LC-MS/MS-based reference intervals for hair cortisol in healthy children.

BACKGROUND: Human scalp hair is a valuable matrix for determining long-term cortisol concentrations, with wide-spread applicability in clinical care as well as research. However, pediatric reference intervals are lacking. The aim of this cross-sectional study is to establish age-adjusted reference intervals for hair cortisol in children and to gain insight into hair growth velocity in children up to 2 years old. METHODS: A total of 625 healthy children were enrolled through recruitment in pregnancy, infant-welfare clinics, and school visits. Scalp hair cortisol levels were measured using liquid chromatography-tandem mass spectrometry. Age-adjusted reference intervals were established in children from birth to 18 years old. Hair growth velocity was determined in children 0-2 years of age by measuring hair length at 4- to 10-week intervals. RESULTS: Hair cortisol levels were high (162.4 pg/mg, 2.5th-97.5th percentile: 28.8-961) after birth with a sharp fall in the first 3 months of life. This is followed by lower values until age 6 and then by graduated and subtle higher values to adult concentrations are reached at the age of 18 years (3.0 pg/mg, 2.5th-97.5th percentile: 0.53-17.8). Average hair growth velocity measured in mm/month was significantly lower in infants 0-6 months of age compared to children 12-24 months (3.5 versus 9.4, P < 0.001). CONCLUSIONS: This is the first study to provide age-adjusted reference intervals for hair cortisol in children from 0-18 years. Higher hair cortisol concentrations in infants might be explained by the significantly lower hair growth rate in the first year of life. The establishment of pediatric hair cortisol reference ranges broadens the potential applications of this biomarker in pediatric clinical care.

Long-term glucocorticoid levels measured in hair in patients with depressive and anxiety disorders.

BACKGROUND: Depressive and anxiety disorders have been linked to a dysregulated hypothalamus-pituitary-adrenal (HPA)-axis. Hair cortisol levels (HairF) reflect integrated long-term cortisol regulation and are therefore promising endocrine markers of chronic (psychological and physical) stress. Our aim was to assess hair cortisol levels in persons with a depressive and/or anxiety disorder and to compare their levels with that of persons in remission and healthy controls. METHODS: Data from 1166 participants of the Netherlands Study of Depression and Anxiety (NESDA) were used, including 266 participants with a recent (1-month) diagnosis of a depressive and/or anxiety disorder, 655 participants with a diagnosis in remission, and 245 healthy controls. HairF was measured in the proximal three cm of scalp hair, using LC-MS/MS. RESULTS: Compared to the healthy controls no differences on HairF or HairE levels were found for depressive and anxiety disorders alone. However the presence of a comorbid depressive and anxiety disorder was significantly associated with increased HairF levels (ß = 0.07; p = .031), as was the severity of depressive symptoms (ß = 0.06; p = .029), but no differences were found on HairE nor the HairF:HairE ratio. CONCLUSIONS: Persons with current diagnosis of comorbid depression and anxiety show moderately higher levels of cortisol than patients with only depression or anxiety, or patients in remission and healthy controls, which may be indicative of a chronic state of hyperactivation of the HPA axis.

Predicting hair cortisol levels with hair pigmentation genes: a possible hair pigmentation bias.

Cortisol concentrations in hair are used to create hormone profiles spanning months. This method allows assessment of chronic cortisol exposure, but might be biased by hair pigmentation: dark hair was previously related to higher concentrations. It is unclear whether this association arises from local effects, such as increased hormone extractability, or whether the association represents systemic differences arising from population stratification. We tested the hypothesis that hair pigmentation gene variants are associated with varying cortisol levels independent of genetic ancestry. Hormone concentrations and genotype were measured in 1674 children from the Generation R cohort at age 6. We computed a polygenic score of hair color based on 9 single nucleotide polymorphisms. This score was used to predict hair cortisol concentrations, adjusted for genetic ancestry, sex, age and corticosteroid use. A 1-standard deviation (SD) higher polygenic score (darker hair) was associated with 0.08 SD higher cortisol levels (SE = 0.03, p = 0.002). This suggests that variation in hair cortisol concentrations is partly explained by local hair effects. In multi-ancestry studies this hair pigmentation bias can reduce power and confound results. Researchers should therefore consider adjusting analyses by reported hair color, by polygenic scores, or by both.

Long-Term Cortisol Concentration in Scalp Hair of Asthmatic Children Using Inhaled Corticosteroids: A Case-Control Study.

INTRODUCTION: Inhaled corticosteroids (ICS) can interfere with the hypothalamic-pituitary-adrenal (HPA) axis and may lead to adrenal insufficiency, resulting in a decrease of cortisol production. Cortisol levels measured in scalp hair provide a marker for long-term cortisol exposure. Data regarding hair cortisol concentration (HCC) in asthmatic children with ICS therapy are scarce. The aim of this study is to compare HCC in asthmatic children under ICS treatment with a healthy control group. METHODS: We set up a case-control study measuring the cortisol concentration in scalp hair in asthmatic children (N = 80) treated with ICS and healthy controls (N = 252) aged 4-18 years. Anthropometric characteristics and hair samples from the posterior vertex were obtained. At least 5 mg of the most proximal 3 cm of hair was used for each hair sample. HCC was analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: HCC did not significantly differ between asthmatic children using ICS and healthy controls (p = 0.950) after adjustment for age, gender, height, body mass index, and socioeconomic status. No correlation was found between budesonide dosage and HCC levels (r = -0.031, p = 0.78). CONCLUSION: There was no evidence of suppression of basal cortisol production, as measured in scalp hair, in asthmatic children using ICS, compared to healthy controls. No conclusions can be drawn on the HPA-axis stress response in these children.

Hair analysis reveals subtle HPA axis suppression associated with use of local corticosteroids: The Lifelines cohort study.

BACKGROUND AND AIMS: Scalp hair is increasingly used to measure the long-term exposure to endogenous glucocorticoids hormones. Long-term cortisone (HairE) and cortisol (HairF) have been associated with obesity, metabolic syndrome, cardiovascular disease and psychopathology. However, little is known about the influence of the use of local corticosteroids and major stressful life events on hair glucocorticoids. MATERIALS AND METHODS: We determined HairE and HairF using liquid chromatography - tandem mass spectrometry in 295 adult participants of the population-based Lifelines cohort study (75% females, median age 42). We collected anthropometry and fasting metabolic laboratory values, questionnaires on hair characteristics, recent use of corticosteroids, and recent major stressful life events. RESULTS: After adjustment for covariates, hair glucocorticoids increased with age, male sex, black or brown hair color, and frequency of sweating on the scalp, and decreased with higher hair washing frequency (P<0.05). HairE was decreased in participants who used systemic corticosteroids (5.4 vs. 8.5pg/mg hair, P=0.041), and in participants who only used local agents such as inhaled, topical and nasal corticosteroids (6.8 vs. 8.5pg/mg, P=0.005). Recent life events were positively associated with HairF after adjustment for age and sex (P=0.026), but this association lost significance after adjustment for hair related characteristics (P>0.05). CONCLUSIONS: HairE can be a useful marker to detect mild adrenal suppression due to corticosteroid use in the general population, even when only inhaled, nasal or topical corticosteroids are used, which suggests that these commonly used agents induce systemic effects.

Mild perinatal adversities moderate the association between maternal harsh parenting and hair cortisol: Evidence for differential susceptibility.

It has been shown that following exposure to mild perinatal adversity, children have greater susceptibility to both the negative and positive aspects of their subsequent environment. In a large population-based cohort study (N = 1,776), we investigated whether mild perinatal adversity moderated the association between maternal harsh parenting and children's hair cortisol levels, a biomarker of chronic stress. Mild perinatal adversity was defined as late preterm birth (gestational age at birth of 34-37 weeks, 6 days) or small for gestational age (birth weight between the 2.5th and 10th percentile for full term gestational age). Harsh parenting was assessed by maternal self-report at 3 years. Children's hair cortisol concentrations were measured from hair samples collected at age 6. There were no significant bivariate associations between mild perinatal adversities and harsh parenting and hair cortisol. However, mild perinatal adversities moderated the association between maternal harsh parenting and hair cortisol levels. Children with mild perinatal adversity had lower cortisol levels if parented more harshly and higher cortisol levels in the absence of harsh parenting than children who did not experience mild perinatal adversity. These results provide further evidence that mild perinatal adversity is a potential marker of differential susceptibility to environmental influences.

No association between hair cortisol or cortisone and brain morphology in children.

Little is known about the relationship between the long-term hypothalamic-pituitary-adrenal (HPA) axis functioning and brain structure in children. Glucocorticoid in hair has emerged as an important biomarker of HPA activity. In this study, we investigated the associations of hair cortisol and cortisone concentrations with brain morphology in young children. We included 219 children aged 6-10 years from the Generation R Study in Rotterdam, the Netherlands. We examined cortisol and cortisone concentrations by hair analysis using liquid chromatography-tandem mass spectrometry, and assessed brain morphometric measures with structural magnetic resonance imaging. The relationships of hair cortisol and cortisone concentrations with brain volumetrics, cortical thickness, cortical surface area and gyrification were analyzed separately after adjustment for several potential confounding factors. We observed a positive association between cortisol concentrations and cortical surface area in the parietal lobe, positive associations of cortisone concentrations with thalamus volume, occipital lobe volume and cortical surface area in the parietal lobe, and a negative association between cortisone concentrations and cortical surface area in the temporal lobe in the regions of interest analyses. A negative association between cortisol or cortisone concentrations and hippocampal volume was observed in children with behavioral problems. The whole brain vertex-wise analyses did however not show any association between cortisol or cortisone concentration and brain morphometric measures after correction for multiple testing. Although some associations are noted in region of interest analyses, we do not observe clear association of hair cortisol or cortisone with brain morphometric measures in typically developing young children.

Is poor neonatal adaptation after exposure to antidepressant medication related to fetal cortisol levels? An explorative study.

BACKGROUND: As a marker for poor neonatal adaptation (PNA) is lacking, the diagnostic process is difficult and includes invasive additional testing. AIMS: In order to develop a marker, it is essential to gain insight into the etiology of PNA. We hypothesized that the fetal cortisol level may play a role in this etiology. STUDY DESIGN: Non-randomized, prospective controlled study. OUTCOME MEASURES: We examined hair cortisol levels of infants exposed and not exposed to selective antidepressants (SADs) during pregnancy. These cortisol levels represent the mean cortisol level during the last trimester of pregnancy. Infants exposed to SADs who developed PNA according to the pediatrician (PNA+, n=25), infants exposed to SADs who did not develop PNA (PNA-, n=40) and infants not exposed to SADs (controls, n=105) were compared. RESULTS: In infants with PNA, hair cortisol levels were higher compared to infants without PNA. However this difference was only statistically significant in female infants (girls B0.33, p=0.04, boys B0.05, p=0.82). There was no correlation between nonspecific distress, measured by the Finnegan score and fetal hair cortisol levels (B-0.15, p=0.30). All analyses were adjusted for type of delivery and gestational age. CONCLUSIONS: Our results suggest that the hypothalamic pituitary adrenal (HPA) axis activity may play a sex-specific role in the development of PNA. As PNA is most likely of a multifactorial origin, it would be interesting to examine other factors possibly involved in the etiology of PNA in future studies, such as (epi) genetics.

Scalp hair 17-hydroxyprogesterone and androstenedione as a long-term therapy monitoring tool in congenital adrenal hyperplasia.

BACKGROUND: Glucocorticoid replacement therapy in congenital adrenal hyperplasia (CAH) is challenging, especially in children, because both over- and under-dosing may have profound and long-lasting adverse effects. Clinical follow-up parameters are largely nonspecific and slow to develop. Steroid concentrations in scalp hair may be a useful monitoring tool, as it provides information on both long-term steroid precursor and glucocorticoid exposure. AIM: We aimed to evaluate scalp hair steroid precursor concentrations as a monitoring tool for treatment follow-up in children with CAH. METHODS: Scalp hair 17-hydroxyprogesterone (17-OHP) and androstenedione concentrations, measured by LC-MS/MS, of children with CAH (N = 26) were correlated with concentrations in serum and saliva, and compared to scalp hair concentrations in patient controls with adrenal insufficiency (AI) (N = 12) and healthy controls (N = 293). RESULTS: Hair cortisol concentrations were higher in children with CAH, compared to both healthy controls (P < 0·001) and patient controls (P = 0·05), and did not differ significantly between patient controls with AI and healthy controls. Concentrations of androstenedione in scalp hair were strongly correlated with concentrations in serum (ρ = 0·72, P < 0·001) and saliva (ρ = 0·82, P = 0·002). This was also seen for 17-OHP in hair with serum (ρ = 0·94, P < 0·001) and saliva (ρ = 0·69, P = 0·009). Both hair 17-OHP and androstenedione were higher in CAH patients (mean concentration 17-OHP 2·9 pg/mg; androstenedione 1·3 pg/mg), when compared to healthy controls (17-OHP 0·44 pg/mg; androstenedione 0·65 pg/mg) and when compared to patients with AI (17-OHP 0·12 pg/mg; androstenedione 0·32 pg/mg). CONCLUSION: This study shows that scalp hair 17-hydroxyprogesterone and androstenedione concentrations seem to be a promising parameter for treatment monitoring in patients with CAH.

Splitting hair for cortisol? Associations of socio-economic status, ethnicity, hair color, gender and other child characteristics with hair cortisol and cortisone.

The aim of this study was to examine associations of SES and ethnicity with hair cortisol and cortisone and to identify potential child and family characteristics that can assist in choosing covariates and potential confounders for analyses involving hair cortisol and cortisone concentrations. Hair samples were collected in 2484 6-year-old children from the Generation R Study, a prospective cohort in Rotterdam, the Netherlands. Measurements for cortisol and cortisone were used as the outcome in regression analyses. Predictors were SES, ethnicity, hair color and child characteristics such as birthweight, gestational age at birth, BMI, disease, allergy, and medication use. Lower family income, more children to be supported by this income, higher BMI and darker hair color were associated with higher hair cortisol and cortisone levels. Boys also showed higher levels. Ethnicity (Dutch and North European descent) was related to lower levels. High amounts of sun in the month of hair collection was related to higher levels of cortisone only. More recent hair washing was related to lower levels of cortisol and cortisone. Gestational age at birth, birth weight, age, medication use, hair washing frequency, educational level of the mother, marital status of the mother, disease and allergy were not associated with cortisol or cortisone levels. Our results serve as a starting point for choosing covariates and confounders in studies of substantive predictors or outcomes. Gender, BMI, income, the number of persons in a household, ethnicity, hair color and recency of hair washing are strongly suggested to take into account.

LC-MS/MS-based method for long-term steroid profiling in human scalp hair.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for quantification of steroids. Human scalp hair provides the possibility to measure long-term retrospective steroid concentrations, which is especially useful for steroids with large time-dependent fluctuations in concentration, such as the glucocorticoid cortisol. AIM: We set up and validated a LC-MS/MS-based method for long-term steroid profiling, quantifying cortisol, cortisone, testosterone, androstenedione, dehydroepiandrosterone sulphate (DHEAS) and 17-α-hydroxyprogesterone (17OHP). METHOD: Hair locks were cut from the posterior vertex of healthy male and female volunteers and washed in isopropanol. Steroids were extracted using methanol, and extract was cleaned up by solid-phase extraction and measured on a Waters XEVO-TQ-S LC-MS/MS. Lower limit of quantification, precision, matrix interference and intra-individual variation were determined. RESULTS: The functional sensitivity of our steroid analysis was <1·3, <9·3, 2·3, <1·3, <15·9, 1·87 pg/mg hair for cortisol, cortisone, testosterone, androstenedione, dehydroepiandrosterone sulphate (DHEAS), and 17OHP, respectively. Measured over a 9-month period, the inter-run CVs were below 16% for all steroids. Intra-individual coefficients of variation were below 15% for all steroids, except 17OHP (19·7%). CONCLUSION: The authors present a LC-MS/MS-based method for long-term steroid profiling in human scalp hair, potentially providing novel insights by a multitude of clinical and research applications in the field of endocrinology.

Validation and reference ranges of hair cortisol measurement in healthy children.

BACKGROUND: Cortisol is produced in a circadian rhythm controlled by the hypothalamus-pituitary-adrenal axis, making it cumbersome to measure long-term cortisol exposure. Hair has proven to be a reliable matrix for long-term cortisol measurement in adults and can be used as diagnostic tool for (cyclic) Cushing's syndrome. The diagnostic applicability in children has not been studied, nor have the effects of development and hair care been evaluated in children. We aimed to establish reference ranges of hair cortisol concentrations (HCC) in healthy children and to evaluate the effects of age, gender, puberty and characteristics of hair care. METHODS: In 128 healthy children aged 4-14 years, HCC were measured in a small 3-cm hair lock from the back of the head. RESULTS: HCC increased with age (p = 0.04) up to age 10 years, with a mean of 5.0, 5.8, 6.8 and 8.5 pg/mg at age 4-5, 6-7, 8-9 and 10-14 years, respectively. Children aged 4-7 years had significantly lower HCC compared to healthy adults (p = 0.007). We did not find any influence of gender, puberty or hair care characteristics on hair cortisol. CONCLUSION: HCC can be reliably measured in childhood, and reference ranges increase with age. HCC in children are not dependent on hair care or hair characteristics.

Increased scalp hair cortisol concentrations in obese children.

CONTEXT: Pathologically increased cortisol exposure induces obesity, but it is not known whether relatively high cortisol within the physiological range is related to childhood obesity. OBJECTIVE: The aim of the study was to compare hair cortisol concentrations between obese and normal-weight children. DESIGN: We performed an observational case-control study. PARTICIPANTS: Twenty obese children (body mass index-SD score [BMI-SDS]>2.3) and 20 age- and sex-matched normal-weight children (BMI-SDS<1.1) aged 8-12 years were recruited. MAIN OUTCOME MEASURES: Scalp hair samples from the posterior vertex were collected, and hair cortisol concentrations were measured using ELISA. Body weight, height, and waist circumference were measured. From the obese children, additional data on blood pressure and blood lipid concentrations were collected. RESULTS: In both groups, five boys and 15 girls were included; their mean age was 10.8±1.3 vs 10.8±1.2 years (obese vs normal weight; not significant). Body weight, BMI, BMI-SDS, and waist circumference were higher in the obese children compared with the normal-weight children (69.8±17.2 vs 35.5±7.2 kg; 29.6±4.9 vs 16.4±1.6 kg/m2; 3.4±0.5 vs -0.2±0.8 SDS; 94±13 vs 62±6 cm; P<.001 all). Hair cortisol concentration was higher in obese than normal-weight children (median [interquartile range], 25 [17, 32] vs 17 [13, 21] pg/mg; P<.05). CONCLUSIONS: Hair cortisol concentration, a measure for long-term cortisol exposure, was higher in obese children than normal-weight children. This suggests long-term activation of the hypothalamus-pituitary-adrenal axis in obese children and may provide a novel target for treatment of obesity in children.

Children's hair cortisol as a biomarker of stress at school entry.

Quantification of cortisol in scalp hair seems a promising measurement for long-term cortisol levels, and thereby a biomarker for stress. We examined hair cortisol concentrations (HCC) in children when first entering elementary school. Participants were 42 children (45% boys) with a mean age of 4.2 years (SD = 0.42 months). Hair samples (≥5 cm) were collected 2 months after school entry. Hair analysis was conducted using two 2-cm long segments, reflecting the first 2 months of school attendance (the scalp-near segment) and 2 months prior to school entry. HCC were higher after school entry than before, especially for fearful children. Alterations in HCC were not moderated by experience in group daycare before school entry. Thus, HCC suggest that starting elementary school is accompanied by increased stress hormone levels in young (in particular fearful) children.

Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.

Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-beta-gal activity. Skin fibroblasts were isolated from young (mean age +/- SD: 25.5 +/- 1.8) and very old (age 90.2 +/- 0.3) subjects. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-beta-gal activity by cytochemistry (X-gal) and by flow cytometry (C(12)FDG). The pH modulator Bafilomycin A1 (Baf A1) was found to be least toxic for fibroblasts and to differentiate best between nonstressed and stressed fibroblast populations. Under nonstressed conditions, fibroblasts from very old subjects showed higher SA-beta-gal activity than fibroblasts from young subjects. This difference was found for both the flow cytometric and cytochemical methods (P = 0.013 and P = 0.056 respectively). Under stress-induced conditions the flow cytometric method but not the cytochemical method revealed significant higher SA-beta-gal activity in fibroblasts from very old compared to young subjects (P = 0.004 and P = 0.635 respectively). We found the modified flow cytometric method measuring SA-beta-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts.