Clinical and laboratory observation

Urinary free cortisol values in normal children and adolescents

Monitoring treatment of children with classic congenital adrenal hyperplasia (CAH) is difficult and biochemical targets are not well defined.

We retrospectively analysed 576 daily urinary steroid hormone metabolite profiles determined by gas chromatography–mass spectrometry of 150 children aged 3.0–17.9 years with classic 21-hydroxylase deficiency (21-OHD) on hydrocortisone and fludrocortisone treatment.

Daily urinary excretion of glucocorticoid-, 17α-hydroxyprogesterone (17-OHP)-, and androgen metabolites as well as growth and weight gain are presented. Children with classic CAH exhibited increased height velocity during prepubertal age, which was then followed by diminished growth velocity during pubertal age until final height was reached. Final height was clearly below the population mean. 11β-Hydroxyandrosterone was the dominant urinary adrenal-derived androgen metabolite in CAH children. Adrenarche is blunted in children with CAH under hydrocortisone treatment and androgen metabolites except 11β-hydroxyandrosterone were suppressed. Cortisol metabolite excretion reflected supraphysiological hydrocortisone treatment dosage, which resulted in higher body-mass-indices in children with CAH.

Reference values of daily urinary steroid metabolite excretions of treated children with CAH allow the clinician to adequately classify the individual patient regarding the androgen-, 17-OHP-, and glucocorticoid status in the context of the underlying disorder. Additionally, urinary 21-OHD-specific reference ranges will be important for research studies in children with CAH.

Blunted diurnal cortisol variation has been associated with overt cardiovascular disease in adults. The relationship between the diurnal cortisol variation and subclinical atherosclerosis in youth has yet to be investigated. The objectives of this study were to (1) determine the relationship between overnight cortisol measures and CIMT in overweight and obese, African-American and Latino children; (2) assess ethnic differences in these relationships; and (3) explore whether overnight cortisol and CIMT relationships were independent of inflammatory markers, C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-∝ (TNF-∝).

One hundred fifty-six overweight and obese African-American and Latino children (ages 8–17, 86 M/70 F, 55 African-American/101 Latino) underwent measures of CIMT by B-mode ultrasound, nocturnal cortisol rise (NCR = salivary cortisol rise from 2200 h to awakening at 0530 h), cortisol awakening response (CAR = salivary cortisol from time of awakening to 30 min later), fasting serum cortisol and overnight urinary free cortisol.

Using linear regression, salivary cortisol_0530 h and NCR were negatively associated with CIMT (β_standardized = −0.215 and −0.220, p < 0.01) independent of age, height, percent body fat, ethnicity and systolic blood pressure. Nocturnal salivary cortisol_2200 h, morning serum cortisol, and overnight urinary free cortisol were not associated with CIMT. Using ANCOVA, participants with LOW NCR (NCR < 0.44 μg/dL, n = 52) had significantly greater CIMT than those with HIGH NCR (NCR ≥ 0.91 μg/dL, n = 52; 0.632 ± 0.008 vs. 0.603 ± 0.008 mm, p = 0.01) after controlling for covariates. Ethnicity was independently associated with CIMT, whereby African-American children had greater CIMT than Latino children (−0.028 ± 0.009, p = 0.006). The relationships between cortisol measures and CIMT did not differ between the two ethnic groups (all p_interaction = 0.28–0.97). CRP, IL-6 and TNF-∝ were not associated with CIMT (p > 0.05). IL-6 was inversely related to NCR (r = −0.186, p = 0.03), but it did not explain the relationship between NCR and CIMT.

Salivary cortisol_0530 h and NCR, but not CAR, nocturnal salivary cortisol _2200 h, morning serum cortisol or overnight urinary free cortisol were associated with CIMT, independent of relevant covariates, including inflammatory factors. A low awakening salivary cortisol or a blunted NCR may be related to increased atherosclerosis risk in overweight and obese minority youth. These findings support adult studies suggesting flattened daytime diurnal cortisol variation impacts cardiovascular disease risk.

Consistent sex differences in regulation of the hypothalamic pituitary adrenocortical (HPA) axis have been shown in animal models and emerge over puberty. However, parallel work in humans is lacking despite implications for elucidating the emergence of sex differences in depression over puberty. We investigated sex differences in HPA response to corticotropin releasing hormone (CRH) challenge over puberty in a carefully screened normative sample.

Participants were 68 healthy children (41% girls), ages 6–16, with no personal or family history of psychiatric disorder. Pubertal maturation was determined by Tanner staging. Following 24 h of adaptation, 9–10 plasma cortisol samples were collected over 30–40 min pre-infusion baseline, 1 μg/kg CRH infusion, and 90–180 min post-infusion recovery. Thirty-seven participants completed 2+ CRH challenges allowing inclusion of cross-sectional and longitudinal data in all analyses. The influence of gender and pubertal maturation on parameters of cortisol response to CRH challenge was investigated using nonlinear mixed model methodology.

Girls showed increasing total cortisol output following CRH challenge over puberty, while boys showed little change in total cortisol output over puberty. Increased cortisol output in girls was explained by slower reactivity and recovery rates leading to prolonged time to reach peak cortisol and delayed return to baseline over puberty. Girls also showed increasing baseline cortisol over puberty, while boys showed declining baseline over puberty.

Results reveal subtle normative sex differences in the influence of pubertal maturation on HPA regulation at the pituitary level. This normative shift may tip the balance towards stress response dysregulation in girls at high risk for depression, and may represent one potential mechanism underlying elevated rates of depression among pubescent girls.

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

The study included 239 AGs (142 females–97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females–41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).

Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.

Le diagnostic du syndrome de Cushing doit être divisé en deux étapes distinctes, le diagnostic positif, clinique et biologique de l’hypercortisolisme, suivi du diagnostic différentiel afin de préciser l’étiologie. Lorsque l’hypercortisolisme est modéré, la première difficulté est de faire la distinction entre hypercorticisme modéré organique et hypercortisolisme fonctionnel ou « pseudo syndrome de Cushing ». Lorsque l’hypercortisolisme est ACTH dépendant, la seconde étape difficile est d’en déterminer l’origine (maladie de Cushing ou sécrétion ectopique d’ACTH). Les investigations biochimiques initiales doivent confirmer que le syndrome de Cushing est réellement présent. Les premiers tests diagnostiques reposent sur l’augmentation de la sécrétion intégrée de cortisol, sur l’absence de rythme circadien, sur l’insensibilité au moins relative du patient au feedback des glucocorticoïdes (exploré par le freinage à la dexaméthasone). L’évaluation du cortisol libre urinaire est un examen incontournable, il est spécifique d’un hypercorticisme organique lorsqu’il est supérieur à 3 N. Par ailleurs, l’étude du cycle nycthéméral du cortisol plasmatique avec plusieurs prélèvements effectués à différents moments du jour et de la nuit est un bon examen en soi puisque l’abolition de la variation nycthémérale de la sécrétion du cortisol permet d’affirmer l’existence d’une hypersécrétion, le point essentiel au diagnostic étant la baisse physiologique du cortisol à minuit, qui est absente ou insuffisante dans les hypercortisolismes. Le dosage du cortisol dans la salive avec prélèvement effectué par le patient lui-même évite le stress d’une prise de sang, ce qui est particulièrement appréciable chez les enfants. Les variations du cortisol salivaire suivent bien sûr un cycle nycthéméral tout à fait parallèle à celui du cortisol plasmatique. En cas d’hypercortisolisme, ce cycle est absent dans la salive ; ainsi le dosage du cortisol salivaire à minuit peut être proposé pour le dépistage d’un hypercorticisme. Après avoir établi le diagnostic d’hypercortisolisme, des taux indosables d’ACTH plasmatique (< 2 pmol/L), orientent vers un syndrome de Cushing ACTH indépendant et par conséquent une atteinte surrénalienne primaire. En présence de taux détectables d’ACTH, les patients avec la maladie de Cushing (syndrome de Cushing hypophysaire), doivent être différenciés de ceux avec le syndrome de sécrétion ectopique d’ACTH, lequel peut être occulte et difficile à localiser. Le recours au cathétérisme des sinus pétreux par une équipe expérimentée permettra de faire le diagnostic étiologique. En conclusion, la variabilité clinique et biologique du syndrome de Cushing impose la mise en place de dosages répétés. L’utilisation du dosage du cortisol dans la salive a de multiples avantages.

The diagnosis of the Cushing's syndrome must be divided into two distinct stages, the clinical and biological diagnosis of the hypercortisolism, follow-up of the differential diagnosis in order to specify the etiology. The initial biochemical investigations must confirm that the Cushing's syndrome is really present. The first diagnostic test is the increase in the cortisol secretion, and the absence of circadian rhythm, the relative insensitivity of the patient to the negative feedback of glucocorticoids (explored by dexamethasone feedback). The evaluation of urinary free cortisol is relevant. It is specific of an organic hypercorticism when the urinary cortisol is above 3 N. In addition, the study of the nychthemeral cycle of plasmatic cortisol with several samples carried out at various times of the day and the night is a good examination since the abolition of the nycthemeral variation of the secretion of cortisol makes possible to affirm the existence of a hypersecretion, the essential point with the diagnosis being the loss of the physiological fall of cortisol at midnight. The measurement of cortisol in the saliva, carried out by the patient himself, avoids the stress of a blood test, which is particularly appreciable in children. Indeed, only the latter can diffuse through the acinous cells of salivary glands. In addition, the variations of salivary cortisol follow a nychthemeral cycle completely parallel with that of plasmatic cortisol. In case of hypercortisolism, this cycle is missing in saliva; thus the proportioning of salivary cortisol at midnight can be proposed for the tracking of a hypercorticism. After having established the diagnosis of hypercortisolism, a dramatic ACTH decrease (ACTH < 2 pmol/L), is evidence for a syndrome of ACTH independent Cushing and consequently a primary adenocortical disease. An imagery with CT and/or IRM makes it possible to delimit with precision the lesion, whose treatment is usually surgical. In the presence of detectable rates of ACTH, the patients with Cushing's disease (syndrome of hypophyseal Cushing), must be differentiated from those with syndrome of ACTH ectopic secretion, which can be occult and difficult to locate.

ACTH-independent macronodular adrenal hyperplasia (AIMAH) is often associated with subclinical cortisol secretion or atypical Cushing's syndrome (CS). We characterized a large series of patients of AIMAH and compared them with patients with other adrenocortical tumors.

We recruited 82 subjects with: 1) AIMAH (n = 16); 2) adrenocortical cortisol-producing adenoma with CS (n = 15); 3) aldosterone-producing adenoma (n = 19); and 4) single adenomas with clinically nonsignificant cortisol secretion (n = 32).

Urinary free cortisol (UFC) and 17-hydroxycorticosteroid (17OHS) were collected at baseline and during dexamethasone testing; aberrant receptor responses was also sought by clinical testing and confirmed molecularly. Peripheral and/or tumor DNA was sequenced for candidate genes.

AIMAH patients had the highest 17OHS excretion, even when UFCs were within or close to the normal range. Aberrant receptor expression was highly prevalent. Histology showed at least two subtypes of AIMAH. For three patients with AIMAH, there was family history of CS; germline mutations were identified in three other patients in the genes for menin (one), fumarate hydratase (one), and adenomatosis polyposis coli (APC) (one); a PDE11A gene variant was found in another. One patient had a GNAS mutation in adrenal nodules only. There were no mutations in any of the tested genes in the patients of the other groups.

AIMAH is a clinically and genetically heterogeneous disorder that can be associated with various genetic defects and aberrant hormone receptors. It is frequently associated with atypical CS and increased 17OHS; UFCs and other measures of adrenocortical activity can be misleadingly normal.