madman
Super Moderator
Accurate measurement of very low circulating estradiol (E2 ) (<5 pg/ml) in postmenopausal women and in mice is essential to investigating sex steroid action in target tissues. However, direct immunoassays are too inaccurate and conventional mass spectrometry-based measurement too insensitive at these serum E2 levels. We report the application of an ultrasensitive method using a novel estrogen-selective derivatization in liquid chromatography-mass spectrometry to measure serum E2, with a detection limit of 0.25 pg/ml in small (0.2 ml) serum volumes that can quantify serum E2 in 98% and serum E1 in 100% of healthy postmenopausal women. Aromatase inhibitor (AI) treatment of postmenopausal women with breast cancer further reduces serum E2 by 85% and serum estrone (E1 ) by 80%. The wide scatter of circulating E2 in AI-treated women suggests that the degree of sustained E2 depletion, now quantifiable, may be an efficacy or safety biomarker of adjuvant AI treatment. This ultrasensitive method can also measure serum E2 in most (65%) females but not in any male mice. Further studies are warranted using this and comparable ultrasensitive liquid chromatography-mass spectrometry estrogen measurements to investigate the relationship of circulating E2 (and E1 ) in male, postmenopausal female, and childhood health where accurate quantification of serum estrogens was not previously feasible. This will focus on the direct impact of estrogens as well as the indirect effects of androgen aromatization on reproductive, bone, and brain tissues and, notably, the efficacy and safety of AIs in adjuvant breast cancer treatment.
The measurement of circulating estradiol in conditions in which its concentrations are very low, such as postmenopausal women receiving aromatase inhibitors (AIs), and in small volume samples such as those from rodents, has, to date, been problematic due to limited sensitivity of available methods.
Recognizing the estrogen dependence of breast cancer led to the development of adjuvant antiestrogen treatments to prevent disease recurrence, thereby prolonging disease-free survival and a potential long-term cure of breast cancer, especially in postmenopausal women with estrogen receptor (ER)-positive disease, the most frequent category of lethal breast cancers [1, 2]. There are 2 classes of antiestrogens, ER blockers (also known as selective ER modulators) and AIs. Estrogen receptor blockers compete with estradiol (E2 ), the major potent bioactive estrogen, for binding to ERs and block estradiol’s growth-promoting effects on breast cancer cells [3]. Aromatase inhibitor drugs are a newer class of antiestrogens that inhibit aromatase, the unique enzyme which converts circulating androgen precursors, testosterone, and androstenedione into corresponding estrogens, estradiol (E2 ), and estrone (E1 ). Despite the success of AI therapy in lowering circulating E2 and E1 concentrations [4], some women experience disease relapse [5] or toxicity (including symptomatic estrogen deficiency) [6]. The mechanisms of AI resistance or adverse effects remain little understood. Important pathophysiological reasons include differences in the efficacy of AI treatment in suppressing E2 synthesis due to medication noncompliance and/or pharmacogenetic differences in metabolism or efficacy of AI drugs or aromatase enzyme activity [7]. Accurate measurement of circulating E2 is required for elucidating whether the degree of E2 depletion determines the efficacy or safety of AI treatment. However, circulating E2 levels are very low in postmenopausal women, making it technically challenging to measure [8, 9]. Consequently, for women taking an AI, undetectable serum E2 concentrations are considered an index of effective AI treatment, but this categorization may be indiscriminate with regard to the extent of E2 depletion [10]. Measurement of serum E2 by direct (non-extraction) E2 immunoassays is unreliable due to their nonspecificity, leading to overestimating concentrations, most prominent at low E2 levels [8, 11]. Conventional liquid chromatography-mass spectrometry (LC-MS) methods have characteristic detection limits for serum E2 of 3–5 pg/ml that are not sufficiently sensitive to quantify serum E2 in all postmenopausal women [8, 9, 12] making them unable to estimate the further depression of serum E2 concentration induced by AI drugs. Similarly, circulating E2 concentrations in rodents are also very low so that measurement is unreliable with direct (non-extraction) estradiol immunoassays [8, 13] and too low (< 3–5 pg/ml) for conventional LC-MS methods [14, 15]. One study of gas chromatography-mass spectrometry using relatively large (250 µl) mouse serum samples reported mean serum E2 of 2.7 pg/ml in female but undetectable levels (< 0.3 pg/ml) in male mice [16].
We reported an ultrasensitive method to measure serum E2 using a novel estrogen selective derivatization featuring the theoretical sensitivity to achieve a serum E2 measurement in postmenopausal women and mice [17]. The present study demonstrates that this ultrasensitive method has sufficient sensitivity so that, using small serum sample volume (0.2 ml in humans, 0.1 ml in mice), it can measure serum E2 in virtually all postmenopausal women as well as quantifying further lowering of serum E2 due to AI treatment. Furthermore, we have applied this ultrasensitive method to measure serum E2, E1, and testosterone (T) in female and male mouse serum samples.
Discussion
The present study demonstrates that the estrogen-selective derivatization using the novel derivatizing regent DMIS allows ultrasensitive measurement by LC-MS of serum E2 and E1 in 98% and 100%, respectively, of healthy postmenopausal women using only a small (0.2 ml) serum sample. This method with a LOD of 0.25 pg/ml (detecting 50 fg E2 on the column with 0.2 ml samples) improves on our nonderivatized LC-MS method featuring LOD limits (3–5 pg/ml), but which can quantify serum E2 in less than half of the same serum samples [12]. Further studies comparing the sensitivity and practical application of this ultrasensitive method with others reporting similar sensitivity [9, 24] to postmenopausal women, notably those on AI treatment, as well as to mice are desirable.
Although AI drugs were introduced primarily for adjuvant treatment of breast cancer, there are other off-label and mostly unproven indications for these drugs including female and male infertility, induction of ovulation, and male hypogonadism. Furthermore, AI drugs may have important side-effects such as bone loss [31] and male sexual dysfunction [37] when used for off-label applications. Appraisal of whether the degree of suppression of circulating E2 is a determinant of the efficacy or side-effects of AI drug treatment in these settings is now feasible with methods such as the ones used presently and others [9] ultrasensitive MS-based estrogen assays.
The measurement of circulating estradiol in conditions in which its concentrations are very low, such as postmenopausal women receiving aromatase inhibitors (AIs), and in small volume samples such as those from rodents, has, to date, been problematic due to limited sensitivity of available methods.
Recognizing the estrogen dependence of breast cancer led to the development of adjuvant antiestrogen treatments to prevent disease recurrence, thereby prolonging disease-free survival and a potential long-term cure of breast cancer, especially in postmenopausal women with estrogen receptor (ER)-positive disease, the most frequent category of lethal breast cancers [1, 2]. There are 2 classes of antiestrogens, ER blockers (also known as selective ER modulators) and AIs. Estrogen receptor blockers compete with estradiol (E2 ), the major potent bioactive estrogen, for binding to ERs and block estradiol’s growth-promoting effects on breast cancer cells [3]. Aromatase inhibitor drugs are a newer class of antiestrogens that inhibit aromatase, the unique enzyme which converts circulating androgen precursors, testosterone, and androstenedione into corresponding estrogens, estradiol (E2 ), and estrone (E1 ). Despite the success of AI therapy in lowering circulating E2 and E1 concentrations [4], some women experience disease relapse [5] or toxicity (including symptomatic estrogen deficiency) [6]. The mechanisms of AI resistance or adverse effects remain little understood. Important pathophysiological reasons include differences in the efficacy of AI treatment in suppressing E2 synthesis due to medication noncompliance and/or pharmacogenetic differences in metabolism or efficacy of AI drugs or aromatase enzyme activity [7]. Accurate measurement of circulating E2 is required for elucidating whether the degree of E2 depletion determines the efficacy or safety of AI treatment. However, circulating E2 levels are very low in postmenopausal women, making it technically challenging to measure [8, 9]. Consequently, for women taking an AI, undetectable serum E2 concentrations are considered an index of effective AI treatment, but this categorization may be indiscriminate with regard to the extent of E2 depletion [10]. Measurement of serum E2 by direct (non-extraction) E2 immunoassays is unreliable due to their nonspecificity, leading to overestimating concentrations, most prominent at low E2 levels [8, 11]. Conventional liquid chromatography-mass spectrometry (LC-MS) methods have characteristic detection limits for serum E2 of 3–5 pg/ml that are not sufficiently sensitive to quantify serum E2 in all postmenopausal women [8, 9, 12] making them unable to estimate the further depression of serum E2 concentration induced by AI drugs. Similarly, circulating E2 concentrations in rodents are also very low so that measurement is unreliable with direct (non-extraction) estradiol immunoassays [8, 13] and too low (< 3–5 pg/ml) for conventional LC-MS methods [14, 15]. One study of gas chromatography-mass spectrometry using relatively large (250 µl) mouse serum samples reported mean serum E2 of 2.7 pg/ml in female but undetectable levels (< 0.3 pg/ml) in male mice [16].
We reported an ultrasensitive method to measure serum E2 using a novel estrogen selective derivatization featuring the theoretical sensitivity to achieve a serum E2 measurement in postmenopausal women and mice [17]. The present study demonstrates that this ultrasensitive method has sufficient sensitivity so that, using small serum sample volume (0.2 ml in humans, 0.1 ml in mice), it can measure serum E2 in virtually all postmenopausal women as well as quantifying further lowering of serum E2 due to AI treatment. Furthermore, we have applied this ultrasensitive method to measure serum E2, E1, and testosterone (T) in female and male mouse serum samples.
Discussion
The present study demonstrates that the estrogen-selective derivatization using the novel derivatizing regent DMIS allows ultrasensitive measurement by LC-MS of serum E2 and E1 in 98% and 100%, respectively, of healthy postmenopausal women using only a small (0.2 ml) serum sample. This method with a LOD of 0.25 pg/ml (detecting 50 fg E2 on the column with 0.2 ml samples) improves on our nonderivatized LC-MS method featuring LOD limits (3–5 pg/ml), but which can quantify serum E2 in less than half of the same serum samples [12]. Further studies comparing the sensitivity and practical application of this ultrasensitive method with others reporting similar sensitivity [9, 24] to postmenopausal women, notably those on AI treatment, as well as to mice are desirable.
Although AI drugs were introduced primarily for adjuvant treatment of breast cancer, there are other off-label and mostly unproven indications for these drugs including female and male infertility, induction of ovulation, and male hypogonadism. Furthermore, AI drugs may have important side-effects such as bone loss [31] and male sexual dysfunction [37] when used for off-label applications. Appraisal of whether the degree of suppression of circulating E2 is a determinant of the efficacy or side-effects of AI drug treatment in these settings is now feasible with methods such as the ones used presently and others [9] ultrasensitive MS-based estrogen assays.
