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Leonard F Blackwell - One of the best experts on this subject based on the ideXlab platform.
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conjugation of Estrone Glucuronide with human lysozyme
Journal of Immunoassay & Immunochemistry, 2015Co-Authors: Delwyn G Cooke, Leonard F BlackwellAbstract:Human milk lysozyme was conjugated with Estrone Glucuronide to give a monoacylated conjugate, two disubstituted isoforms, and one trisubstituted isoform in 99.4% yield. The conjugates were pure and highly inhibited (>98%) by the anti-Estrone Glucuronide antibody. The clearing curves were biphasic for all four conjugates but a 3 min initial rate assay was established and used to measure a normal menstrual cycle profile of Estrone Glucuronide excretion rates. The marked differences between the hen egg white and human milk lysozyme conjugates show that near identical tertiary structures do not necessarily imply similar physical, chemical, biochemical, and kinetic behavior.
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monitoring of ovarian activity by measurement of urinary excretion rates of Estrone Glucuronide and pregnanediol Glucuronide using the ovarian monitor part ii reliability of home testing
Human Reproduction, 2012Co-Authors: Leonard F Blackwell, Delwyn G Cooke, Pilar Vigil, Barbara Gross, Catherine Darcangues, J B BrownAbstract:Background The UNDP/WHO/World Bank/Special Programme of Research, Development and Research Training in Human Reproduction (Geneva) set up a study to determine whether it is feasible for women to monitor their ovarian activity reliably by home testing. Daily self-monitoring of urinary hormone metabolites for menstrual cycle assessment was evaluated by comparison of results obtained with the Home Ovarian Monitor by untrained users both at home and in study centres. Methods Women collected daily data for urinary Estrone Glucuronide (E1G) and pregnanediol Glucuronide (PdG) for two cycles, then the procedure was repeated in the women's local centre (in Chile, Australia or New Zealand) giving a total of 113 duplicate cycles. The tests were performed without the benefit of replicates or quality controls. The home and centre cycles were normalized and compared to identify assay errors, and the resulting home and centre menstrual cycle profiles were averaged. Results Reliable mean cycle profiles were obtained with the home and centre excretion rates agreeing to within 36 ± 21 nmol/24 h for E1G and 0.77 ± 0.28 µmol/24 h for baseline PdG values (1-5 µmol/24 h). The cycles had a mean length of 28.1 ± 3.1 days (n = 112; 5th and 95th percentiles: 24 and 35 days, respectively), a mean follicular phase of 14.8 ± 3.1 days (n = 107; 5th and 95th percentiles: 11 and 21 days) and a mean luteal phase length of 13.3 ± 1.5 days (n = 106; 5th and 95th percentiles: 11 and 17 days), calculated from the day of the LH peak. Conclusions The study confirmed that the Ovarian Monitor pre-coated assay tubes worked well even in the hands of lay users, without standard curves, quality controls or replicates. Point-of-care monitoring to give reliable fertility data is feasible.
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validation of a reference elisa for Estrone Glucuronide using urine samples normalized by dilution to a constant rate of urine production
Steroids, 2007Co-Authors: Delwyn G Cooke, Jan E Binnie, Leonard F BlackwellAbstract:Abstract A direct enzyme linked immunosorbent assay (ELISA) system has been optimized as a reference method for the measurement of first statistically significant rises in Estrone Glucuronide excretion rates in human urine by analysing samples pre-diluted at the time of the collection by the women subjects to a constant urine production rate of 150 mL/h. Validation was achieved by correlation of the individual menstrual cycle profiles with the corresponding Estrone Glucuronide excretion rates determined by radioimmunoassay (RIA) on the same urine samples for a total of 221 samples from nine cycles. The pre-dilution procedure removed random variations due to fluctuations in the daily rate of urine excretion and minimized between sample matrix effects. When the ELISA data were correlated with the RIA data, Deming regression gave a slope of 1.20 ± 0.03 and an intercept of 4.6 ± 1.8 nmol/24 h ( r = 0.944) and a random experimental error of 14.2 nmol/24 h. The major difference in the measurements was a proportional error of 20%, which was present in either the ELISA or RIA methods or in both. Comparison of the standard normal variate transformation of the ELISA and RIA data gave hormonal profiles of the individual menstrual cycles ( N = 9) that overlapped almost perfectly. Statistically significant rises or falls in the magnitude of the excretion rate in one profile were mirrored faithfully in the other.
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use of defined Estrone Glucuronide hen egg white lysozyme conjugates as signal generators in homogeneous enzyme immunoassays for urinary Estrone Glucuronide
Journal of Immunoassay & Immunochemistry, 2003Co-Authors: Delwyn G Cooke, Simone Flight, Mark C Smales, Leonard F BlackwellAbstract:Abstract Three structurally characterized Estrone Glucuronide–lysozyme conjugates, E1 (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7 M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-Estrone Glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary Estrone Glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguisha...
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characterization of lysozyme Estrone Glucuronide conjugates the effect of the coupling reagent on the substitution level and sites of acylation
Bioconjugate Chemistry, 1999Co-Authors: Christopher Mark Smales, Christopher H Moore, Leonard F BlackwellAbstract:Estrone Glucuronide conjugates of hen egg white lysozyme were prepared by the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugates, but the active ester procedure gave a more diverse range of products, making it less suitable for preparing conjugates for homogeneous enzyme immunoassay. Conjugation of lysozyme with Estrone Glucuronide by the mixed anhydride procedure gave one major derivative exclusively acylated at lysine residue 33 whereas conjugation by the active ester method gave six derivatives which were acylated at one or more of lysine residues 33, 97, and 116. None of the lysine residues 1, 13, and 96, or the N-terminal α-amino group, were acylated in any of the conjugates isolated. The correlation of the conjugate structures with the protein environments of the amino groups in the crystal structure of lysozyme suggested that the sites of acylation were determined not only by the chemical nature of the acylating reagent but also by the surface accessib...
Nancy Fugate Woods - One of the best experts on this subject based on the ideXlab platform.
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abdominal pain during the menopause transition and early postmenopause observations from the seattle midlife women s health study
Women's Midlife Health, 2019Co-Authors: Nini G L Callan, Ellen Sullivan Mitchell, Margaret M Heitkemper, Nancy Fugate WoodsAbstract:To assess the relationship between abdominal pain severity during the menopausal transition (MT) and age, MT stage, reproductive biomarkers, stress biomarkers, and stress perceptions. Women ages 35–55 were recruited from multiethnic neighborhoods in the greater Seattle area from 1990 to 1992, for an original study cohort of 508. From 1990 to 2013, a subset of this cohort consented to ongoing annual data collection by annual health questionnaire, health diary, and daily menstrual calendar. Beginning in 1997, a portion of these women also provided a first morning voided urine specimen to be assayed for levels of Estrone Glucuronide (E1G), follicle stimulating hormone (FSH), testosterone, cortisol, norepinephrine, and epinephrine. To identify how changes in abdominal pain severity changed over time in relation to age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions, mixed effects modeling was used. In a univariate model, E1G (p = 0.02) and testosterone (p = 0.02) were significantly and negatively related to abdominal pain severity, while perceived stress (p = 0.06), tension (p < 0.001), and anxiety (p < 0.001) were significantly and positively associated. In a multivariate model, increasing age (p = 0.001) and E1G (p = 0.04) were negatively associated with abdominal pain severity, and anxiety (p = 0.00) positively associated. Testosterone did not improve the fit to the final model, nor did tension or perceived stress. These results suggest that age, anxiety, and E1G each show a significant association with abdominal pain severity in the MT. In contrast, stress perception, tension, testosterone, stress biomarkers, and MT stage do not. These factors should be evaluated further in research on abdominal pain experienced during the MT and early postmenopause years.
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constipation and diarrhea during the menopause transition and early postmenopause observations from the seattle midlife women s health study
Menopause, 2018Co-Authors: Nini G L Callan, Ellen Sullivan Mitchell, Margaret M Heitkemper, Nancy Fugate WoodsAbstract:OBJECTIVE To assess the relationship of constipation and diarrhea severity during the menopause transition (MT) with age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions. METHODS From 1990 to 1992, women aged 35 to 55 years were recruited from the greater Seattle area; 291 of them consented to ongoing (1990-2013) annual data collection by daily menstrual calendar, health diary, and annual health questionnaire. A subset (n = 131) provided a first morning voided urine specimen (1997-2013). These were assayed for levels of E1G, follicle-stimulating hormone, testosterone, cortisol, norepinephrine, and epinephrine. Mixed-effects modeling was used to identify how changes in constipation and diarrhea severity over time related to age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions. RESULTS In a univariate model, age, late reproductive (LR) stage, tension, and anxiety were all significantly and positively related to constipation severity, whereas cortisol was significantly and negatively associated. In a multivariate model, only tension and cortisol remained significant predictors of constipation severity (P < 0.05). In a univariate model, age, LR stage, and Estrone Glucuronide were significantly and negatively associated with diarrhea severity, whereas tension, anxiety, and perceived stress were significantly and positively related. In a multivariate model, only tension and age remained significant predictors of diarrhea severity. CONCLUSIONS Key reproductive hormones do not play a significant role in constipation or diarrhea severity in the MT. In contrast, stress perception, tension, anxiety, and cortisol do. These factors should be evaluated in further research involving constipation and diarrhea.
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pain symptoms during the menopausal transition and early postmenopause
Climacteric, 2010Co-Authors: Ellen Sullivan Mitchell, Nancy Fugate WoodsAbstract:Aim The aim of this study was to describe changes in levels of back pain and joint pain during the stages of menopausal transition stages and early postmenopause, including the effects of age, menopausal transition-related factors (Estrone, follicle stimulating hormone (FSH), testosterone), menopausal transition stages, symptoms (hot flush, sleep, mood, cognitive), health-related factors (body mass index, alcohol use, smoking, well-being), stress-related factors (perceived stress, history of sexual abuse, cortisol, catecholamines) and social factors (partner and parenting status, education). Methods A subset was studied of the participants in the Seattle Midlife Women’s Health Study who provided data during the late reproductive, early and late menopausal transition stages or postmenopause (n ¼292) including menstrual calendars for staging the menopausal transition, annual health reports completed between 1990 and 2006, and morning urine samples assayed for Estrone Glucuronide (E1G), testosterone, FSH, cortisol and catecholamines, and symptom diary ratings several times each year. Multilevel modeling with an R program was used to test patterns of pain symptoms related to age, menopausal transition-related factors, symptoms, health-related, stress-related and social factors, with as many as 6812 observations. Age was centered at 47.4 years. Results Women experienced a slight, non-significant rise in back pain with age and a significant increase in back pain during the early (p ¼0.003) and late menopausal transition stages (p ¼0.002) and early postmenopause (p ¼0.02), but urinary E1G, FSH and testosterone levels were unrelated. Of the stress-related factors, perceived stress (p ¼0.01) and lower overnight urinary cortisol levels were associated with more severe back pain (p ¼0.03); history of sexual abuse and catecholamines did not have a significant effect. Those most troubled by symptoms of hot flushes, depressed mood, anxiety, night-time awakening, and difficulty concentrating reported significantly greater back pain (all p50.0001). Of the health-related factors, having worse perceived health (p50.0001), exercising more (p ¼0.005), using analgesics (p50.0001), and having a higher body mass index (p50.0001) were associated with more back pain, but alcohol use and smoking did not have significant effects. Of the social factors, only having a more formal education (p ¼0.004) was associated with less back pain; parenting, having a partner, and employment were not significant. Factors associated with joint pain included age (p50.0001), but not menopausal transition-related factors. Symptoms of hot flushes, night-time awakening, depressed mood, and difficulty concentrating were each significantly associated with joint pain (p50.0001). Poorer perceived health, more exercise, higher body mass index, and greater analgesic use were all associated similarly with joint pain. History of sexual abuse was the only stress-related factor significantly related to joint pain severity (p ¼0.024).
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sexual desire during the menopausal transition and early postmenopause observations from the seattle midlife women s health study
Journal of Womens Health, 2010Co-Authors: Nancy Fugate Woods, Ellen Sullivan Mitchell, Kathy Smith-di JulioAbstract:Abstract Aims: To describe levels of sexual desire across the menopausal transition (MT) and early postmenopause (PM), including effects of age, MT-related factors, health, stress, symptoms (hot flash, sleep, mood), and social opportunity factors. Methods: A subset of Seattle Midlife Women's Health Study (SMWHS) participants who provided data during the early reproductive, early and late menopausal transition stages, or postmenopause (n = 286), including menstrual calendars for staging the MT, annual health reports between 1990 and 2005, and morning urine samples assayed for Estrone Glucuronide (E1G), testosterone (T), and follicle-stimulating hormone (FSH) was included. Multilevel modeling using the R program was used to test factors related to sexual desire. Results: Women experienced a significant decrease in sexual desire during the late MT stage (p < 0.01) and early PM (p < 0.0001). Those with higher urinary E1G and T reported significantly higher levels of sexual desire, whereas those with higher FS...
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cortisol levels during the menopausal transition and early postmenopause
Menopause, 2009Co-Authors: Nancy Fugate Woods, Ellen Sullivan Mitchell, Kathleen SmithdijulioAbstract:AbstractObjective:Cortisol levels rise among some women during the late stage of the menopausal transition (MT), but we know little about changes in cortisol levels in relation to menopause-related factors (MT stage, urinary Estrone Glucuronide [E1G], testosterone, follicle-stimulating hormone [FSH]
Ellen Sullivan Mitchell - One of the best experts on this subject based on the ideXlab platform.
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abdominal pain during the menopause transition and early postmenopause observations from the seattle midlife women s health study
Women's Midlife Health, 2019Co-Authors: Nini G L Callan, Ellen Sullivan Mitchell, Margaret M Heitkemper, Nancy Fugate WoodsAbstract:To assess the relationship between abdominal pain severity during the menopausal transition (MT) and age, MT stage, reproductive biomarkers, stress biomarkers, and stress perceptions. Women ages 35–55 were recruited from multiethnic neighborhoods in the greater Seattle area from 1990 to 1992, for an original study cohort of 508. From 1990 to 2013, a subset of this cohort consented to ongoing annual data collection by annual health questionnaire, health diary, and daily menstrual calendar. Beginning in 1997, a portion of these women also provided a first morning voided urine specimen to be assayed for levels of Estrone Glucuronide (E1G), follicle stimulating hormone (FSH), testosterone, cortisol, norepinephrine, and epinephrine. To identify how changes in abdominal pain severity changed over time in relation to age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions, mixed effects modeling was used. In a univariate model, E1G (p = 0.02) and testosterone (p = 0.02) were significantly and negatively related to abdominal pain severity, while perceived stress (p = 0.06), tension (p < 0.001), and anxiety (p < 0.001) were significantly and positively associated. In a multivariate model, increasing age (p = 0.001) and E1G (p = 0.04) were negatively associated with abdominal pain severity, and anxiety (p = 0.00) positively associated. Testosterone did not improve the fit to the final model, nor did tension or perceived stress. These results suggest that age, anxiety, and E1G each show a significant association with abdominal pain severity in the MT. In contrast, stress perception, tension, testosterone, stress biomarkers, and MT stage do not. These factors should be evaluated further in research on abdominal pain experienced during the MT and early postmenopause years.
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constipation and diarrhea during the menopause transition and early postmenopause observations from the seattle midlife women s health study
Menopause, 2018Co-Authors: Nini G L Callan, Ellen Sullivan Mitchell, Margaret M Heitkemper, Nancy Fugate WoodsAbstract:OBJECTIVE To assess the relationship of constipation and diarrhea severity during the menopause transition (MT) with age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions. METHODS From 1990 to 1992, women aged 35 to 55 years were recruited from the greater Seattle area; 291 of them consented to ongoing (1990-2013) annual data collection by daily menstrual calendar, health diary, and annual health questionnaire. A subset (n = 131) provided a first morning voided urine specimen (1997-2013). These were assayed for levels of E1G, follicle-stimulating hormone, testosterone, cortisol, norepinephrine, and epinephrine. Mixed-effects modeling was used to identify how changes in constipation and diarrhea severity over time related to age, MT stage, reproductive biomarkers, stress-related biomarkers, and stress-related perceptions. RESULTS In a univariate model, age, late reproductive (LR) stage, tension, and anxiety were all significantly and positively related to constipation severity, whereas cortisol was significantly and negatively associated. In a multivariate model, only tension and cortisol remained significant predictors of constipation severity (P < 0.05). In a univariate model, age, LR stage, and Estrone Glucuronide were significantly and negatively associated with diarrhea severity, whereas tension, anxiety, and perceived stress were significantly and positively related. In a multivariate model, only tension and age remained significant predictors of diarrhea severity. CONCLUSIONS Key reproductive hormones do not play a significant role in constipation or diarrhea severity in the MT. In contrast, stress perception, tension, anxiety, and cortisol do. These factors should be evaluated in further research involving constipation and diarrhea.
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pain symptoms during the menopausal transition and early postmenopause
Climacteric, 2010Co-Authors: Ellen Sullivan Mitchell, Nancy Fugate WoodsAbstract:Aim The aim of this study was to describe changes in levels of back pain and joint pain during the stages of menopausal transition stages and early postmenopause, including the effects of age, menopausal transition-related factors (Estrone, follicle stimulating hormone (FSH), testosterone), menopausal transition stages, symptoms (hot flush, sleep, mood, cognitive), health-related factors (body mass index, alcohol use, smoking, well-being), stress-related factors (perceived stress, history of sexual abuse, cortisol, catecholamines) and social factors (partner and parenting status, education). Methods A subset was studied of the participants in the Seattle Midlife Women’s Health Study who provided data during the late reproductive, early and late menopausal transition stages or postmenopause (n ¼292) including menstrual calendars for staging the menopausal transition, annual health reports completed between 1990 and 2006, and morning urine samples assayed for Estrone Glucuronide (E1G), testosterone, FSH, cortisol and catecholamines, and symptom diary ratings several times each year. Multilevel modeling with an R program was used to test patterns of pain symptoms related to age, menopausal transition-related factors, symptoms, health-related, stress-related and social factors, with as many as 6812 observations. Age was centered at 47.4 years. Results Women experienced a slight, non-significant rise in back pain with age and a significant increase in back pain during the early (p ¼0.003) and late menopausal transition stages (p ¼0.002) and early postmenopause (p ¼0.02), but urinary E1G, FSH and testosterone levels were unrelated. Of the stress-related factors, perceived stress (p ¼0.01) and lower overnight urinary cortisol levels were associated with more severe back pain (p ¼0.03); history of sexual abuse and catecholamines did not have a significant effect. Those most troubled by symptoms of hot flushes, depressed mood, anxiety, night-time awakening, and difficulty concentrating reported significantly greater back pain (all p50.0001). Of the health-related factors, having worse perceived health (p50.0001), exercising more (p ¼0.005), using analgesics (p50.0001), and having a higher body mass index (p50.0001) were associated with more back pain, but alcohol use and smoking did not have significant effects. Of the social factors, only having a more formal education (p ¼0.004) was associated with less back pain; parenting, having a partner, and employment were not significant. Factors associated with joint pain included age (p50.0001), but not menopausal transition-related factors. Symptoms of hot flushes, night-time awakening, depressed mood, and difficulty concentrating were each significantly associated with joint pain (p50.0001). Poorer perceived health, more exercise, higher body mass index, and greater analgesic use were all associated similarly with joint pain. History of sexual abuse was the only stress-related factor significantly related to joint pain severity (p ¼0.024).
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sexual desire during the menopausal transition and early postmenopause observations from the seattle midlife women s health study
Journal of Womens Health, 2010Co-Authors: Nancy Fugate Woods, Ellen Sullivan Mitchell, Kathy Smith-di JulioAbstract:Abstract Aims: To describe levels of sexual desire across the menopausal transition (MT) and early postmenopause (PM), including effects of age, MT-related factors, health, stress, symptoms (hot flash, sleep, mood), and social opportunity factors. Methods: A subset of Seattle Midlife Women's Health Study (SMWHS) participants who provided data during the early reproductive, early and late menopausal transition stages, or postmenopause (n = 286), including menstrual calendars for staging the MT, annual health reports between 1990 and 2005, and morning urine samples assayed for Estrone Glucuronide (E1G), testosterone (T), and follicle-stimulating hormone (FSH) was included. Multilevel modeling using the R program was used to test factors related to sexual desire. Results: Women experienced a significant decrease in sexual desire during the late MT stage (p < 0.01) and early PM (p < 0.0001). Those with higher urinary E1G and T reported significantly higher levels of sexual desire, whereas those with higher FS...
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cortisol levels during the menopausal transition and early postmenopause
Menopause, 2009Co-Authors: Nancy Fugate Woods, Ellen Sullivan Mitchell, Kathleen SmithdijulioAbstract:AbstractObjective:Cortisol levels rise among some women during the late stage of the menopausal transition (MT), but we know little about changes in cortisol levels in relation to menopause-related factors (MT stage, urinary Estrone Glucuronide [E1G], testosterone, follicle-stimulating hormone [FSH]
Delwyn G Cooke - One of the best experts on this subject based on the ideXlab platform.
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conjugation of Estrone Glucuronide with human lysozyme
Journal of Immunoassay & Immunochemistry, 2015Co-Authors: Delwyn G Cooke, Leonard F BlackwellAbstract:Human milk lysozyme was conjugated with Estrone Glucuronide to give a monoacylated conjugate, two disubstituted isoforms, and one trisubstituted isoform in 99.4% yield. The conjugates were pure and highly inhibited (>98%) by the anti-Estrone Glucuronide antibody. The clearing curves were biphasic for all four conjugates but a 3 min initial rate assay was established and used to measure a normal menstrual cycle profile of Estrone Glucuronide excretion rates. The marked differences between the hen egg white and human milk lysozyme conjugates show that near identical tertiary structures do not necessarily imply similar physical, chemical, biochemical, and kinetic behavior.
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monitoring of ovarian activity by measurement of urinary excretion rates of Estrone Glucuronide and pregnanediol Glucuronide using the ovarian monitor part ii reliability of home testing
Human Reproduction, 2012Co-Authors: Leonard F Blackwell, Delwyn G Cooke, Pilar Vigil, Barbara Gross, Catherine Darcangues, J B BrownAbstract:Background The UNDP/WHO/World Bank/Special Programme of Research, Development and Research Training in Human Reproduction (Geneva) set up a study to determine whether it is feasible for women to monitor their ovarian activity reliably by home testing. Daily self-monitoring of urinary hormone metabolites for menstrual cycle assessment was evaluated by comparison of results obtained with the Home Ovarian Monitor by untrained users both at home and in study centres. Methods Women collected daily data for urinary Estrone Glucuronide (E1G) and pregnanediol Glucuronide (PdG) for two cycles, then the procedure was repeated in the women's local centre (in Chile, Australia or New Zealand) giving a total of 113 duplicate cycles. The tests were performed without the benefit of replicates or quality controls. The home and centre cycles were normalized and compared to identify assay errors, and the resulting home and centre menstrual cycle profiles were averaged. Results Reliable mean cycle profiles were obtained with the home and centre excretion rates agreeing to within 36 ± 21 nmol/24 h for E1G and 0.77 ± 0.28 µmol/24 h for baseline PdG values (1-5 µmol/24 h). The cycles had a mean length of 28.1 ± 3.1 days (n = 112; 5th and 95th percentiles: 24 and 35 days, respectively), a mean follicular phase of 14.8 ± 3.1 days (n = 107; 5th and 95th percentiles: 11 and 21 days) and a mean luteal phase length of 13.3 ± 1.5 days (n = 106; 5th and 95th percentiles: 11 and 17 days), calculated from the day of the LH peak. Conclusions The study confirmed that the Ovarian Monitor pre-coated assay tubes worked well even in the hands of lay users, without standard curves, quality controls or replicates. Point-of-care monitoring to give reliable fertility data is feasible.
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validation of a reference elisa for Estrone Glucuronide using urine samples normalized by dilution to a constant rate of urine production
Steroids, 2007Co-Authors: Delwyn G Cooke, Jan E Binnie, Leonard F BlackwellAbstract:Abstract A direct enzyme linked immunosorbent assay (ELISA) system has been optimized as a reference method for the measurement of first statistically significant rises in Estrone Glucuronide excretion rates in human urine by analysing samples pre-diluted at the time of the collection by the women subjects to a constant urine production rate of 150 mL/h. Validation was achieved by correlation of the individual menstrual cycle profiles with the corresponding Estrone Glucuronide excretion rates determined by radioimmunoassay (RIA) on the same urine samples for a total of 221 samples from nine cycles. The pre-dilution procedure removed random variations due to fluctuations in the daily rate of urine excretion and minimized between sample matrix effects. When the ELISA data were correlated with the RIA data, Deming regression gave a slope of 1.20 ± 0.03 and an intercept of 4.6 ± 1.8 nmol/24 h ( r = 0.944) and a random experimental error of 14.2 nmol/24 h. The major difference in the measurements was a proportional error of 20%, which was present in either the ELISA or RIA methods or in both. Comparison of the standard normal variate transformation of the ELISA and RIA data gave hormonal profiles of the individual menstrual cycles ( N = 9) that overlapped almost perfectly. Statistically significant rises or falls in the magnitude of the excretion rate in one profile were mirrored faithfully in the other.
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use of defined Estrone Glucuronide hen egg white lysozyme conjugates as signal generators in homogeneous enzyme immunoassays for urinary Estrone Glucuronide
Journal of Immunoassay & Immunochemistry, 2003Co-Authors: Delwyn G Cooke, Simone Flight, Mark C Smales, Leonard F BlackwellAbstract:Abstract Three structurally characterized Estrone Glucuronide–lysozyme conjugates, E1 (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7 M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-Estrone Glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary Estrone Glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguisha...
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use of ion exchange and hydrophobic interaction chromatography for the rapid purification of lysozyme Estrone Glucuronide conjugates
Journal of Chromatography B: Biomedical Sciences and Applications, 1994Co-Authors: Christopher Mark Smales, Delwyn G Cooke, Leonard F BlackwellAbstract:Estrone Glucuronide conjugates of hen egg white lysozyme were prepared by both the mixed-anhydride and active-ester coupling procedures. Both methods gave good yields of conjugate but the active-ester procedure gave a more diverse range of products consistent with a greater acylating ability. Unreacted lysozyme which was present in all cases was removed by a combination of cation-exchange chromatography on a Pharmacia Mono-S column and hydrophobic-interaction chromatography on an Alkyl Superose column. The conjugate families were more hydrophobic than native lysozyme. The chromatographic behaviour of the reaction mixtures on Mono S columns under non-denaturing conditions was complex as a result of hydrophobic effects and only at pH values above 7.0 did the conjugates elute in the order of their overall charges. At pH values below 6.0 the conjugates, although less charged than lysozyme, eluted last on salt gradients. In contrast when denaturing 7 M urea buffers were used the conjugates eluted in the order of their electrostatic charges and reproducible patterns were obtained which served as an excellent analytical system for lysozyme-steroid Glucuronide conjugates. The purified conjugate material from the active-ester reaction gave over 90% inhibition of the lytic activity in the presence of an Estrone Glucuronide antibody. When used in a homogeneous enzyme immunoassay system the levels of urinary Estrone Glucuronide encountered in a normal menstrual cycle were easily measured.
Frank Dudbridge - One of the best experts on this subject based on the ideXlab platform.
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cytochrome p450 allele cyp3a7 1c associates with adverse outcomes in chronic lymphocytic leukemia breast and lung cancer
Cancer Research, 2016Co-Authors: Nichola Johnson, Nick Orr, Paolo De Ieso, Gabriele Migliorini, Peter Broderick, Daniel Catovsky, Athena Matakidou, Timothy Eisen, Christy Goldsmith, Frank DudbridgeAbstract:CYP3A enzymes metabolize endogenous hormones and chemotherapeutic agents used to treat cancer, thereby potentially affecting drug effectiveness. Here, we refined the genetic basis underlying the functional effects of a CYP3A haplotype on urinary Estrone Glucuronide (E1G) levels and tested for an association between CYP3A genotype and outcome in patients with chronic lymphocytic leukemia (CLL), breast, or lung cancers. The most significantly associated SNP was rs45446698, an SNP that tags the CYP3A7*1C allele; this SNP was associated with a 54% decrease in urinary E1G levels. Genotyping this SNP in 1,008 breast cancer, 1,128 lung cancer, and 347 CLL patients, we found that rs45446698 was associated with breast cancer mortality (HR, 1.74; P = 0.03), all-cause mortality in lung cancer patients (HR, 1.43; P = 0.009), and CLL progression (HR, 1.62; P = 0.03). We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (Pinteraction = 0.06). The CYP3A7*1C allele, which results in adult expression of the fetal CYP3A7 gene, is likely to be the functional allele influencing levels of circulating endogenous sex hormones and outcome in these various malignancies. Further studies confirming these associations and determining the mechanism by which CYP3A7*1C influences outcome are required. One possibility is that standard chemotherapy regimens that include CYP3A substrates may not be optimal for the approximately 8% of cancer patients who are CYP3A7*1C carriers.
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genetic variation at cyp3a is associated with age at menarche and breast cancer risk a case control study
Breast Cancer Research, 2014Co-Authors: Nichola Johnson, Frank Dudbridge, Nick Orr, Lorna Gibson, Michael E Jones, Minouk J Schoemaker, Elizabeth Folkerd, Ben P Haynes, John L Hopper, Melissa C SoutheyAbstract:Introduction: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary Estrone Glucuronide levels and a modest reduction in risk of breast cancer in women age = 15 years (ORhet = 0.84, 95% CI 0.75, 0.94; ORhom = 0.81, 95% CI 0.51, 1.30; P-trend = 0.002) but not for those who had their menarche age <= 11 years (ORhet = 1.06, 95% CI 0.95, 1.19, ORhom = 1.07, 95% CI 0.67, 1.72; P-trend = 0.29). Conclusions: To our knowledge rs10235235 is the first single nucleotide polymorphism to be associated with both breast cancer risk and age at menarche consistent with the well-documented association between later age at menarche and a reduction in breast cancer risk. These associations are likely mediated via an effect on circulating hormone levels.