A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography–mass spectrometry

Li, Junyi; Oberly, Patrick J.; Poloyac, Samuel M.; Gibbs, Robert G.

doi:10.1016/j.jsbmb.2016.04.013

Cited by 18 publications

(8 citation statements)

References 40 publications

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“…Our UPLC-MS/MS method has been optimized in response to clinical and research needs and thus has evolved over time. It has allowed us to accurately and precisely quantify estrogen and/or progestogen levels in pre-peri-and post-menopausal women (16,(36)(37)(38)(39), men (36), children and young female rats (40). It is also notable that our method is not limited to the sex-steroid hormones reported in this manuscript.…”

Section: Discussionmentioning

confidence: 99%

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

et al. 2019

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Objective: With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC-MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. Study Design: We developed and validated a UPLC-MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. Results: Each UPLC-MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25-50,000, 2.5-2,000 (1-2,000 for estrogens-only analysis) and 5-2,000 pg/ml, respectively. Both the accuracy and precision were below ±15% not only for routine validation (intraday and interday), but for long-term (> two years) assay robustness with external controls, thereby, demonstrating the utility of this method for

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Section: Discussionmentioning

confidence: 99%

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

et al. 2019

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“…Serum levels of 17β-estradiol (E2), testosterone (T) and and rostenedione (AD) were quantified by UPLC-MS/MS as described [29]. Briefly, for E2 analysis, samples were spiked with internal standard 25 μl 2,4,16,16,17-d5-17 beta-estradiol (1 ng/ml in methanol).…”

Section: Methodsmentioning

confidence: 99%

Use of the REVERT® total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states

Kirshner

Gibbs

2018

Molecular and Cellular Endocrinology

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Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, β-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, β-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT total protein stain can be used in Western blot analysis of brain tissue homogenates to control for variations in loading and protein transfer, and provides significant advantages over the use of housekeeping proteins for quantifying changes in the levels of multiple target proteins.

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“…Serum levels of E2 were quantified as recently described [30]. Briefly, samples were spiked with internal standard 25μl 2,4,16,16,17-d5–17 beta-estradiol (1 ng/ml in methanol).…”

Section: Methodsmentioning

confidence: 99%

Comparison of transitional vs surgical menopause on monoamine and amino acid levels in the rat brain

Long

Yao

et al. 2018

Molecular and Cellular Endocrinology

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Loss of ovarian function has important effects on neurotransmitter production and release with corresponding effects on cognitive performance. To date, there has been little direct comparison of the effects of surgical and transitional menopause on neurotransmitter pathways in the brain. In this study, effects on monoamines, monoamine metabolites, and the amino acids tryptophan (TRP) and tyrosine (TYR) were evaluated in adult ovariectomized (OVX) rats and in rats that underwent selective and gradual ovarian follicle depletion by daily injection of 4-vinylcyclohexene-diepoxide (VCD). Tissues from the hippocampus (HPC), frontal cortex (FCX), and striatum (STR) were dissected and analyzed at 1- and 6-weeks following OVX or VCD treatments. Tissues from gonadally intact rats were collected at proestrus and diestrus to represent neurochemical levels during natural states of high and low estrogens. In gonadally intact rats, higher levels of serotonin (5-HT) were detected at proestrus than at diestrus in the FCX. In addition, the ratio of 5-hydroxyindoleacetic acid (5-HIAA)/5HT in the FCX and HPC was lower at proestrus than at diestrus, suggesting an effect on 5-HT turnover in these regions. No other significant differences between proestrus and diestrus were observed. In OVX- and VCD-treated rats, changes were observed which were both brain region- and time point-dependent. In the HPC levels of norepinephrine, 5-HIAA, TRP and TYR were significantly reduced at 1 week, but not 6 weeks, in both OVX and VCD-treated rats relative to proestrus and diestrus. In the FCX, dopamine levels were elevated at 6 weeks after OVX relative to diestrus. A similar trend was observed at 1 week (but not 6 weeks) following VCD treatment. In the STR, norepinephrine levels were elevated at 1 week following OVX, and HVA levels were elevated at 1 week, but not 6 weeks, following VCD treatment, relative to proestrus and diestrus. Collectively, these data provide the first comprehensive analysis comparing the effects of two models of menopause on multiple neuroendocrine endpoints in the brain. These effects likely contribute to effects of surgical and transitional menopause on brain function and cognitive performance that have been reported.

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A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography–mass spectrometry

Cited by 18 publications

References 40 publications

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

Use of the REVERT® total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states

Comparison of transitional vs surgical menopause on monoamine and amino acid levels in the rat brain

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