Higher Insulin Resistance and Adiposity in Postmenopausal Women With Breast Cancer Treated With Aromatase Inhibitors.

CONTEXT: Aromatase deficiency causes obesity and insulin resistance in aromatase knockout mice and humans with rare mutations of the aromatase gene (CYP19). Aromatase inhibitors are a commonly prescribed therapy for postmenopausal breast cancer. OBJECTIVE: We hypothesized that aromatase inhibitors induce obesity and insulin resistance when used in treatment of breast cancer. DESIGN: Case-control study. SETTING: University teaching hospital. PARTICIPANTS: Patients with postmenopausal breast cancer (n = 20) treated with aromatase inhibitors and 20 age-matched control subjects. MAIN OUTCOME MEASURES: The primary outcome measure was insulin sensitivity index - Matsuda, derived from a 75-g oral glucose tolerance test. Body composition was assessed by dual energy x-ray absorptiometry and biopsy specimens of subcutaneous adipose tissue obtained for assessment of mRNA transcript levels. Data are reported as mean ± SEM (patients receiving inhibitors vs control group, respectively). RESULTS: Aromatase inhibitor therapy was associated with significantly lower insulin sensitivity (5.15 ± 0.45 vs 6.80 ± 0.64; P = 0.041), higher peak insulin concentration after oral glucose tolerance test (693.4 ± 78.6 vs 527.6 ± 85.5 pmol/L; P = 0.035), greater percentage of body fat (38.4% ± 1.0% vs 34.6% ± 1.3%; P = 0.026), and higher plasma leptin concentration (23.5 ± 2.8 vs 15.5 ± 2.3 ng/mL; P = 0.035). CONCLUSION: Women who received aromatase inhibitors for postmenopausal breast cancer had greater percentage body fat and insulin resistance compared with control subjects with no history of breast cancer.

Aromatase Inhibition Reduces Insulin Sensitivity in Healthy Men.

CONTEXT: Deficiency of aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is associated with insulin resistance in humans and mice. OBJECTIVE: We hypothesized that pharmacological aromatase inhibition results in peripheral insulin resistance in humans. DESIGN: This was a double-blind, randomized, controlled, crossover study. SETTING: The study was conducted at a clinical research facility. PARTICIPANTS: Seventeen healthy male volunteers (18-50 y) participated in the study. INTERVENTION: The intervention included oral anastrozole (1 mg daily) and placebo, each for 6 weeks with a 2-week washout period. MAIN OUTCOME MEASURE: Glucose disposal and rates of lipolysis were measured during a stepwise hyperinsulinemic euglycemic clamp. Data are mean (SEM). RESULTS: Anastrozole therapy resulted in significant estradiol suppression (59.9 ± 3.6 vs 102.0 ± 5.7 pmol/L, P = < .001) and a more modest elevation of total T (25.8 ± 1.2 vs 21.4 ± 0.7 nmol/L, P = .003). Glucose infusion rate, during the low-dose insulin infusion, was lower after anastrozole administration (12.16 ± 1.33 vs 14.15 ± 1.55 µmol/kg·min, P = .024). No differences in hepatic glucose production or rate of lipolysis were observed. CONCLUSION: Aromatase inhibition reduces insulin sensitivity, with respect to peripheral glucose disposal, in healthy men. Local generation and action of estradiol, at the level of skeletal muscle, is likely to be an important determinant of insulin sensitivity.

Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry.

Estrogens circulate at concentrations less than 20pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the "reagent" group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify "FMP" derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2pg on-column and the method was linear from 1-400pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24h at 10°C (7-9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5mL) 135-473, 193-722pmol/L; male plasma (1mL) 25-111, 60-180pmol/L and post-menopausal female plasma (2mL), 22-78, 29-50pmol/L. Thus FMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.

Simultaneous pharmacokinetic and pharmacodynamic analysis of 5α-reductase inhibitors and androgens by liquid chromatography tandem mass spectrometry.

Benign prostatic hyperplasia and prostate cancer can be treated with the 5α-reductase inhibitors, finasteride and dutasteride, when pharmacodynamic biomarkers are useful in assessing response. A novel method was developed to measure the substrates and products of 5α-reductases (testosterone, 5α-dihydrotestosterone (DHT), androstenedione) and finasteride and dutasteride simultaneously by liquid chromatography tandem mass spectrometry, using an ABSciex QTRAP(®) 5500, with a Waters Acquity™ UPLC. Analytes were extracted from serum (500 µL) via solid-phase extraction (Oasis(®) HLB), with (13)C3-labelled androgens and d9-finasteride included as internal standards. Analytes were separated on a Kinetex C18 column (150 × 3 mm, 2.6 µm), using a gradient run of 19 min. Temporal resolution of analytes from naturally occurring isomers and mass +2 isotopomers was ensured. Protonated molecular ions were detected in atmospheric pressure chemical ionisation mode and source conditions optimised for DHT, the least abundant analyte. Multiple reaction monitoring was performed as follows: testosterone (m/z 289 → 97), DHT (m/z 291 → 255), androstenedione (m/z 287 → 97), dutasteride (m/z 529 → 461), finasteride (m/z 373 → 317). Validation parameters (intra- and inter-assay precision and accuracy, linearity, limits of quantitation) were within acceptable ranges and biological extracts were stable for 28 days. Finally the method was employed in men treated with finasteride or dutasteride; levels of DHT were lowered by both drugs and furthermore the substrate concentrations increased.