Yoon Ju Bae

Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair

Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC-MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS(3) (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100mL of human plasma/serum, saliva, urine and 10-20mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC-MS/MS or LC-MS(3). Steroids can be analyzed in 4min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS(3) detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37pmol/L (estradiol) up to 3.1nmol/L (DHEAS). General accuracy was 89-107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (-71% mean), aldosterone (-40% mean) and plasma aldosterone (-45% mean). The comparison of MS(2) and MS(3) quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS(3) was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS(3) can be superior to MS(2) regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC-MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair.

24-Month Use of Once-Weekly GH, LB03002, in Prepubertal Children With GH Deficiency

BACKGROUND Sustained-release GH formulations may provide a strategy for improving treatment compliance and persistence in GH-deficient patients. OBJECTIVE The aim of the study was to examine efficacy and safety of LB03002, a sustained-release GH formulation for once-weekly administration. DESIGN We conducted a phase III, 12-month, multinational, randomized, open-label, comparator-controlled trial with a 12-month uncontrolled extension. PATIENTS Prepubertal GH treatment-naive GH-deficient children (mean age, 7.8 y) participated in the study. INTERVENTION We administered once-weekly LB03002 (n=91) or daily GH (n=87) for 1 year, followed by once-weekly LB03002 for all patients for another year (LB03002 throughout, n=87; switched to LB03002, n=80). OUTCOME MEASURES Height, height velocity (HV), IGF-1, GH antibodies, and adverse events were determined throughout. Primary analysis was noninferiority of LB03002 vs daily GH at 1 year by analysis of covariance. RESULTS Mean±SD HV during year 1 was 11.63±2.60 cm/y with LB03002, and 11.97±3.09 cm/y with daily GH, with increases from baseline of 8.94±2.91 and 9.04±3.19 cm/y, respectively. The least square mean HV difference for LB03002 - daily GH was -0.43 cm/y (99% confidence interval, -1.45 to 0.60 cm/y). Mean HV also remained above baseline in year 2 (8.33±1.92 cm/y in the LB03002 throughout group, and 7.28±2.34 cm/y in the switched to LB03002 group). Injection site reactions occurred more frequently in LB03002-treated patients but were considered mild to moderate in >90% of cases. CONCLUSIONS Growth response with once-weekly LB03002 in GH-deficient children is comparable to that with daily GH, achieving expected growth rates for 24 months. Once-weekly LB03002 is a strong candidate for long-term GH replacement in GH-deficient children.

Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications.

Corticosteroid-binding globulin (CBG) is the principal transport protein of glucocorticoids. Approximately 80-90% of serum cortisol binds to CBG with high affinity and only about 5% of cortisol remain unbound and is considered biologically active. CBG seems to modulate and influence the bioavailability of cortisol to local tissues. In this review, we will discuss physicochemical properties of CBG and structure of CBG in the mechanisms of binding and release of cortisol. This review describes several factors affecting CBG functions, such as genetic factors or temperature. Furthermore, clinical implications of CBG abnormalities and the measurement of CBG and its use for assessment of free cortisol levels are described in this review.

The hyporeactivity of salivary cortisol at stress test (TSST-C) in children with internalizing or externalizing disorders is contrastively associated with α-amylase

BACKGROUND Stress biomarkers of the autonomic nervous system and hypothalamic-pituitary-adrenal axis (HPA-axis) can be measured via alpha-amylase (AA) and cortisol and cortisone in saliva. Objectives were to determine 1) the response patterns of cortisol, cortisone, and AA under both circadian conditions and the Trier Social Stress Test for Children (TSST-C), 2) which reactivity index is most suitable to differentiate internalizing or externalizing disorders from controls, and to explore 3) the interaction between AA and cortisol in the presence of internalizing or externalizing disorders. METHODS Saliva samples (n = 2893) from children with internalizing (n = 55) or externalizing disorders (n = 33) and healthy children (n = 81) were analyzed for cortisol, cortisone, and AA under circadian conditions and TSST-C. RESULTS Circadian rhythm of three biomarkers did not differ between diagnostic groups. Age and gender were significant predictors for cortisol and awakening time influenced all three biomarkers significantly. TSST-C responses appeared sequentially in the order of AA, cortisol, and cortisone. Trajectories of cortisol and cortisone responses, not in AA, were significantly lower in children with internalizing or externalizing disorders than in healthy children. Cortisol percentage increase appeared to be the most suitable reactivity index to detect the difference between the diagnostic groups. Internalizing disorders had a negative association between AA decrease and cortisol increase (β = -.199, p < .05, R(2) = .304). Externalizing disorders had a positive association between AA baseline and cortisol increase (β = .229, p < .05, R(2) = .304). CONCLUSION An altered HPA-axis response during stress might result from chronic allostatic load in internalizing disorders and underaroused stress response system in externalizing disorders.

Multicenter performance evaluation of a second generation cortisol assay.

Abstract Background: Untreated disorders of the adrenocortical system, such as Cushing’s or Addison’s disease, can be fatal, and accurate quantification of a patient’s cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva. Methods: Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals. Results: The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5–95th percentile) were 166.1–507 nmol/L and afternoon concentrations were 73.8–291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. Conclusions: The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.

Vitamin D in health and disease: the global threat of vitamin D deficient rickets

Nowadays, infectious diseases are less prevalent in many countries around the world and chronic diseases including obesity, diabetes, behavioral problems and nutritional deficiencies are thought to have become the major and eminent health burdens in both the adult and the young population. Among these, vitamin D deficiency poses a major health problem in children and adolescents globally. In fact, even in countries with high sun and specifically high ultraviolet B (UVB) exposure such as Brasil and Australia vitamin D deficiency rickets has become very prevalent. In Australia this has been attributed to the use of sun protecting ointments and changes in life styles that lead to lesser sun exposure especially in children and young people [1]. In populations with dark skin living, for example, in Africa or the Middle East, the prevalence of vitamin D insufficiency with 25-hydroxyvitamin D (25(OH)D) levels ranging between 15 and 20 ng/ mL (37.5–50 nmol/L) ranges from 5% to 80%. Risk factors to develop nutritional rickets include traditional dress and avoidance of sunlight exposure, and also multiple dietary factors as a result of specific cultural beliefs in many of the affected countries as nutritional rickets may also be caused by inadequate dietary intake of calcium when, for example, milk and milk products are consumed less or are not available [2]. As a result of vitamin D and/or calcium deficiency severe bone disease, stunted growth and even the emergence of autoimmune disease and alterations of the immune system are being observed in many populations.

Vitamin D and calcium in the human breast milk

Vitamin D and calcium in the human milk is essential for the growth and the prevention of rickets in infants. In this review, we will discuss the physiology and the functions of vitamin D and calcium and the mechanisms of vitamin D and calcium transfer into the human breast milk. This review describes the recommended intake of vitamin D and calcium for infants and lactating mothers and the factors influencing the content of vitamin D and calcium in human milk. Furthermore, the measurement of vitamin D compounds and calcium in human breast milk is described in this review.

Salivary cortisone, as a biomarker for psychosocial stress, is associated with state anxiety and heart rate

BACKGROUND Stress activates the central nervous, the autonomic nervous, and the endocrine system. This study aimed to (1) test the usability of salivary cortisone in a standardized psychosocial stressor, (2) create a comprehensive profile of hormonal responses to determine laboratory parameters with high discriminatory power, and (3) analyze their association with psychometric and autonomic stress measures. METHODS Healthy young men (18-35 years) completed either the Trier Social Stress Test (TSST) (n = 33) or a Placebo-TSST (n = 34). Blood and saliva were collected at 14 time points along with state-anxiety (STAI) and heart rate. Serum steroids (cortisol*, cortisone*, dehydroepiandrosterone-sulfate, androstenedione*, progesterone*, 17-hydroxyprogesterone*, testosterone, estradiol*, aldosterone*), salivary cortisol* and cortisone*, copeptin*, adrenocorticoptropic hormone*, corticosteroid-binding globulin, and salivary alpha-amylase* were analyzed. We used mixed-design ANOVAs to test group differences, receiver operator characteristic (ROC) curve analyses to assess the discriminatory power of each measure, and Spearman correlation analyses to probe the association between measures. RESULTS The largest area under the ROC curve was observed in salivary cortisone at 20 min after the end of the TSST (AUC = 0.909 ± 0.044, p < 0.0001). Significant time-by-group interactions were found in the parameters marked with * above, indicating stress-induced increases. The peak response of salivary cortisone was significantly associated with those of STAI (rho = 0.477, p = 0.016) and heart rate (rho = 0.699, p < 0.0001) in the TSST group. CONCLUSION Our study found salivary cortisone to be a stress biomarker with high discriminatory power and significant correlations with subjective and autonomic stress measures. Our results can inform future stress studies of sampling time for different laboratory parameters.

Increased estrogen level can be associated with depression in males

BACKGROUND Several studies have shown a positive association between depression and obesity; however the underlying mechanisms are not fully understood. It is not known if this association is driven by altered sex hormone levels in men due to increased BMI. PATIENTS AND METHODS Data were obtained from the LIFE-Adult-Study, a population-based cohort study. A total of 3925 men (2244<60years and 1681>60years) were included into analyses. Associations between BMI, sex hormones and depressive symptomatology according to CES-D score were evaluated. RESULTS Obese men had compared to normal weight controls lower total testosterone (12.6±4.7 vs 19.4±5.5 nmol/L, p<0.001 in <60years, and 13.8±6.9 vs 18.3±5.9 nmol/L, p<0.001 in >60years group) and free testosterone (249.0±73.9 vs 337.2±82.0pmol/L, p<0.001, and 217.8±71.2 vs 263.4±72.2pmol/L, p<0.001), and increased estradiol in older group only (97.3±43.0 vs 82.3±34.2pmol/L, p<0.001 in obese). Men <60years old with depressive symptomatology had higher estradiol levels compared to those without depressive symptomatology (96.3±40.7 vs 84.4±36.6pmol/L, p<0.001), however no association with BMI was observed. CONCLUSIONS Selected sex hormone parameters were significantly different in overweight and obese compared to normal weight males and certain differences could be seen between younger and older males. Depressive symptomatology was associated with increased estradiol levels in younger men, regardless of BMI.

Stress biomarkers in minimally invasive and conventional colorectal resections

Abstract Introduction: Any surgical procedure develops a stress situation for the patient, which can modulate the individual outcome. At present, there is only limited information about stress response in colorectal resections by laparoscopic compared to conventional surgery. Therefore, our objectives were the feasibility and the investigation of stress biomarkers including copeptin and steroid hormones before, during and after colorectal surgery. Methods: Eleven patients underwent minimally invasive and ten patients conventionally open colorectal surgery. Blood samples were collected before, during and 24 h after surgery and copeptin, NT-proBNP, cortisol, cortisone, interleukin-6 and glucose were analyzed. Results: Both, minimally invasive and conventional-open colorectal surgery caused a fast but heterogeneous response of stress biomarkers. However, the postoperative decrease of cortisol, cortisone and glucose differed between both groups. The stress biomarkers decreased faster down to baseline after minimally invasive surgery, while in open surgery cortisol, cortisone and glucose did not return to baseline within 24 h after operation. Conclusions: We show in this feasibility study for the first time an increase of copeptin in combination with glucocorticoids as stress biomarkers by open surgery compared to minimally invasive procedures in patients undergoing colorectal surgery. Exceeding an individual threshold of ‘stress burden’ may have unfavorable effects on the long-time clinical outcome.