Nelson Vergel
Founder, ExcelMale.com
Estradiol
1 pg/mL = 3.671 pmol/L
SENSITIVE ESTRADIOL TESTING USED LIQUID CHROMATOGRAPHY/ MASS SPECTROMETRY INSTEAD OF IMMUNOASSAY (ria)
Handelsman DJ, Wartofsky L. Requirement for Mass Spectrometry Sex Steroid Assays in the Journal of Clinical Endocrinology and Metabolism. Journal of Clinical Endocrinology & Metabolism 2013;98(10):3971-3. http://jcem.endojournals.org/content/98/10/3971.full
Huhtaniemi IT, Tajar A, Lee DM, et al. Comparison of serum testosterone and estradiol measurements in 3174 European men using platform immunoassay and mass spectrometry; relevance for the diagnostics in aging men. European Journal of Endocrinology 2012;166(6):983-91. http://www.eje-online.org/content/166/6/983.full
Background The limitations of serum testosterone and estradiol (E2) measurements using non-extraction platform immunoassays (IAs) are widely recognized. Switching to more specific mass spectrometry (MS)-based methods has been advocated, but directly comparative data on the two methods are scarce.
Methods We compared serum testosterone and E2 measurements in a large sample of middle-aged/elderly men using a common platform IA and a gas chromatography (GC)–MS method, in order to assess their limitations and advantages, and to diagnose male hypogonadism. Of subjects from the European Male Aging Study (n=3174; age 40–79 years), peripheral serum testosterone and E2 were analyzed using established commercial platform IAs (Roche Diagnostics E170) and in-house GC–MS methods.
Results Over a broad concentration range, serum testosterone concentration measured by IA and MS showed high correlation (R=0.93, P<0.001), which was less robust in the hypogonadal range (<11 nmol/l; R=0.72, P<0.001). The IA/MS correlation was weaker in E2 measurements (R=0.32, P<0.001, at E2<40.8 pmol/l, and R=0.74, P<0.001, at E2 >40.8 pmol/l). Using MS as the comparator method, IA ascertained low testosterone compatible with hypogonadism (<11 nmol/l), with 75% sensitivity and 96.3% specificity. The same parameters with IA for the detection of low E2 (<40.7 pmol/l) were 13.3 and 99.3%, and for high E2 (>120 pmol/l) 88.4 and 88.6%.
Conclusion A validated platform IA is sufficient to detect subnormal testosterone concentrations in the diagnosis of male hypogonadism. The IA used for E2 measurements showed poor correlation with MS and may only be suitable for the detection of high E2 in men.
Santen RJ, Demers L, Ohorodnik S, et al. Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy. Steroids 2007;72(8):666-71. http://www.sciencedirect.com/science/article/pii/S0039128X0700089X
Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.
Hsing AW, Stanczyk FZ, Belanger A, et al. Reproducibility of Serum Sex Steroid Assays in Men by RIA and Mass Spectrometry. Cancer Epidemiology Biomarkers & Prevention 2007;16(5):1004-8. http://cebp.aacrjournals.org/content/16/5/1004.full
There is an increasing trend to apply gas chromatography combined with mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) assay methods to large-scale epidemiologic studies for the measurement of serum sex steroids. These methods are generally considered the gold standard for sex steroid measurements because of their accuracy, sensitivity, turnaround time, and ability to assess a more complete panel of steroid metabolites in the same run. In this report, we evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility, of steroid hormone measurements determined by GC-MS and LC-MS/MS assays and RIA and compared measurements among these methods.
Specifically, 282 overnight fasting serum samples from 20 male volunteers were analyzed for 12 steroid metabolites by GC-MS or LC-MS/MS in one lab over a 4-month period. Six of the analytes were also measured by RIA in another lab. Unconjugated hormones, including testosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione, androst-5-ene-3β,17β-diol, estrone, and estradiol, were measured by GC-MS, whereas conjugated hormones, including DHEA sulfate, androsterone glucuronide, 5α-androstane-3α,17β-diol 3-glucuronide, 5α-androstane-3α,17β-diol 17-glucuronide, and estrone sulfate, were measured by LC-MS/MS. A subset of these hormones, including testosterone, dihydrotestosterone, androstenedione, 5α-androstane-3α,17β-diol 17-glucuronide, estrone, and estradiol, were also measured by RIA following extraction and chromatography.
We used the coefficient of variation (CV) and the intraclass correlation coefficient (ICC) to assess within- and between-batch assay variations. For the 12 analytes measured by GC-MS or LC-MS/MS, CVs and ICCs for within- and between-batch measurements were similar, with CVs ranging from 6.1% to 21.4% and ICCs ranging from 87.6% to 99.2%. The six analytes measured by RIA had good CVs and ICCs, with CVs <10% and ICCs >70% (range, 71.7-99.7%). For the six metabolites that were measured by both methods, the CVs were similar, whereas the ICCs were generally higher with the GC-MS method. The absolute values for each analyte measured by RIA and GC-MS differed, with RIAs usually yielding markedly higher levels than GC-MS, although the Pearson and Spearman correlation coefficients for these six analytes were near one and all were significant (P < 0.001). Our results show that RIA, GC-MS, and LC-MS/MS assays for androgens and estrogens in the two labs included in the study have good reproducibility, as measured by small CVs (<15%) and high ICCs (>80%), with the exception of estradiol (71.7%) when measured by RIA.
Despite substantial differences in absolute measurements of sex steroid hormones by RIA and MS methods, correlations between the two assays for the six sex steroids measured in the two labs were high (>0.9). However, it is important for future large epidemiologic studies to incorporate MS with high reproducibility and specificity to measure a more complete profile of androgen and estrogen metabolites to clarify the role of sex steroids in prostate cancer.
Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. Challenges to the Measurement of Estradiol: An Endocrine Society Position Statement. Journal of Clinical Endocrinology & Metabolism 2013;98(4):1376-87. http://jcem.endojournals.org/content/98/4/1376.full
Objective: The objective of the study was to evaluate the current state of clinical assays for estradiol in the context of their applications.
Participants: The participants were appointed by the Council of The Endocrine Society and charged with attaining the objective using published data and expert opinion.
Evidence: Data were gathered from published sources via online databases (principally PubMed, Ovid MEDLINE, Google Scholar), and the clinical and laboratory experience of the participants.
Consensus Process: The statement was an effort of the committee and was reviewed by each member. The Clinical Affairs Committee, the Council of The Endocrine Society, and JCEM reviewers reviewed the manuscript and made recommendations.
Conclusions: The measurement of estradiol in biological fluids is important in human biology from cradle to grave. In addition to its centrality in sexual development, it has significant effects on skin, blood vessels, bone, muscle, coagulation, hepatic cells, adipose tissue, the kidney, the gastrointestinal tract, brain, lung, and pancreas. Alterations in its plasma concentration have been implicated in coronary artery disease, stroke, and breast cancer. Although modern immunoassays and liquid chromatography/tandem mass spectrometry-based methods for estradiol are reasonably well suited to the diagnosis and management of infertility (nonetheless, imprecision and method-to-method differences remain problematic), the very low concentrations that appear to be crucial in nonreproductive tissues are a separate and more difficult issue. Such levels of estradiol are too low to be routinely measured accurately or precisely, and further evolution of analytical methods and the way in which estradiol is standardized is needed.
Ohlsson C, Nilsson ME, Tivesten Ã, et al. Comparisons of Immunoassay and Mass Spectrometry Measurements of Serum Estradiol Levels and Their Influence on Clinical Association Studies in Men. Journal of Clinical Endocrinology & Metabolism. http://jcem.endojournals.org/content/98/6/E1097.full
Context: Immunoassay-based techniques, routinely used to measure serum estradiol (E2), are known to have reduced specificity, especially at lower concentrations, when compared with the gold standard technique of mass spectrometry (MS). Different measurement techniques may be responsible for the conflicting results of associations between serum E2 and clinical phenotypes in men.
Objective: Our objective was to compare immunoassay and MS measurements of E2 levels in men and evaluate associations with clinical phenotypes.
Design and Setting: Middle-aged and older male subjects participating in the population-based Osteoporotic Fractures in Men (MrOS) Sweden study (n = 2599), MrOS US (n = 688), and the European Male Aging Study (n = 2908) were included.
Main Outcome Measures: Immunoassay and MS measurements of serum E2 were compared and related to bone mineral density (BMD; measured by dual energy x-ray absorptiometry) and ankle-brachial index.
Results: Within each cohort, serum E2 levels obtained by immunoassay and MS correlated moderately (Spearman rank correlation coefficient rS 0.53–0.76). Serum C-reactive protein (CRP) levels associated significantly (albeit to a low extent, rS = 0.29) with immunoassay E2 but not with MS E2 levels. Similar associations of immunoassay E2 and MS E2 were seen with lumbar spine and total hip BMD, independent of serum CRP. However, immunoassay E2, but not MS E2, associated inversely with ankle-brachial index, and this correlation was lost after adjustment for CRP.
Conclusions: Our findings suggest interference in the immunoassay E2 analyses, possibly by CRP or a CRP-associated factor. Although associations with BMD remain unaffected, this might imply for a reevaluation of previous association studies between immunoassay E2 levels and inflammation-related outcomes.
Khosla S, Amin S, Singh RJ, Atkinson EJ, Melton LJ, 3rd, Riggs BL. Comparison of sex steroid measurements in men by immunoassay versus mass spectroscopy and relationships with cortical and trabecular volumetric bone mineral density. Osteoporos Int 2008;19(10):1465-71. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2636568/
In men, measurement of serum testosterone and estradiol levels with immunoassays correlated with mass spectroscopic measurements, and correlations of sex steroids with volumetric bone mineral density were similar.
INTRODUCTION: While immunoassays have been used extensively for measurement of serum testosterone (T) and estradiol (E(2)) levels, there is concern about their specificity, particularly at low E(2) levels as present in men.
METHODS: We compared T and E(2) measured by mass spectroscopy to levels measured by immunoassay in men (n = 313, age 22 to 91 years) and related these to volumetric bone mineral density (vBMD) at various skeletal sites.
RESULTS: Serum T and non-SHBG bound (or bioavailable) T levels by immunoassay correlated well with the corresponding mass spectroscopy measurements (R = 0.90 and 0.95, respectively, P < 0.001); the correlations for serum E(2) measured using the two techniques were less robust (R = 0.63 for total E(2) and 0.84 for bioavailable E(2), P < 0.001). Overall relationships between serum bioavailable T and E(2) levels with vBMD at various skeletal sites were similar for the immunoassay and mass spectroscopic measures.
CONCLUSIONS: Although E(2) levels with immunoassay correlate less well with the mass spectroscopic measurements than do the T measurements in men, our findings indicate that the fundamental relationships observed previously between vBMD and the sex steroids by immunoassay are also present with the mass spectroscopic measurements.
Stanczyk FZ, Jurow J, Hsing AW. Limitations of Direct Immunoassays for Measuring Circulating Estradiol Levels in Postmenopausal Women and Men in Epidemiologic Studies. Cancer Epidemiology Biomarkers & Prevention 2010;19(4):903-6. http://cebp.aacrjournals.org/content/19/4/903.full
Serum estradiol (E2) serves as an important diagnostic marker in a variety of clinical conditions. In epidemiologic studies, E2 is commonly used to define the etiologic role of estrogen in hormone-related cancers and chronic conditions. Having an accurate and reliable E2 assay is of critical importance in these studies, especially when measuring the very low E2 levels (<30 pg/mL) common in postmenopausal women and men, and for discerning the relatively small (usually <20%) case-control differences in E2 levels. Because E2 is metabolized to >100 metabolites in the body, some of which cross-react with E2 antibodies, direct RIAs without purification steps lack specificity for E2 and can substantially overestimate E2 levels. Although direct E2 RIAs using commercial kits are simpler, less time consuming, and less expensive and require less sample volume than conventional RIAs with preceding purification steps, their lack of sensitivity and specificity makes them invalid for measuring circulating E2 levels in epidemiologic studies of postmenopausal women or men. Instead, we recommend the use of a well-validated RIA with purification steps to improve sensitivity and specificity and to help achieve the necessary accuracy and reliability needed for epidemiologic studies.
Trabado S, Maione L, Salenave S, et al. Estradiol levels in men with congenital hypogonadotropic hypogonadism and the effects of different modalities of hormonal treatment. Fertil Steril 2011;95(7):2324-9, 9 e1-3. http://www.fertstert.org/article/S0015-0282(11)00521-8/abstract
OBJECTIVE: To evaluate the degree of E2 deficiency in male congenital hypogonadotropic hypogonadism (CHH), and its response to different hormonal treatments. DESIGN: Retrospective and prospective studies.
SETTING: Academic institution.
PATIENT(S): Untreated or treated CHH, healthy men, untreated men with Klinefelter syndrome (KS).
INTERVENTION(S): Serum sex hormone-binding globulin (SHBG) and total E2 (TE2) as well as bioavailable (BE2) and free (FE2) levels were measured and determined.
MAIN OUTCOME MEASURE(S): Total, bioavailable, and free testosterone, TE2, BE2, FE2 were compared in normal men, untreated and treated CHH and in untreated KS.
RESULT(S): TE2, BE2, and FE2 levels were very significantly lower in untreated patients with CHH (n=91) than in controls (n=63) and in patients with KS (n=45). The TE2 correlated positively with serum total T in patients with CHH. The TE2 also correlated very positively with serum LH in the combined population of patients with CHH and healthy men, suggesting that low E2 levels in CHH are due to severe LH-driven T deficiency. All fractions of circulating E2 were very significantly higher in patients with CHH receiving T enanthate (n=101) or the FSH-hCG combination (n=88) than in untreated patients with CHH. Contrary to dihydrotestosterone (DHT), both T enanthate and combined FSH-hCG therapy significantly and prospectively increased TE2 levels in patients with CHH.
CONCLUSION(S): Contrary to KS, the male hypogonadism observed in CHH is associated with profound E2 deficiency, which can be overcome by aromatizable androgen or combined gonadotropin therapy.
1 pg/mL = 3.671 pmol/L
Lower limit | Upper limit | Unit |
50 | 200 | pmol/L |
14 | 55 | pg/m |
SENSITIVE ESTRADIOL TESTING USED LIQUID CHROMATOGRAPHY/ MASS SPECTROMETRY INSTEAD OF IMMUNOASSAY (ria)
Handelsman DJ, Wartofsky L. Requirement for Mass Spectrometry Sex Steroid Assays in the Journal of Clinical Endocrinology and Metabolism. Journal of Clinical Endocrinology & Metabolism 2013;98(10):3971-3. http://jcem.endojournals.org/content/98/10/3971.full
Huhtaniemi IT, Tajar A, Lee DM, et al. Comparison of serum testosterone and estradiol measurements in 3174 European men using platform immunoassay and mass spectrometry; relevance for the diagnostics in aging men. European Journal of Endocrinology 2012;166(6):983-91. http://www.eje-online.org/content/166/6/983.full
Background The limitations of serum testosterone and estradiol (E2) measurements using non-extraction platform immunoassays (IAs) are widely recognized. Switching to more specific mass spectrometry (MS)-based methods has been advocated, but directly comparative data on the two methods are scarce.
Methods We compared serum testosterone and E2 measurements in a large sample of middle-aged/elderly men using a common platform IA and a gas chromatography (GC)–MS method, in order to assess their limitations and advantages, and to diagnose male hypogonadism. Of subjects from the European Male Aging Study (n=3174; age 40–79 years), peripheral serum testosterone and E2 were analyzed using established commercial platform IAs (Roche Diagnostics E170) and in-house GC–MS methods.
Results Over a broad concentration range, serum testosterone concentration measured by IA and MS showed high correlation (R=0.93, P<0.001), which was less robust in the hypogonadal range (<11 nmol/l; R=0.72, P<0.001). The IA/MS correlation was weaker in E2 measurements (R=0.32, P<0.001, at E2<40.8 pmol/l, and R=0.74, P<0.001, at E2 >40.8 pmol/l). Using MS as the comparator method, IA ascertained low testosterone compatible with hypogonadism (<11 nmol/l), with 75% sensitivity and 96.3% specificity. The same parameters with IA for the detection of low E2 (<40.7 pmol/l) were 13.3 and 99.3%, and for high E2 (>120 pmol/l) 88.4 and 88.6%.
Conclusion A validated platform IA is sufficient to detect subnormal testosterone concentrations in the diagnosis of male hypogonadism. The IA used for E2 measurements showed poor correlation with MS and may only be suitable for the detection of high E2 in men.
Santen RJ, Demers L, Ohorodnik S, et al. Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy. Steroids 2007;72(8):666-71. http://www.sciencedirect.com/science/article/pii/S0039128X0700089X
Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.
Hsing AW, Stanczyk FZ, Belanger A, et al. Reproducibility of Serum Sex Steroid Assays in Men by RIA and Mass Spectrometry. Cancer Epidemiology Biomarkers & Prevention 2007;16(5):1004-8. http://cebp.aacrjournals.org/content/16/5/1004.full
There is an increasing trend to apply gas chromatography combined with mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) assay methods to large-scale epidemiologic studies for the measurement of serum sex steroids. These methods are generally considered the gold standard for sex steroid measurements because of their accuracy, sensitivity, turnaround time, and ability to assess a more complete panel of steroid metabolites in the same run. In this report, we evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility, of steroid hormone measurements determined by GC-MS and LC-MS/MS assays and RIA and compared measurements among these methods.
Specifically, 282 overnight fasting serum samples from 20 male volunteers were analyzed for 12 steroid metabolites by GC-MS or LC-MS/MS in one lab over a 4-month period. Six of the analytes were also measured by RIA in another lab. Unconjugated hormones, including testosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione, androst-5-ene-3β,17β-diol, estrone, and estradiol, were measured by GC-MS, whereas conjugated hormones, including DHEA sulfate, androsterone glucuronide, 5α-androstane-3α,17β-diol 3-glucuronide, 5α-androstane-3α,17β-diol 17-glucuronide, and estrone sulfate, were measured by LC-MS/MS. A subset of these hormones, including testosterone, dihydrotestosterone, androstenedione, 5α-androstane-3α,17β-diol 17-glucuronide, estrone, and estradiol, were also measured by RIA following extraction and chromatography.
We used the coefficient of variation (CV) and the intraclass correlation coefficient (ICC) to assess within- and between-batch assay variations. For the 12 analytes measured by GC-MS or LC-MS/MS, CVs and ICCs for within- and between-batch measurements were similar, with CVs ranging from 6.1% to 21.4% and ICCs ranging from 87.6% to 99.2%. The six analytes measured by RIA had good CVs and ICCs, with CVs <10% and ICCs >70% (range, 71.7-99.7%). For the six metabolites that were measured by both methods, the CVs were similar, whereas the ICCs were generally higher with the GC-MS method. The absolute values for each analyte measured by RIA and GC-MS differed, with RIAs usually yielding markedly higher levels than GC-MS, although the Pearson and Spearman correlation coefficients for these six analytes were near one and all were significant (P < 0.001). Our results show that RIA, GC-MS, and LC-MS/MS assays for androgens and estrogens in the two labs included in the study have good reproducibility, as measured by small CVs (<15%) and high ICCs (>80%), with the exception of estradiol (71.7%) when measured by RIA.
Despite substantial differences in absolute measurements of sex steroid hormones by RIA and MS methods, correlations between the two assays for the six sex steroids measured in the two labs were high (>0.9). However, it is important for future large epidemiologic studies to incorporate MS with high reproducibility and specificity to measure a more complete profile of androgen and estrogen metabolites to clarify the role of sex steroids in prostate cancer.
Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. Challenges to the Measurement of Estradiol: An Endocrine Society Position Statement. Journal of Clinical Endocrinology & Metabolism 2013;98(4):1376-87. http://jcem.endojournals.org/content/98/4/1376.full
Objective: The objective of the study was to evaluate the current state of clinical assays for estradiol in the context of their applications.
Participants: The participants were appointed by the Council of The Endocrine Society and charged with attaining the objective using published data and expert opinion.
Evidence: Data were gathered from published sources via online databases (principally PubMed, Ovid MEDLINE, Google Scholar), and the clinical and laboratory experience of the participants.
Consensus Process: The statement was an effort of the committee and was reviewed by each member. The Clinical Affairs Committee, the Council of The Endocrine Society, and JCEM reviewers reviewed the manuscript and made recommendations.
Conclusions: The measurement of estradiol in biological fluids is important in human biology from cradle to grave. In addition to its centrality in sexual development, it has significant effects on skin, blood vessels, bone, muscle, coagulation, hepatic cells, adipose tissue, the kidney, the gastrointestinal tract, brain, lung, and pancreas. Alterations in its plasma concentration have been implicated in coronary artery disease, stroke, and breast cancer. Although modern immunoassays and liquid chromatography/tandem mass spectrometry-based methods for estradiol are reasonably well suited to the diagnosis and management of infertility (nonetheless, imprecision and method-to-method differences remain problematic), the very low concentrations that appear to be crucial in nonreproductive tissues are a separate and more difficult issue. Such levels of estradiol are too low to be routinely measured accurately or precisely, and further evolution of analytical methods and the way in which estradiol is standardized is needed.
Ohlsson C, Nilsson ME, Tivesten Ã, et al. Comparisons of Immunoassay and Mass Spectrometry Measurements of Serum Estradiol Levels and Their Influence on Clinical Association Studies in Men. Journal of Clinical Endocrinology & Metabolism. http://jcem.endojournals.org/content/98/6/E1097.full
Context: Immunoassay-based techniques, routinely used to measure serum estradiol (E2), are known to have reduced specificity, especially at lower concentrations, when compared with the gold standard technique of mass spectrometry (MS). Different measurement techniques may be responsible for the conflicting results of associations between serum E2 and clinical phenotypes in men.
Objective: Our objective was to compare immunoassay and MS measurements of E2 levels in men and evaluate associations with clinical phenotypes.
Design and Setting: Middle-aged and older male subjects participating in the population-based Osteoporotic Fractures in Men (MrOS) Sweden study (n = 2599), MrOS US (n = 688), and the European Male Aging Study (n = 2908) were included.
Main Outcome Measures: Immunoassay and MS measurements of serum E2 were compared and related to bone mineral density (BMD; measured by dual energy x-ray absorptiometry) and ankle-brachial index.
Results: Within each cohort, serum E2 levels obtained by immunoassay and MS correlated moderately (Spearman rank correlation coefficient rS 0.53–0.76). Serum C-reactive protein (CRP) levels associated significantly (albeit to a low extent, rS = 0.29) with immunoassay E2 but not with MS E2 levels. Similar associations of immunoassay E2 and MS E2 were seen with lumbar spine and total hip BMD, independent of serum CRP. However, immunoassay E2, but not MS E2, associated inversely with ankle-brachial index, and this correlation was lost after adjustment for CRP.
Conclusions: Our findings suggest interference in the immunoassay E2 analyses, possibly by CRP or a CRP-associated factor. Although associations with BMD remain unaffected, this might imply for a reevaluation of previous association studies between immunoassay E2 levels and inflammation-related outcomes.
Khosla S, Amin S, Singh RJ, Atkinson EJ, Melton LJ, 3rd, Riggs BL. Comparison of sex steroid measurements in men by immunoassay versus mass spectroscopy and relationships with cortical and trabecular volumetric bone mineral density. Osteoporos Int 2008;19(10):1465-71. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2636568/
In men, measurement of serum testosterone and estradiol levels with immunoassays correlated with mass spectroscopic measurements, and correlations of sex steroids with volumetric bone mineral density were similar.
INTRODUCTION: While immunoassays have been used extensively for measurement of serum testosterone (T) and estradiol (E(2)) levels, there is concern about their specificity, particularly at low E(2) levels as present in men.
METHODS: We compared T and E(2) measured by mass spectroscopy to levels measured by immunoassay in men (n = 313, age 22 to 91 years) and related these to volumetric bone mineral density (vBMD) at various skeletal sites.
RESULTS: Serum T and non-SHBG bound (or bioavailable) T levels by immunoassay correlated well with the corresponding mass spectroscopy measurements (R = 0.90 and 0.95, respectively, P < 0.001); the correlations for serum E(2) measured using the two techniques were less robust (R = 0.63 for total E(2) and 0.84 for bioavailable E(2), P < 0.001). Overall relationships between serum bioavailable T and E(2) levels with vBMD at various skeletal sites were similar for the immunoassay and mass spectroscopic measures.
CONCLUSIONS: Although E(2) levels with immunoassay correlate less well with the mass spectroscopic measurements than do the T measurements in men, our findings indicate that the fundamental relationships observed previously between vBMD and the sex steroids by immunoassay are also present with the mass spectroscopic measurements.
Stanczyk FZ, Jurow J, Hsing AW. Limitations of Direct Immunoassays for Measuring Circulating Estradiol Levels in Postmenopausal Women and Men in Epidemiologic Studies. Cancer Epidemiology Biomarkers & Prevention 2010;19(4):903-6. http://cebp.aacrjournals.org/content/19/4/903.full
Serum estradiol (E2) serves as an important diagnostic marker in a variety of clinical conditions. In epidemiologic studies, E2 is commonly used to define the etiologic role of estrogen in hormone-related cancers and chronic conditions. Having an accurate and reliable E2 assay is of critical importance in these studies, especially when measuring the very low E2 levels (<30 pg/mL) common in postmenopausal women and men, and for discerning the relatively small (usually <20%) case-control differences in E2 levels. Because E2 is metabolized to >100 metabolites in the body, some of which cross-react with E2 antibodies, direct RIAs without purification steps lack specificity for E2 and can substantially overestimate E2 levels. Although direct E2 RIAs using commercial kits are simpler, less time consuming, and less expensive and require less sample volume than conventional RIAs with preceding purification steps, their lack of sensitivity and specificity makes them invalid for measuring circulating E2 levels in epidemiologic studies of postmenopausal women or men. Instead, we recommend the use of a well-validated RIA with purification steps to improve sensitivity and specificity and to help achieve the necessary accuracy and reliability needed for epidemiologic studies.
Trabado S, Maione L, Salenave S, et al. Estradiol levels in men with congenital hypogonadotropic hypogonadism and the effects of different modalities of hormonal treatment. Fertil Steril 2011;95(7):2324-9, 9 e1-3. http://www.fertstert.org/article/S0015-0282(11)00521-8/abstract
OBJECTIVE: To evaluate the degree of E2 deficiency in male congenital hypogonadotropic hypogonadism (CHH), and its response to different hormonal treatments. DESIGN: Retrospective and prospective studies.
SETTING: Academic institution.
PATIENT(S): Untreated or treated CHH, healthy men, untreated men with Klinefelter syndrome (KS).
INTERVENTION(S): Serum sex hormone-binding globulin (SHBG) and total E2 (TE2) as well as bioavailable (BE2) and free (FE2) levels were measured and determined.
MAIN OUTCOME MEASURE(S): Total, bioavailable, and free testosterone, TE2, BE2, FE2 were compared in normal men, untreated and treated CHH and in untreated KS.
RESULT(S): TE2, BE2, and FE2 levels were very significantly lower in untreated patients with CHH (n=91) than in controls (n=63) and in patients with KS (n=45). The TE2 correlated positively with serum total T in patients with CHH. The TE2 also correlated very positively with serum LH in the combined population of patients with CHH and healthy men, suggesting that low E2 levels in CHH are due to severe LH-driven T deficiency. All fractions of circulating E2 were very significantly higher in patients with CHH receiving T enanthate (n=101) or the FSH-hCG combination (n=88) than in untreated patients with CHH. Contrary to dihydrotestosterone (DHT), both T enanthate and combined FSH-hCG therapy significantly and prospectively increased TE2 levels in patients with CHH.
CONCLUSION(S): Contrary to KS, the male hypogonadism observed in CHH is associated with profound E2 deficiency, which can be overcome by aromatizable androgen or combined gonadotropin therapy.
