Hong N. Bui

Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay.

BACKGROUND Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose. METHODS Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination. RESULTS Performance of the ID-LC-MS/MS method was concordant with a published reference method (y=1.007x-0.056 nmol/L; r=0.9998). Comparison of the immunoassay to ID-LC-MS/MS yielded y=1.095x+0.104 nmol/L (r=0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC-MS/MS method. The reference interval in women was 0.30-1.69 nmol/L (8.7-48.7 ng/dL) for ID-LC-MS/MS and 0.50-2.00 nmol/L (14.4-57.7 ng/dL) for the immunoassay. CONCLUSION The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-to-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle.

Serum testosterone levels measured by isotope dilution-liquid chromatography–tandem mass spectrometry in postmenopausal women versus those in women who underwent bilateral oophorectomy

Background Differentiation between subtle changes in low serum testosterone concentrations, common in women and children, is not possible with current commercially available assays. The objectives of the study were to develop a method based on stable isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with adequate sensitivity and specificity and to investigate the applicability of this assay in serum samples from pre- and postmenopausal women. Methods For 16 women, testosterone levels were measured in blood samples drawn two years before and after physiological menopause, and for eight women in samples drawn before and after bilateral oophorectomy. Testosterone was extracted from serum, derivatized and analysed on an LC-MS/MS. Results The developed ID-LC-MS/MS method allowed for specific and reproducible measurement of testosterone. Comparison with stable isotope dilution-gas chromatography coupled to mass spectrometry detection by Deming regression analysis gave a slope of 1.025 and an intercept of 0.055 nmol/L (r = 0.9998). A significant decrease was found in testosterone concentrations before and after bilateral oophorectomy (P = 0.02), whereas no significant difference was found before and after natural menopause (P = 0.4). Conclusions The ID-LC-MS/MS assay measures serum testosterone with acceptable accuracy and is useful in female samples, supporting the conclusion that the postmenopausal ovary contributes to circulating testosterone. To our knowledge, our analytical method compares favourably to similar published methods in terms of sensitivity. The sensitivity and specificity of this method comply with the reference method for measurement of testosterone in serum samples of women, children and men suffering from hypogonadism and can also be used for men with testosterone in the reference range.

Accuracy of First and Second Generation Testosterone Assays and Improvement through Sample Extraction

To the Editor: The monitoring of antiandrogen treatment in patients with prostate cancer, investigation of hyperandrogenism in women, and evaluation of infants with ambiguous genitalia require accurate measurement of low testosterone concentrations. However, testosterone immunoassays have been shown to be inaccurate and often to overestimate testosterone concentrations in the low range (1). A working group of the Endocrine Society recently reviewed this concern and presented several recommendations to ensure the accuracy of future testosterone testing for improvement of diagnosis and treatment of disease (2). In the present study we evaluated the current situation with regard to the accuracy of 7 testosterone immunoassays, including 2 second generation assays, by comparison with isotope-dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS). In addition, we investigated the possible improvement of these immunoassays by diethyl ether sample extraction. Serum from 50 men, 50 women, and 16 children (age 4–16 years) was collected, divided into aliquots, and stored (−20 °C) until analysis. All investigations conformed to the ethics standards of the Helsinki Declaration. Total serum testosterone was measured singly …

Lower Testosterone Levels With Luteinizing Hormone-Releasing Hormone Agonist Therapy Than With Surgical Castration: New Insights Attained by Mass Spectrometry

PURPOSE Androgen deprivation therapy by bilateral orchiectomy (surgical castration) or luteinizing hormone-releasing hormone agonist therapy (medical castration) is recommended for advanced or metastatic prostate cancer. Both methods aim at reducing serum testosterone concentrations to a castrate level which is currently defined as less than 50 ng/dl. The results of previous studies are based on testosterone immunoassays that have insufficient accuracy in the low range. In this study we reevaluated serum testosterone concentrations in men on androgen deprivation therapy using isotope dilution-liquid chromatography-tandem mass spectrometry, an accurate method of measuring testosterone in the castrate range. MATERIALS AND METHODS Subjects underwent surgical castration (34) or received a luteinizing hormone-releasing hormone agonist (32). Serum samples were obtained more than 3 months after surgery or initiation of luteinizing hormone-releasing hormone agonist therapy. Testosterone levels were determined using isotope dilution-liquid chromatography-tandem mass spectrometry. Dihydroepiandrosterone sulfate, androstenedione, sex hormone-binding globulin and inhibin B levels were determined. RESULTS All subjects had serum testosterone values less than 50 ng/dl and 97% had testosterone concentrations less than 20 ng/dl. Medically castrated men had significantly lower testosterone levels (median 4.0 ng/dl, range less than 2.9 to 20.2) than those surgically castrated (median 9.2 ng/dl, range less than 2.9 to 28.8, p <0.001). No difference was found in dehydroepiandrosterone sulfate, androstenedione and sex hormone-binding globulin levels between the groups, whereas inhibin B levels were significantly higher in the luteinizing hormone-releasing hormone agonist treated group. CONCLUSIONS Using an accurate technique for testosterone measurement, subjects on luteinizing hormone-releasing hormone agonist therapy had significantly lower testosterone concentrations than men who underwent surgical castration. The clinical relevance of these findings remains to be determined.

Intraprostatic testosterone and dihydrotestosterone. Part I: concentrations and methods of determination in men with benign prostatic hyperplasia and prostate cancer.

Whats known on the subject? and What does the study add?

Intraprostatic testosterone and dihydrotestosterone. Part II: concentrations after androgen hormonal manipulation in men with benign prostatic hyperplasia and prostate cancer

Whats known on the subject? and What does the study add?

Reduction in 24-Hour Plasma Testosterone Levels in Subjects Who Showered 15 or 30 Minutes After Application of Testosterone Gel

Study Objective. To investigate whether showering, to prevent the involuntary transfer of testosterone to others through skin contact, either 15 or 30 minutes after application of testosterone gel would significantly affect plasma testosterone levels.

Salivary testosterone in female-to-male transgender adolescents during treatment with intra-muscular injectable testosterone esters.

INTRODUCTION In our hospital, female-to-male (FtM) transgender adolescents from the age of 16 are treated with two- or four-weekly intra-muscular injections of testosterone-esters. Some patients treated with four-weekly injections have complaints of fatigue and experience mood swings towards the end of the inter-injection period, which calls for an evaluation of the time-course of testosterone levels between injections. Evaluation of salivary testosterone is a practical approach for sequential measurements. Since only ∼2% of total serum testosterone is present in saliva, a sensitive assay is necessary. The objective was to develop an isotope dilution-liquid chromatography-tandem mass spectrometry method (ID-LC-MS/MS) for salivary testosterone measurements and to evaluate the testosterone profiles after testosterone-ester mixture injections in FtM-adolescents. EXPERIMENTAL FtM treated with 125 mg/2 weeks or with 250 mg/4 weeks depots of testosterone-ester mixture collected saliva at different time intervals. Salivary testosterone was measured by a thoroughly validated ID-LC-MS/MS assay. RESULTS An ID-LC-MS/MS method for measuring salivary testosterone was developed with adequate accuracy and specificity. The reference range was established at 135-400 pmol/L. Testosterone levels peaked supra-physiologically immediately post-injection, and decreased to levels within the male reference range after nine days in all patients. 250 mg/4 weeks depots resulted in values below the reference range at the end of the 4 weeks. DISCUSSION The development of an adequate ID-LC-MS/MS method for measuring salivary testosterone allowed us to investigate the testosterone profile in FtM-adolescents after testosterone-esters mixture injections. These injections lead to extreme concentrations which may affect the wellbeing of the patients.

Testosterone, free testosterone, and free androgen index in women: Reference intervals, biological variation, and diagnostic value in polycystic ovary syndrome

OBJECTIVE The objective of our study was to determine reference intervals and biologic variation for testosterone (T), free testosterone (fT), and free androgen index (FAI) in women with accurate methods and to test the discriminative value of these parameters in a polycystic ovary syndrome (PCOS)-population. METHODS Serum was obtained daily during a normal menstrual cycle from 25 healthy women (677 data-points). A single serum sample was obtained from 44 PCOS-patients. T was measured by LC–MS/MS and by Architect® 2nd generation T Immunoassay. Sex hormone-binding globulin was measured to calculate fT and FAI. Results: Reference intervals which were established in healthy women with an ovulatory menstrual cycle were T = 0.3-1.6 nmol/L and 0.5-2.0 nmol/L, fT = 5.2-26 pmol/L and 7.2-33 pmol/L, and FAI = 0.4-2.9 and 0.6-4.4, by LC-MS/MS and immunoassay, respectively. T, fT and FAI were higher in PCOS patients than in controls (p b 0.0001). The areas under the curve of receiver operator characteristic (ROC) plots were not different for T, fT, or FAI when T was measured by LC–MS/MS versus immunoassay based on prediction of PCOS. FAI and fT were the strongest predictors of PCOS. CONCLUSIONS When based upon the appropriate reference intervals and ROC analysis, LC-MS/MS and second generation immunoassay have equivalent clinical utility for the diagnosis of PCOS.

Relationship Between Body Mass Index and Serum Testosterone Concentration in Patients Receiving Luteinizing Hormone-releasing Hormone Agonist Therapy for Prostate Cancer

OBJECTIVE To evaluate the relationship between the body mass index (BMI) and serum testosterone concentrations in men receiving luteinizing hormone-releasing hormone (LHRH) agonist therapy for prostate cancer. MATERIALS AND METHODS A total of 66 white men were included in the present study. All subjects had received LHRH agonist therapy for ≥ 3 months. The BMI was calculated, and the subjects were classified as normal weight (i.e. BMI <25 kg/m(2)), overweight (BMI 25-30 kg/m(2)), or obese (BMI >30 kg/m(2)). The serum testosterone concentration was determined using the highly sensitive isotope dilution-liquid chromatography-tandem mass spectrometry technique. The sex hormone-binding globulin level was determined using an immunometric assay, and the free serum testosterone concentration was calculated. RESULTS The median serum testosterone concentration of the patients with a BMI <25 kg/m(2) was 5.5 ng/dL. The patients with a BMI of 25-30 kg/m(2) had a median serum testosterone concentration of 3.8 ng/dL. Those patients with a BMI >30 kg/m(2) had a median concentration of 5.7 ng/dL. No significant difference in the serum testosterone concentrations among the 3 groups was found. The sex hormone-binding globulin levels declined with an increasing BMI. The concentration of free testosterone was significantly greater in the obese men. CONCLUSION Using an ultrasensitive technique of serum testosterone measurement, the present data have shown that no difference exists in the serum testosterone concentration in the castrate range among normal weight, overweight, and obese patients receiving LHRH agonist therapy for prostate cancer. From our findings and current knowledge, more stringent follow-up or changes in dosage or dosage intervals of LHRH agonist therapy in those with a greater or high BMI is not warranted.