and FAI
20 Testosterone in hair (pg/mg)
Testosterone in hair (pg/mg)
16
Cortisol / Testosterone (hair)
Testosterone in hair (pg/mg)
r = –0.47 P< 0.05
0
Cortisol / Testosterone (hair)
Hair T and BMI 20
40 20 0
0
20
Age (years)
30 BMI (kg/m2)
40
r = –0.31 p=NS
60 40 20 0
0
50
100
Free Androgen Index
Fig. 2 There was a significant negative correlation between hair testosterone and age and body mass index (BMI), and stronger correlations between the hair cortisol/testosterone (C/T) ratio and age and BMI. (n = 26 subjects; Spearman correlation test).
nate from conversion of cortisone by 11β-hydroxysteroid dehydrogenase activity in visceral adipose tissue (Andrew et al., 2005). It is also known that obese men excrete a greater proportion of glucocorticoids as metabolites of cortisone rather than cortisol which may result in a discrepancy between cortisol levels in hair and urine. In obesity, there may be an increased production of cortisol together with enhanced clearance by tissues other than the kidneys (Andrew et al., 1998). It is therefore possible that hair levels of cortisol provide a more sensitive measure for tissue exposure to cortisol than measurement of urinary excretion. We did not ▶ Fig. 1), a findfind a correlation between hair cortisol and age (● ing in line with previous research (Raul et al., 2004; Dettenborn et al., 2010; Manenschijn et al., 2011). In contrast, a recent study showed a quadratic relationship between hair cortisol levels and age (Dettenborn et al., 2012). In this study, hair cortisol levels were elevated among the young and elderly compared to adults; however, the number of participants in the young and elderly categories was relatively small. Further studies are required to confirm the relationship between hair cortisol levels and age and between hair cortisol levels and obesity, to allow improved understanding of the pathophysiology involved. In the present study, we found a trend toward correlation between hair testosterone and the Free Androgen Index, while there was no such trend for total testosterone in serum. This supports the hypothesis that hair testosterone levels may more closely reflect bioavailable than total testosterone levels. The lack of significance between hair testosterone and the Free Androgen Index may be due to the limited power of this study.
Hair testosterone levels were negatively correlated with age, BMI, and waist circumference. The decline of hair testosterone with age is in agreement with the well-established decline of testosterone with age. The annual decrease of total testosterone is estimated to vary between 0.8 and 1.6 % in cross-sectional and longitudinal studies (Harman et al., 2001). Little consensus exists among clinicians as to what constitutes a normal sex hormone profile for an aging male (Lamberts et al., 1997), and currently there is no universal recommendation for testosterone replacement therapy. One of the issues is that within individuals, the correlation between age and change in testosterone over time is relatively poor (Lamberts et al., 1997). Some studies suggest that the diurnal rhythm of testosterone disappears with aging (Bremner et al., 1983), but in other studies this was found in less half of aging men (Plymate et al., 1989). It is currently unknown whether hair testosterone levels reflect the differences in testosterone secretion among individuals with, and without testosterone rhythm. Obesity is associated with decreased levels of total testosterone and SHBG (Harman et al., 2001). The effect of obesity on free or bioavailable testosterone levels is less clear. Some studies do not show a relation between obesity and free testosterone levels (Harman et al., 2001), while other investigators demonstrate diminished free testosterone with increasing total or abdominal obesity in men (Svartberg et al., 2004). In a relatively small sample, we found a clear reduction in hair testosterone levels in relation to increased waist circumference and BMI. It should be kept in mind that epidemiological studies are based on single hormone samples obtained in the morning, while hair testosterone
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Hair T and Age 20
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have sufficient hair at the vertex posterior and do not have cultural/religious objections to taking a hair sample. It is unknown if hair growth rate affects hair levels of cortisol or testosterone and hormonal levels may be affected by the intermittent growth of hair follicles. In summary, the current study indicates that hair levels of cortisol and testosterone may reflect average levels of free/bioavailable hormones over several months. Further studies are required to confirm these findings, and assess possible applications in research and clinical practice.
Acknowledgements
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This study was supported by the Physicians’ Services Incorporation Foundation and Canadian Institutes of Health Research. We thank Grace Walsh for her invaluable assistance.
Disclosure: Nothing to declare. Affiliations Department of Physiology and Pharmacology, Western University, London Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, Canada 3 Department of Biochemistry, Western University, London, Ontario 4 Department of Medicine, Western University, London, Ontario 5 Department of Paediatrics, Western University, London, Ontario 6 Ivey Chair in Molecular Toxicology, Western University, London, Ontario 7 Lawson Health Research Institute, Western University, London, Ontario 1
2
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samples reflect the integral of free levels throughout several months. Our study suggests that testosterone measurements that assess testosterone levels integrated over a several month period may be more reflective of the true gonadal status than single morning measurements. Moreover, studies requiring 24-h sampling are quite labor intensive and necessitate hospitalization. As hair testosterone levels are hypothesized to measure free hormone levels integrated over several months, the measurement of testosterone in hair may be useful in larger scale studies on testosterone and gonadal status. The PSS score is used to assess subjective stress over the last 4 weeks. In the present pilot study, we did not find any significant correlation between PSS and cortisol, testosterone or their ratio in hair. Increased hair cortisol may be a marker for increased cardiovascular risk. We have previously demonstrated that increased hair cortisol is associated with increased risk for myocardial infarcts in men (Pereg et al., 2011). Another study in a group of 283 community-dwelling, elderly participants found that high hair cortisol levels were associated with a history of cardiovascular disease, but not non-cardiovascular diseases (Manenschijn et al. 2013). Further, high urinary cortisol secretion strongly predicts cardiovascular death among persons both with and without preexisting cardiovascular diseases (Vogelzangs et al., 2010). A low testosterone level is associated with increased carotid intimamedia thickness in men with low-grade inflammation (Soisson et al., 2012) and impaired endothelial function (Empen et al., 2012). Although an analysis of the Framingham population did not find an association between low testosterone and cardiovascular risk (Haring et al., 2013), a low serum testosterone was associated with increased risk of all-cause mortality (Haring et al., 2010). In light of the association between high cortisol and increased cardiovascular risk factors and all-cause mortality, coupled with the association between low testosterone and allcause mortality and possibly cardiovascular risk, the ratio of cortisol over testosterone might be particularly interesting. With respect to hair measurements, we found stronger correlations between this ratio and age, BMI and waist circumference as compared to correlations for cortisol or testosterone in hair separately. The clinical significance of the cortisol over testosterone ratio in serum has been demonstrated in the Caerphilly study, showing a strong positive correlation between this ratio and both the insulin resistance syndrome and incident ischemic heart disease (Smith et al., 2005). Based on these data it is possible that the hair cortisol-testosterone ratio may be useful in population studies investigating the risk of metabolic syndrome and cardiovascular events. Measurement of hormone levels in hair has several advantages. Its collection is non-invasive, does not require health care workers, and can be conducted at any time of the day. Samples can be stored at room temperature and be sent by mail. Further, levels reflect average hormone levels over a period of months and years, as opposed to blood and saliva samples that reflect one moment in time, and urine that reflects levels during 1 day. A unique feature of the measurement of hormones in hair is that the hormone levels are not affected by acute stress and hence may potentially be used even at or immediately after a major event, such as a myocardial infarction, which would cause acute changes in hormonal levels and thus not represent the hormonal milieu before the event. There are several potential limitations to measurement of endogenous hormones in hair. It is limited to individuals who
14 Lamberts SW, van den Beld AW, van der Lely AJ. The endocrinology of aging. Science 1997; 278: 419–424 15 Manenschijn L, Koper JW, Lamberts SW et al. Evaluation of a method to measure long term cortisol levels. Steroids 2011; 76: 1032–1036 16 Manenschijn L, Schaap L, van Schoor NM et al. High long-term cortisol levels, measured in scalp hair, are associated with a history of cardiovascular disease. J Clin Endocrinol Metab 2013; 98: 2078–2083 17 Manenschijn L, van Kruysbergen RG, de Jong FH et al. Shift work at young age is associated with elevated long-term cortisol levels and body mass index. J Clin Endocrinol Metab 2011; 96: E1862–E1865 18 Morley JE, Charlton E, Patrick P et al. Validation of a screening questionnaire for androgen deficiency in aging males. Metabolism 2000; 49: 1239–1242 19 Pereg D, Gow R, Mosseri M et al. Hair cortisol and the risk for acute myocardial infarction in adult men. Stress 2011; 14: 73–81 20 Plymate SR, Tenover JS, Bremner WJ. Circadian variation in testosterone, sex hormone-binding globulin, and calculated non-sex hormonebinding globulin bound testosterone in healthy young and elderly men. J Androl 1989; 10: 366–371 21 Purnell JQ, Brandon DD, Isabelle LM et al. 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. J Clin Endocrinol Metab 2004; 89: 281–287 22 Raul JS, Cirimele V, Ludes B et al. Detection of physiological concentrations of cortisol and cortisone in human hair. Clin Biochem 2004; 37: 1105–1111 23 Russell E, Koren G, Rieder M et al. Hair cortisol as a biological marker of chronic stress: Current status, future directions and unanswered questions. Psychoneuroendocrinol 2012; 37: 589–601 24 Sauve B, Koren G, Walsh G et al. Measurement of cortisol in human hair as a biomarker of systemic exposure. Clin Invest Med 2007; 30: E183–E191
25 Smith GD, Ben-Shlomo Y, Beswick A et al. Cortisol, testosterone, and coronary heart disease: Prospective evidence from the caerphilly study. Circulation 2005; 112: 332–340 26 Soisson V, Brailly-Tabard S, Empana JP et al. Low plasma testosterone and elevated carotid intima-media thickness: Importance of low-grade inflammation in elderly men. Atherosclerosis 2012; 223: 244–249 27 Stalder T, Steudte S, Alexander N et al. Cortisol in hair, body mass index and stress-related measures. Biol Psychol 2012; 90: 218–223 28 Svartberg J, von Muhlen D, Sundsfjord J et al. Waist circumference and testosterone levels in community dwelling men. The Tromso study. Eur J Epidemiol 2004; 19: 657–663 29 Thomson S, Koren G, Van Steen V et al. Testosterone concentrations in hair of hypogonadal men with and without testosterone replacement therapy. Ther Drug Monit 2009; 31: 779–782 30 Thomson S, Koren G, Fraser LA et al. Hair analysis provides a historical record of cortisol levels in cushing’s syndrome. Exp Clin Endocrinol Diabetes 2009; 118: 133–138 31 Vila R, Granada ML, Guitierrez RM et al. Urinary free cortisol excretion pattern in morbid obese women. Endocr Res 2001; 27: 261–268 32 Villain M, Cirimele V, Kintz P. Hair analysis in toxicology. Clin Chem Lab Med 2004; 42: 1265–1272 33 Vogelzangs N, Beekman AT, Milaneschi Y et al. Urinary cortisol and sixyear risk of all-cause and cardiovascular mortality. J Clin Endocrinol Metab 2010; 95: 4959–4964 34 Walker RF, Riad-Fahmy D, Read GF. Adrenal status assessed by direct radioimmunoassay of cortisol in whole saliva or parotid saliva. Clin Chem 1978; 24: 1460–1463 35 Yamada J, Stevens B. Hair cortisol as a potential biologic marker of chronic stress in hospitalized neonates. Neonatology 2007; 92: 42–49 36 Zelissen PM, Koppeschaar HP, Erkelens DW et al. Beta-endorphin and adrenocortical function in obesity. Clin Endocrinol 1991; 35: 369–372
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
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362 Article
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Authors
J. Chan1, B. Sauvé2, S. Tokmakejian3, G. Koren2, 4, 5, 6, S. Van Uum4, 7
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ cortisol ● ▶ testosterone ● ▶ ratio ● ▶ hair ● ▶ obesity ●
Summary
received 13.12.2013 first decision 25.02.2014 accepted 03.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1374609 Exp Clin Endocrinol Diabetes 2014; 122: 356–362 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence S. Van Uum, MD, PhD, FRCPC Associate Professor Endocrinology and Metabolism Department of Medicine, Western University St. Joseph’s Health Care, Room B5-120 268 Grosvenor St. London Ontario N6A 4V2 Canada Tel.: + 1/519/646 6170 Fax: + 1/519/646 6058 [email protected]. on.ca
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Objective: Hair analysis has been demonstrated to accurately reflect exposure to drug abuse, environmental toxins and exogenous hormones. We tested the feasibility of measuring cortisol and testosterone in hair of healthy and obese subjects. Measurements: A modified immunoassay (ELISA) originally developed for saliva was used. Hair, urine and blood samples were collected from young nonobese and obese patients. Perceived stress (PSS) was measured using a validated questionnaire. Results: There was no difference in PSS between non-obese and obese subjects. Hair cortisol levels were significantly correlated with weight (r = 0.27, p < 0.05) and systolic blood pressure (r = 0.28, p < 0.05), while the correlation with BMI did not reach statistical significance (p = 0.063). Hair cortisol levels did not correlate with age or urinary cortisol. There was a negative correlation between hair testosterone and age
Introduction
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Proper assessment of endogenous production of cortisol and testosterone is essential for diagnosis of insufficient or overproduction of these hormones, and for monitoring of therapy. Measurement of total hormone concentrations in serum includes the large fractions of cortisol and testosterone that are bound to cortisol-binding globulin (90 %) (Dunn et al., 1981), and sex-hormone binding globulin (25–50 %), respectively. To better assess biologically active hormone fractions, methods have been developed to measure free cortisol, free testosterone and bioavailable testosterone (consisting of 1–2 % free and 50–75 % albumin bound). These methods are often technically demanding or have limited reliability. Saliva samples do more closely reflect free hormone levels (Walker et al., 1978). However, both saliva and
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
(r = − 0.47, p < 0.05) and BMI (r = − 0.40, p < 0.05). The correlation between hair testosterone and free androgen index (FAI) did not reach statistical significance (p = 0.098). The ratio of hair cortisol over hair testosterone (C/T) was higher in the obese group than in the young non-obese group. The C/T ratio correlated positively with age (r = 0.56, p < 0.01), waist circumference (r = 0.63, p < 0.01) and BMI (r = 0.62, p < 0.01), while the correlation between C/T ratio and FAI did not reach statistical significance. Conclusion: Hair cortisol levels increase, while hair testosterone levels decrease with obesity. The hair C/T ratio was significantly correlated with age, BMI and waist circumference better than hair cortisol or testosterone alone. As hair collection is non-invasive and is not influenced by moment-to-moment variations, the measurement of hormones in hair is a useful tool in research and possibly clinical practice.
blood samples often need to be taken at a specific time of the day, and repeat sampling may be required due to significant diurnal variations. Another limitation of these methods is that they do not assess bioactive hormone levels over a prolonged period of time. Hair analysis has been demonstrated to accurately reflect exposure to drug abuse, environmental toxins and exogenous hormones (Raul et al., 2004; Villain et al., 2004). Over the last few years, we and other groups have investigated hair as a matrix to measure cortisol and testosterone for assessment of systemic exposure to these hormones in chronic stress, endocrine and neuropsychiatric disorders, and in assessing cardiovascular health risks (Russell et al., 2012). Davenport et al. demonstrated in macaque monkeys that prolonged stress significantly increased cortisol levels in hair in parallel to the increase in
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Measurement of Cortisol and Testosterone in Hair of Obese and Non-Obese Human Subjects
salivary cortisol levels (Davenport et al., 2006). In humans we have demonstrated a significant correlation between hair cortisol levels and cortisol exposure in patients with Cushing syndrome and adrenal insufficiency (Thomson et al., 2010; Gow et al., 2011). In 2009, our group demonstrated that hair testosterone levels of hypogonadal men receiving testosterone injections were significantly higher than those not receiving treatment (Thomson et al., 2009). Importantly, hair testosterone of men treated with testosterone was not significantly different from eugonadal men. These findings suggest that testosterone can be measured in the hair as a matrix useful for monitoring testosterone therapy. We hypothesized that hair levels of cortisol and testosterone reflect free or bioactive hormone levels over prolonged periods of time, and may provide further insight into tissue exposure to these hormones in relation to clinical status. In the present explorative study, we aimed to determine hair levels of cortisol and testosterone in obese and non-obese individuals and to compare them to hormonal levels in serum and urine.
Methods
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Participants Non-obese (BMI < 30 mg/kg2) and obese (BMI ≥ 30 mg/kg2) subjects were recruited via advertising in local media. Exclusion criteria included use of any glucocorticosteroid treatment (either systemic or topical) within the last 6 months, dyed hair, and diagnosis of Cushing’s or Addison’s disease, use of testosterone medications or other drugs or medical conditions that affect testosterone secretion. All participants gave written informed consent before enrollment in the study. The study was approved by the local Research Ethics Board.
Procedures All participants were seen by a research nurse between 0730 h and 1000 h. Data on medical and surgical history, current intake of drugs, and use of alcohol and nicotine were obtained. All subjects filled out the Perceived Stress Scale questionnaire, a psychological instrument to quantify perceived stress for the period of 4–8 weeks before the date of administration (Cohen et al., 1983). All male participants also filled out the Saint Louis University Androgen Deficiency in the Aging Male (ADAM) questionnaire which has been developed and validated for screening of hypogonadism and low bioavailable testosterone levels (Morley et al., 2000). Blood pressure, weight, height and waist circumference were measured. A scalp hair sample was obtained as per guidelines established previously by the Motherisk Laboratory (Sauve et al., 2007; Yamada et al., 2007). Briefly, at least 20 mg (20–30 pieces) of hair is obtained from the vertex posterior using fine-tipped surgical scissors, as close to the scalp as possible. All volunteers collected 24-h urine for cortisol and creatinine, and a venous blood sample was taken for cortisol in all participants, and for total testosterone and SHBG in all male participants. A saliva sample was taken for cortisol measurement. All samples for steroid measurements in serum and urine were frozen at − 79 °C until analyzed.
and cortisol exposure was accurately weighed. 2 ml of methanol was added and the hair was minced finely with scissors and incubated for 16 h at 50 °C at 100 RPM. The next day, the methanol was transferred into a clean tube and evaporated to dryness using nitrogen gas. For measurement of cortisol and testosterone testing in hair we used a modified immunoassay (ELISA) from Alpco Diagnostics (Salem, NH, USA) originally developed for assaying these hormones in saliva. The reagents are commercially available (Alpco, Inc), and for quantitation we used calibrators prepared in house by spiking hair extract with the pure hormones to yield concentrations between 5–100 pg/mg of hair. The cortisol recovery was 94 % at 60 ng/ml, and 88 % at 6 ng/ml. The CV for cortisol measurement was 7 % at a normal concentration (64 ng/g) and 6 % at a high concentration (609 ng/g), the CV for between day measurements was 14 %. According to the manufacturer, the lowest level of detection for cortisol is 1.14 ng/mL, and we found intra-assay CVs of 7.2 % and 6.0 % for hair cortisol concentrations of 20 and 600 pg/mg, respectively13. For the saliva assay of testosterone, the lower limit of detection was 1.0 pg/mL, and the coefficient of variation was reported as 9 % (information manufacturer). Based on our method using a minimum of 10 mg of hair, 1 mL methanol, and concentrating the extract, the minimum detectable testosterone concentration in hair was approximately 0.05 pg/g (Thomson et al., 2009). Salivary cortisol was measured using the same salivary ELISA kit (Alpco, Inc.) as per manufacturer’s instructions as previously described. Serum cortisol was measured by Chemiluminescent Immunoassay (Siemens Centaur Analyzer) using trilevel serum quality control material for monitoring performance. Urinary cortisol was first extracted by Methylene Chloride, then constituted and tested on the same analyzer with the trilevel serum control. A urine extraction control was included in the extraction and testing. Testosterone was measured on the Centaur analyzer and SHBG was measured on the Immulite 2000 chemiluminescent analyzer (Diagnostic Products Corporation).
Statistical analysis All data are presented as median (range) unless indicated otherwise. A BMI of 30 kg/m2 was used as cut-off between non-obese and obese participants. Comparisons between groups were analyzed by the Mann-Whitney U test. Correlation coefficients were calculated using Spearman’s rank correlation. Descriptive statistics were calculated in both the arithmetic (natural) and logtransformed (geometric) scales, with the distribution analyzed for normality using the Shapiro-Wilkes test. Statistical significance was accepted at P < 0.05.
Results
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Participants In total 57 subjects were recruited, including 39 non-obese (20 women and 19 men) and 18 obese (11 women and 7 men) indi▶ Table viduals. Their baseline characteristics are presented in ● 1. Most participants were Caucasian. Blood pressure was higher in the obese group as compared to the non-obese group.
Cortisol Measurement of cortisol and testosterone Between 20–50 mg of hair from the 3 cm closest to the scalp, representing approximately 3 months of systemic testosterone
Cortisol levels in hair, urine, saliva or serum did not differ between ▶ Table 1). Therefore, for corthe non-obese and obese groups (● ▶ Fig. 1 relation studies, we used data for all subjects combined. ●
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
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Table 1 Baseline characteristics and results of cortisol measurements in non-obese and obese subjects. Obese
subjects n = 39
subjects n = 18
19/20 38 (20–76) 170 (127–187) 71 (45–92) 82 (60–106) 24 (18–29) 110 (92–138) 70 (56–82) 12 (3–31) 37 (2–213) 222 (25–545) 7.6 (2.3–12.8)# 46 (27–200)
7/11 48 (28–67)a 164 (158–185) 93 (76–30)a 106 (85–125)a 32 (30–50)a 119 (102–138)a 76 (64–88)a 18 (1–40) 37 (6–64) 196 (74–438) 4.6 (6.8–26.3)* 57 (33–424)
BMI = Body Mass Index; BP = blood pressure; PSS = Perceived Stress Scale. Data are presented as median (range). #n = 27; * n = 11 due to insufficient quantity. a P < 0.05 compared to non-obese subjects (Mann-Whitney U test)
r = 0.09 p = NS
400 300 200 100 0
0
20
40 60 Age (years)
Cortisol in Hair and Urine
Hair Cortisol and Weight
Cortisol in hair (pg/mg)
Cortisol in hair (pg/mg)
Hair Cortisol and Age
To assess the relation between testosterone levels in hair and age and obesity, we divided the 26 male participants in 3 groups: young non-obese participants (18–50 years), non-obese participants over 50 years of age, and obese subjects (BMI ≥ 30 mg/kg2). The baseline characteristics and results of hormonal measure▶ Table 3. Hair testosterone levels in ments are presented in ● non-obese subjects over 50 years of age were significantly lower than the levels in the 18–50 year old group. A similar difference was found for the Free Androgen Index, but not for total serum testosterone levels. The Free Androgen Index is a ratio used to determine abnormal androgen levels. It is calculated by measuring total testosterone concentration divided by sex hormonebinding globulin concentration multiplied by 100. In the obese group, total serum testosterone concentration and SHBG were lower than in both other groups, but neither the Free Androgen Index nor the hair testosterone levels were different compared to the non-obese groups. The ratio of hair cortisol over hair testosterone was higher in the obese group than in the young non-obese group, and a statistically non-significant trend toward a higher ratio in the nonobese subjects over 50 years as compared to the 18–50 year non-obese group (P = 0.1). The correlations between clinical and laboratory parameters were analyzed for the non-obese and obese male participants ▶ Table 4). Hair testosterone levels were negatively combined (● correlated with age, BMI and waist circumference, while the correlation between hair testosterone levels and the Free Androgen ▶ Fig. 2, Index did not reach statistical significance (P = 0.098) (●
80
r = 0.27 P< 0.05
400
Cortisol in hair (pg/mg)
male/female age (years) height (cm) weight (kg) waist (cm) BMI (kg/m2) systolic BP (mmHg) diastolic BP (mmHg) PSS score urinary cortisol (μmol/24 h) plasma cortisol (nmol/L) saliva cortisol (pmol/L) hair cortisol (pg/mg)
Non-obese
Testosterone
300 200 100 0
40
60
80 100 Weight (kg)
120
r = 0.11 p= NS
400 300 200 100 0
0
50 100 150 200 250 Urinary Cortisol (µmol/day)
Fig. 1 The relation between hair cortisol levels and age, weight and urinary cortisol. There was a significant positive correlation between hair cortisol levels and weight (n = 57 subjects; Spearman correlation test).
Table 2 Correlation coefficients for cortisol studies in all subjects (n = 57).
urine cortisol serum cortisol saliva cortisol age weight waist BMI SBP DBP PSS score
Hair Cortisol
Urine Cortisol
Serum Cortisol
0.11 0.16 0.27 0.04 0.27b 0.19 0.25 0.28b 0.20 0.01
− 0.08 0.21 0.14 0.15 0.06 0.03 0.01 − 0.04 − 0.08
0.54a − 0.23 − 0.13 − 0.15 − 0.09 − 0.15 − 0.23 − 0.21
Saliva Cortisol
0.03 0.17 0.07 0.15 0.01 0.04 − 0.41
Age
Weight
Waist
BMI
SBP
DBP
0.29b 0.37a 0.3b 0.24 0.32b − 0.09
0.94a 0.90a 0.59a 0.48a 0.17
0.88a 0.55a 0.48a 0.22
0.55a 0.47a 0.27b
0.74a − 0.04
0.12
BMI = Body Mass Index, SBP = Systolic Blood Pressure, DBP = Diastolic Blood Pressure. a P < 0.01, b P < 0.05 (Spearman’s rank correlation)
Chan J et al. Measurement of Cortisol and … Exp Clin Endocrinol Diabetes 2014; 122: 356–362
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shows the relation between cortisol levels in hair, for all subjects combined, and age, weight and 24-h urinary cortisol excretion. The correlations between cortisol measurements and clinical ▶ Table 2. Hair cortisol levels were parameters are presented in ● significantly correlated with weight and systolic blood pressure, while the correlation with BMI did not reach statistical significance (P = 0.063), possibly due to limited power.
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Table 3 Clinical characteristics and laboratory results for testosterone studies. Non-obese males ≤ 50 yr
Non-obese males > 50 yr
14 27 (20–50) 75 (55–92) 84 (70–99) 24 (19–29) 112 (92–122) 70 (60–80) 12 (7–31) 17 (11–33 ) 29 (14–56) 58 (39–109) 49 (27–199) 8.0 (4.2–11) 6.5 (4.2–58.8)
Obese males (BMI ≥ 30)
5 68 (54–76)a 84 (71–88) 99 (86–106)a 26 (22–29) 110 (92–138) 70 (60–82) 12 (6–22) 14 (12–20) 39 (35–48)a 36 (25–56)a 58 (37–91) 5.5 (2.7–6.7)a 10.6 (6.0–23.3)
7 51 (32–67)a,b 100 (93–117)b 106 (100–117)a,b 33 (31–38)a,b 120 (108–132)a 72 (68–82) 11 (1–27) 9 (4–13)a,b 16 (10–36)a,b 48 (24–75) 66 (39–424) 5.6 (2.4–17) 14.0 (8.0–46.0)a
BP = blood pressure; PSS = Perceived Stress Scale; SHBG = Sex Hormone Binding Globulin. Data are presented as median (range). a P < 0.05 vs. non-obese young males, b P < 0.05 vs. non-obese males > 50 years (Mann-Whitney U-test)
Table 4 Correlations coefficients for testosterone studies (all 26 males).
BMI waist serum test SHBG FAI PSS score hair cort hair test cort/test hair
Age
BMI
Waist
0.51a 0.66a − 0.53a 0.27 − 0.74a − 0.09 0.28 − 0.47b 0.56a
0.93a − 0.72a − 0.42b − 0.31 − 0.01 0.28 − 0.40b 0.62a
− 0.75a − 0.31 − 0.41b − 0.04 0.20 − 0.56a 0.63a
Serum Test
0.49b 0.46b − 0.02 − 0.40b 0.14 − 0.47b
SHBG
− 0.46b − 0.08 − 0.20 − 0.19 − 0.11
FAI
− 0.07 − 0.08 0.33 − 0.31
PSS score
Hair Cort
Hair Test
0.01 0.08 0.02
0.11 0.71a
− 0.54a
Cort = cortisol, Test = testosterone, FAI = Free Androgen Index a
P < 0.01 bP < 0.05 Spearman’s rank correlation
top panels). The ratio of cortisol over testosterone in hair was ▶ Fig. 2, bottom panels), positively correlated with age and BMI (● ▶ Table 4). and negatively correlated with serum testosterone (● To assess the relation between subjective androgen deficiency and testosterone levels in hair, we compared subjects who scored positive on the ADAM questionnaire (n = 11) with those who did not (n = 15). ADAM positive subjects differed from ADAM negative subjects with respect to age (51;22–76 vs. 32;20–67 years, P < 0.01), diastolic blood pressure (76;60–82 vs. 68;60–82 mmHg, P < 0.05) and increased subjective stress (PSS score (21;6–31 vs. 10;1–16, P < 0.05)), while no difference was found regarding weight, waist circumference, BMI, systolic blood pressure or saliva cortisol and urinary cortisol. ADAM positive subjects had lower serum testosterone (12;4–17 vs. 16;7– 33 nmol/L P < 0.05) and lower Free Androgen Index (40;25–69 vs. 58;24–109, P < 0.01). When we compared hair concentrations between ADAM positive and ADAM negative subjects, we found no difference in hair cortisol (63;39–424 vs. 51;27–114 pg/mg, P = NS) or hair testosterone (5.5;2.4–16.9 vs. 6.4;4.7–11.0 pg/mg, P < 0.05), but the ratio of cortisol over testosterone was significantly higher in the ADAM positive group then in the ADAM negative group (16.4;6.8–58.8 vs. 6.2;4.2–17.7, P = 0.002).
Discussion
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In this pilot study we find that hair cortisol levels are positively correlated to weight and systolic blood pressure, and that hair
testosterone levels are negatively correlated with age, BMI and ▶ Fig. 1, 2). The ratio of cortisol over testowaist circumference (● sterone in hair was positively correlated with age, BMI and waist circumference, while a negative correlation was found with ▶ Fig. 2). serum testosterone levels (● We recognize that this study included only a small number of participants, and that patient groups were not matched for comparison analyses. As most participants were Caucasian, the results can not necessarily be extrapolated to non-Caucasian subjects. However, as this is the first study measuring cortisol and testosterone in hair in relation to obesity, the results provide important data to be used for design and power calculations for future studies. In this light, it is important to determine if these results corroborate established relationships shown in serum, saliva and urine. In the present study, we found a positive correlation between hair cortisol levels and body weight, and a trend (P = 0.063) for a positive correlation with BMI, while there was no correlation between cortisol in hair and urine. Our trend for a positive correlation between hair cortisol levels and BMI is in line with 2 recent studies showing a significant positive correlation between these 2 parameters (Manenschijn et al., 2011; Stalder et al., 2012). Previous studies, using cortisol measurement in serum or urine to assess cortisol production, have found increased (Zelissen et al., 1991), similar (Andrew et al., 1998) or decreased (Vila et al., 2001) cortisol production in obesity. However, a study using stable isotope techniques demonstrated an increase of cortisol production in proportion to body weight and fat mass (Purnell et al., 2004). The increase in cortisol may origi-
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Parameter no. age (years) weight (kg) waist (cm) BMI (kg/m2) systolic BP (mmHg) diastolic BP (mmHg) PSS score serum testosterone (nmol/L) SHBG (nmol/L) free androgen index hair cortisol (pg/mg) hair testosterone (pg/mg) cortisol/testosterone (hair)
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12 8 4
20
40
60
20
0
20
30
40
8 4 0
0
50
100
Free Androgen Index
Hair C/T and Age
Hair C/T and BMI
Hair C/T and FAI
40
60
80
r = 0.62 P< 0.01
60
Cortisol / Testosterone (hair)
20
0
4
12
BMI (kg/m2)
40
0
8
r = 0.33 p=NS
16
Age (years)
r = 0.56 p< 0.01
60
12
0
80
r = –0.40 P< 0.05
16
Hair T and FAI
20 Testosterone in hair (pg/mg)
Testosterone in hair (pg/mg)
16
Cortisol / Testosterone (hair)
Testosterone in hair (pg/mg)
r = –0.47 P< 0.05
0
Cortisol / Testosterone (hair)
Hair T and BMI 20
40 20 0
0
20
Age (years)
30 BMI (kg/m2)
40
r = –0.31 p=NS
60 40 20 0
0
50
100
Free Androgen Index
Fig. 2 There was a significant negative correlation between hair testosterone and age and body mass index (BMI), and stronger correlations between the hair cortisol/testosterone (C/T) ratio and age and BMI. (n = 26 subjects; Spearman correlation test).
nate from conversion of cortisone by 11β-hydroxysteroid dehydrogenase activity in visceral adipose tissue (Andrew et al., 2005). It is also known that obese men excrete a greater proportion of glucocorticoids as metabolites of cortisone rather than cortisol which may result in a discrepancy between cortisol levels in hair and urine. In obesity, there may be an increased production of cortisol together with enhanced clearance by tissues other than the kidneys (Andrew et al., 1998). It is therefore possible that hair levels of cortisol provide a more sensitive measure for tissue exposure to cortisol than measurement of urinary excretion. We did not ▶ Fig. 1), a findfind a correlation between hair cortisol and age (● ing in line with previous research (Raul et al., 2004; Dettenborn et al., 2010; Manenschijn et al., 2011). In contrast, a recent study showed a quadratic relationship between hair cortisol levels and age (Dettenborn et al., 2012). In this study, hair cortisol levels were elevated among the young and elderly compared to adults; however, the number of participants in the young and elderly categories was relatively small. Further studies are required to confirm the relationship between hair cortisol levels and age and between hair cortisol levels and obesity, to allow improved understanding of the pathophysiology involved. In the present study, we found a trend toward correlation between hair testosterone and the Free Androgen Index, while there was no such trend for total testosterone in serum. This supports the hypothesis that hair testosterone levels may more closely reflect bioavailable than total testosterone levels. The lack of significance between hair testosterone and the Free Androgen Index may be due to the limited power of this study.
Hair testosterone levels were negatively correlated with age, BMI, and waist circumference. The decline of hair testosterone with age is in agreement with the well-established decline of testosterone with age. The annual decrease of total testosterone is estimated to vary between 0.8 and 1.6 % in cross-sectional and longitudinal studies (Harman et al., 2001). Little consensus exists among clinicians as to what constitutes a normal sex hormone profile for an aging male (Lamberts et al., 1997), and currently there is no universal recommendation for testosterone replacement therapy. One of the issues is that within individuals, the correlation between age and change in testosterone over time is relatively poor (Lamberts et al., 1997). Some studies suggest that the diurnal rhythm of testosterone disappears with aging (Bremner et al., 1983), but in other studies this was found in less half of aging men (Plymate et al., 1989). It is currently unknown whether hair testosterone levels reflect the differences in testosterone secretion among individuals with, and without testosterone rhythm. Obesity is associated with decreased levels of total testosterone and SHBG (Harman et al., 2001). The effect of obesity on free or bioavailable testosterone levels is less clear. Some studies do not show a relation between obesity and free testosterone levels (Harman et al., 2001), while other investigators demonstrate diminished free testosterone with increasing total or abdominal obesity in men (Svartberg et al., 2004). In a relatively small sample, we found a clear reduction in hair testosterone levels in relation to increased waist circumference and BMI. It should be kept in mind that epidemiological studies are based on single hormone samples obtained in the morning, while hair testosterone
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have sufficient hair at the vertex posterior and do not have cultural/religious objections to taking a hair sample. It is unknown if hair growth rate affects hair levels of cortisol or testosterone and hormonal levels may be affected by the intermittent growth of hair follicles. In summary, the current study indicates that hair levels of cortisol and testosterone may reflect average levels of free/bioavailable hormones over several months. Further studies are required to confirm these findings, and assess possible applications in research and clinical practice.
Acknowledgements
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This study was supported by the Physicians’ Services Incorporation Foundation and Canadian Institutes of Health Research. We thank Grace Walsh for her invaluable assistance.
Disclosure: Nothing to declare. Affiliations Department of Physiology and Pharmacology, Western University, London Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, Canada 3 Department of Biochemistry, Western University, London, Ontario 4 Department of Medicine, Western University, London, Ontario 5 Department of Paediatrics, Western University, London, Ontario 6 Ivey Chair in Molecular Toxicology, Western University, London, Ontario 7 Lawson Health Research Institute, Western University, London, Ontario 1
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References 1 Andrew R, Phillips DI, Walker BR. Obesity and gender influence cortisol secretion and metabolism in man. J Clin Endocrinol Metab 1998; 83: 1806–1809 2 Andrew R, Westerbacka J, Wahren J et al. The contribution of visceral adipose tissue to splanchnic cortisol production in healthy humans. Diabetes 2005; 54: 1364–1370 3 Bremner WJ, Vitiello MV, Prinz PN. Loss of circadian rhythmicity in blood testosterone levels with aging in normal men. J Clin Endocrinol Metab 1983; 56: 1278–1281 4 Cohen S, Kamarck T, Mermelstein R. A global measure of perceived stress. J Health Soc Behav 1983; 24: 385–396 5 Davenport MD, Tiefenbacher S, Lutz CK et al. Analysis of endogenous cortisol concentrations in the hair of rhesus macaques. Gen Comp Endocrinol 2006; 147: 255–261 6 Dettenborn L, Tietze A, Bruckner F et al. Higher cortisol content in hair among long-term unemployed individuals compared to controls. Psychoneuroendocrinol 2010; 35: 1404–1409 7 Dettenborn L, Tietze A, Kirschbaum C et al. The assessment of cortisol in human hair: Associations with sociodemographic variables and potential confounders. Stress 2012; 15: 578–588 8 Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981; 53: 58–68 9 Empen K, Lorbeer R, Dorr M et al. Association of testosterone levels with endothelial function in men: Results from a population-based study. Arterioscler Thromb Vasc Biol 2012; 32: 481–486 10 Gow R, Koren G, Rieder M et al. Hair cortisol content in patients with adrenal insufficiency on hydrocortisone replacement therapy. Clin Endocrinol 2011; 74: 687–693 11 Haring R, Volzke H, Steveling A et al. Low serum testosterone levels are associated with increased risk of mortality in a population-based cohort of men aged 20–79. Eur Heart J 2010; 31: 1494–1501 12 Haring R, Teng Z, Xanthakis V et al. Association of sex steroids, gonadotrophins, and their trajectories with clinical cardiovascular disease and all-cause mortality in elderly men from the framingham heart study. Clin Endocrinol 2013; 78: 629–634 13 Harman SM, Metter EJ, Tobin JD et al. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. baltimore longitudinal study of aging. The J Clin Endocrinol Metab 2001; 86: 724–731
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samples reflect the integral of free levels throughout several months. Our study suggests that testosterone measurements that assess testosterone levels integrated over a several month period may be more reflective of the true gonadal status than single morning measurements. Moreover, studies requiring 24-h sampling are quite labor intensive and necessitate hospitalization. As hair testosterone levels are hypothesized to measure free hormone levels integrated over several months, the measurement of testosterone in hair may be useful in larger scale studies on testosterone and gonadal status. The PSS score is used to assess subjective stress over the last 4 weeks. In the present pilot study, we did not find any significant correlation between PSS and cortisol, testosterone or their ratio in hair. Increased hair cortisol may be a marker for increased cardiovascular risk. We have previously demonstrated that increased hair cortisol is associated with increased risk for myocardial infarcts in men (Pereg et al., 2011). Another study in a group of 283 community-dwelling, elderly participants found that high hair cortisol levels were associated with a history of cardiovascular disease, but not non-cardiovascular diseases (Manenschijn et al. 2013). Further, high urinary cortisol secretion strongly predicts cardiovascular death among persons both with and without preexisting cardiovascular diseases (Vogelzangs et al., 2010). A low testosterone level is associated with increased carotid intimamedia thickness in men with low-grade inflammation (Soisson et al., 2012) and impaired endothelial function (Empen et al., 2012). Although an analysis of the Framingham population did not find an association between low testosterone and cardiovascular risk (Haring et al., 2013), a low serum testosterone was associated with increased risk of all-cause mortality (Haring et al., 2010). In light of the association between high cortisol and increased cardiovascular risk factors and all-cause mortality, coupled with the association between low testosterone and allcause mortality and possibly cardiovascular risk, the ratio of cortisol over testosterone might be particularly interesting. With respect to hair measurements, we found stronger correlations between this ratio and age, BMI and waist circumference as compared to correlations for cortisol or testosterone in hair separately. The clinical significance of the cortisol over testosterone ratio in serum has been demonstrated in the Caerphilly study, showing a strong positive correlation between this ratio and both the insulin resistance syndrome and incident ischemic heart disease (Smith et al., 2005). Based on these data it is possible that the hair cortisol-testosterone ratio may be useful in population studies investigating the risk of metabolic syndrome and cardiovascular events. Measurement of hormone levels in hair has several advantages. Its collection is non-invasive, does not require health care workers, and can be conducted at any time of the day. Samples can be stored at room temperature and be sent by mail. Further, levels reflect average hormone levels over a period of months and years, as opposed to blood and saliva samples that reflect one moment in time, and urine that reflects levels during 1 day. A unique feature of the measurement of hormones in hair is that the hormone levels are not affected by acute stress and hence may potentially be used even at or immediately after a major event, such as a myocardial infarction, which would cause acute changes in hormonal levels and thus not represent the hormonal milieu before the event. There are several potential limitations to measurement of endogenous hormones in hair. It is limited to individuals who
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