Harmonization of LC-MS/MS Measurements of Plasma Free Normetanephrine, Metanephrine, and 3-Methoxytyramine.

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

The free androgen index is inaccurate in women when the SHBG concentration is low.

OBJECTIVE/CONTEXT: The free androgen index (FAI) is known to give erroneous results in men, but it is still a commonly used test for the investigation of hyperandrogenism in women. This study aimed to compare the results of the FAI with the gold standard equilibrium dialysis method for free testosterone in women. DESIGN/PATIENTS: Free serum testosterone T (ED-T) and total serum T (T) were measured by equilibrium dialysis and LC-MS/MS in patients with polycystic ovarian syndrome (n = 130), normal female controls (n = 53) and normal males (n = 120). Calculated free T (cFT) and free androgen index (FAI) were also measured in these patients. In addition, cFT was retrospectively calculated in 4223 female patients with a normal T (<1.6 nmol/L) routinely investigated for hyperandrogenism. RESULTS: The cFT showed good agreement with measured ED-T, and the ratio cFT/ED-T was stable across all SHBG concentrations. In contrast, the FAI/ED-T ratio and the FAI/cFT ratio increased when the concentration of SHBG fell below 30 nmol/L. CONCLUSIONS: The FAI is not a reliable indicator of free T when the SHBG concentration is low and would give misleading information in a large number of women being investigated for hyperandrogenism.

Salivary androgens in adolescence and their value as a marker of puberty: results from the SCAMP cohort.

Context: Salivary androgens represent non-invasive biomarkers of puberty that may have utility in clinical and population studies. Objective: To understand normal age-related variation in salivary sex steroids and demonstrate their correlation to pubertal development in young adolescents. Design, setting and participants: School-based cohort study of 1495 adolescents at two time points for collecting saliva samples approximately 2 years apart. Outcome measures: The saliva samples were analyzed for five androgens (testosterone, androstenedione (A4), 17-hydroxyprogesterone, 11-ketotestosterone and 11ß-hydroxyandrostenedione) using liquid chromatography-mass spectrometry; in addition, salivary dehydroepiandrosterone (DHEA) and oestradiol (OE2) were analysed by ELISA. The pubertal staging was self-reported using the Pubertal Development Scale (PDS). Results: In 1236 saliva samples from 903 boys aged between 11 and 16 years, salivary androgens except DHEA exhibited an increasing trend with an advancing age (ANOVA, P < 0.001), with salivary testosterone and A4 concentration showing the strongest correlation (r = 0.55, P < 0.001 and r = 0.48, P < 0.001, respectively). In a subgroup analysis of 155 and 63 saliva samples in boys and girls, respectively, morning salivary testosterone concentrations showed the highest correlation with composite PDS scores and voice-breaking category from PDS self-report in boys (r = 0.75, r = 0.67, respectively). In girls, salivary DHEA and OE2 had negligible correlations with age or composite PDS scores. Conclusion: In boys aged 11-16 years, an increase in salivary testosterone and A4 is associated with self-reported pubertal progress and represents valid non-invasive biomarkers of puberty in boys.

Measurement of saliva tacrolimus levels in pediatric renal transplant recipients.

The aim of this study was to investigate whether a strong and clinically applicable correlation exists between saliva and whole-blood tacrolimus levels measured by high-performance liquid chromatography-tandem mass spectrometry. A high degree of correlation would potentially allow pain-free saliva sample collection to replace blood sampling for the measurement of tacrolimus levels. Enrolled in the study were 37 children (24 boys) aged 8-18 years [median (IQR) 16.2 (12.9-17.5) years] attending the renal transplant clinic at the Royal Manchester Children's Hospital and 77 paired blood saliva samples were collected. The mean (SD) saliva tacrolimus level was 0.14 (0.16), range 0-0.7 µg/l. In ten cases, tacrolimus was not detected in the saliva despite being present in blood. The ratio of blood-to-saliva tacrolimus levels varied from 2.6 to 550. The Pearson product-moment correlation suggested a weak linear relationship between tacrolimus levels in blood and saliva with a coefficient 0.36. Individual patients did not demonstrate consistent tacrolimus blood/saliva ratios with a mean correlation of 0.08. Additional experiments excluded saliva contamination with blood and sample collection and storage conditions as causes of poor correlation. The measurement of saliva tacrolimus levels in place of or as an adjunct to blood sampling therefore cannot be recommended.

Salivary Profiles of 11-oxygenated Androgens Follow a Diurnal Rhythm in Patients With Congenital Adrenal Hyperplasia.

CONTEXT: Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD). OBJECTIVE: To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30). OUTCOME MEASURES: Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11ß-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). RESULTS: Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls. CONCLUSIONS: Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

Neurokinin 3 Receptor Antagonists Do Not Increase FSH or Estradiol Secretion in Menopausal Women.

BACKGROUND: Neurokinin 3 receptor (NK3R) antagonism is a promising novel treatment for menopausal flashes. However, to avoid adverse hormonal effects it is clinically important to first confirm whether gonadotropin and estradiol concentrations change as a result of their administration. METHODS: Single-center, randomized, double-blind, placebo-controlled, crossover trial of an oral NK3R antagonist (MLE4901) in 28 women aged 40 to 62 years, experiencing >7 hot flashes/24 h; some bothersome or severe (Clinicaltrials.gov, NCT02668185). Weekly serum gonadotropins and estradiol levels were measured using commercially available automated immunoassays a priori. Serum estradiol was also measured post hoc using a highly sensitive direct assay by liquid chromatography tandem mass spectrometry. Hormone levels were compared by the paired sample t tests or by the Wilcoxon matched-pairs signed rank test, as appropriate for the distribution of the data. RESULTS: Mean (standard deviation) serum follicle-stimulating hormone (FSH) concentration was not significantly increased when taking MLE4901 (72.07 ± 19.81 IU/L) compared to placebo (70.03 ± 19.56 IU/L), P = .26. Serum estradiol was also not significantly altered, irrespective of which assay method was used (median interquartile range of serum estradiol by immunoassay: placebo 36 ± 3 pmol/L, MLE4901 36 ± 1 pmol/L, P = .21; median serum highly sensitive estradiol: placebo 12 ± 16 pmol/L, MLE4901 13 ± 15 pmol/L, P = .70). However, mean (standard deviation) serum luteinizing hormone concentration significantly decreased with MLE4901 (27.63 ± 9.76 IU/L) compared to placebo (30.26 ± 9.75 IU/L), P = .0024. IMPLICATION: NK3R antagonists do not increase serum estradiol or FSH despite their reduction in hot flashes. This is clinically significant and highly reassuring for women who have a contraindication to conventional hormone therapy such as prior/existing breast cancer and/or thromboembolism.

Assessment of free testosterone concentration.

Testosterone (T) is strongly bound to sex hormone binding globulin and measurement of free T may be more appropriate than measuring total serum T, according to the free hormone theory. This view remains controversial and it has its detractors who claim that little extra benefit is gained than simply measuring total T, but it is endorsed by recent clinical practice guidelines for investigation of androgen disorders in both men and women. Free T measurement is very challenging. The gold standard equilibrium dialysis methods are too complex for use in routine clinical laboratories, assays are not harmonized and consequently there are no common reference intervals to aid result interpretation. The algorithms derived for calculating free T are inaccurate because they were founded on faulty models of testosterone binding to SHBG, however they can still give clinically useful results. To negate the effects of differences in binding protein constants, some equations for free T have been derived from accurate measurement of testosterone in large population studies, however a criticism is that the equations may not hold true in different patient populations. The free androgen index is not recommended for use in men because of inaccuracy at extremes of SHBG concentration, and in women it can also give inaccurate results when SHBG concentrations are low. If the free hormone hypothesis is to be believed, then calculated free testosterone may offer the best way forward but better equations are needed to improve accuracy and these should be derived from detailed knowledge of testosterone binding to SHBG. There is still much work to be done to improve harmonization of T and SHBG assays between laboratories because these can have a profound effect on the equations used to calculate free testosterone.

Measurement of Salivary Adrenal-Specific Androgens as Biomarkers of Therapy Control in 21-Hydroxylase Deficiency.

BACKGROUND: Monitoring of hormonal control represents a key part of the management of congenital adrenal hyperplasia (CAH). Monitoring strategies remain suboptimal because they rely on frequent blood tests and are not specific for adrenal-derived hormones. Recent evidence suggests the crucial role of adrenal-specific 11-oxygenated-C19 androgens in the pathogenesis of CAH. OBJECTIVE: To establish a correlation between plasma and salivary adrenal-specific androgens in CAH as a noninvasive monitoring strategy. DESIGN: This prospective cross-sectional study recruited patients between 2015 and 2018. SETTING: Multicenter study including 13 tertiary centers in the United Kingdom. PARTICIPANTS: Seventy-eight children with CAH and 62 matched healthy controls. METHODS: Using liquid chromatography-tandem mass spectrometry, plasma and salivary concentrations of five steroids were measured: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). The correlation between plasma and salivary steroids was analyzed to assess their use in clinical practice. RESULTS: Strong correlations between plasma and salivary steroid concentrations in patients with CAH were detected: 17OHP (rs = 0.871; P < 0.001), A4 (rs = 0.931; P < 0.001), T (rs = 0.867; P < 0.001), 11OH4A (rs = 0.876; P < 0.001), and 11KT (rs = 0.944; P < 0.001). These results were consistent for patient subgroups based on sex and age. Analysis of patient subgroups based on 17OHP concentrations established clear correlations between plasma and salivary concentrations of the adrenal-specific androgen 11KT. CONCLUSIONS: The current study identified tight correlations between plasma and saliva for the adrenal-derived 11-oxygenated C19 androgen 11KT, as well as 17OHP and A4, which are widely used for monitoring treatment in CAH. This combination of steroid hormones will serve as an improved noninvasive salivary test for disease monitoring in patients with CAH.

Salivary cortisol and cortisone in the clinical setting.

PURPOSE OF REVIEW: A resurgence of interest in salivary biomarkers has generated evidence for their value in assessing adrenal function. The advantages of salivary measurements include only free hormone is detected, samples can be collected during normal daily routines and stress-induced cortisol release is less likely to occur than during venepuncture. We review the use of salivary biomarkers to diagnose and monitor patients for conditions of cortisol excess and deficiency and discuss the value of measuring salivary cortisone versus salivary cortisol. RECENT FINDINGS: Developments in laboratory techniques have enabled the measurement of salivary hormones with a high level of sensitivity and specificity. In states of altered cortisol binding, salivary biomarkers are more accurate measures of adrenal reserve than serum cortisol. Salivary cortisone is a superior marker of serum cortisol compared with salivary cortisol, specifically when serum cortisol is low and during hydrocortisone therapy when contamination of saliva may result in misleading salivary cortisol concentrations. SUMMARY: Salivary cortisol and cortisone can be used to assess cortisol excess, deficiency and hydrocortisone replacement, with salivary cortisone having the advantage of detection when serum cortisol levels are low and there is no interference from oral hydrocortisone.

Age-specific reference ranges for serum testosterone and androstenedione concentrations in women measured by liquid chromatography-tandem mass spectrometry.

OBJECTIVE: RIA-based sex hormone measurements offer only limited precision and specificity in the low concentration range of women. Therefore, we aimed to establish age-specific reference ranges for serum sex hormone concentrations in women using mass spectrometry and quantile regression. METHODS AND RESULTS: Data from 985 women aged 20-80 yr, recruited for the prospective Study of Health in Pomerania, were included in the analyses. Quantile regressions models were performed to calculate the age-specific 2.5th and 97.5th percentiles for sex hormone concentrations in women. Serum total testosterone (TT) and androstenedione (AD) concentrations were measured by liquid chromatography-tandem mass spectrometry. Measured concentrations of SHBG and TT were used to calculate free testosterone (free T). TT, AD, and free T concentrations showed a distinct age-related decline across 10-yr age groups (one way ANOVA P < 0.001). Sex hormone reference ranges for TT, AD, and free T were determined across each single year of age and for 10-yr age groups. Reference ranges over the whole age range of 20-80 yr were 0.35-1.97 nmol/liter for TT, 0.89-4.56 nmol/liter for AD, and 0.0025-0.0253 nmol/liter for free T. Separate reference ranges were provided for pre- and postmenopausal women as well as after inclusion of women using oral contraceptives or hormone therapy (n = 1357). CONCLUSION: This is the first study to establish age-specific reference ranges for liquid chromatography-tandem mass spectrometry-measured TT and AD and calculated free T concentrations based on quantile regression analyses, accurately accounting for the observed low concentration range and the strong age dependency of these sex hormones in women.