A novel imaging classification system for the neuroendoscopic treatment of chronic subdural hematoma.

Chronic subdural hematoma (CSDH) is common, especially in patients over 50 years of age, and represents about 10% of all intracranial hematomas. The pathogenesis, diagnosis, and treatment of CSDH are controversial. The purpose of this study was to document the clinical application of a novel imaging classification system for the neuroendoscopic treatment of CSDH. This was a prospective study of sixty patients who underwent neuroendoscopic CSDH treatment beginning in January 2017, with a 6-month follow-up. Hematomas were classified into two types based on imaging features: simple (type I) and complex (type II). Complex type was further subclassified as septated (type II-A), stratified (type II-B), recurrent (type II-C), thin-layer (type II-D), bilateral (type II-E), or mixed (type II-F). Most hematomas were located on the left side. Type II hematomas had fibrous septa and bridging veins in the cavities. Bender classification and Glasgow Outcome Scale (GOS) scores were improved after neuroendoscopic surgery and hematoma thickness was improved significantly in all CSDHs on days 1, 7, and 14 after surgery (all P<0.05). Lung infection, pneumocephalus, and seizures occurred in 17, 12, and 8 patients, respectively. Neither a recurrence of symptoms nor CSDH occurred based on the analysis of images. All patients recovered well and none suffered additional bleeding, recurrence, or intracranial infection. This novel imaging classification for CSDH provides a useful guide for the successful neuroendoscopic treatment of CSDH.

High expression of TXNDC11 indicated unfavorable prognosis of glioma.

BACKGROUND: Thioredoxin domain containing 11 (TXNDC11) has been implicated in numerous cancers. Nevertheless, the function of TXNDC11 in glioma is not well described. This study aimed to assess clinical significance of TXNDC11 in glioma based on bioinformatics analysis and immunohistochemical (IHC) staining. METHODS: GEPIA2, The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases were employed to detect the levels of TXNDC11 transcript in glioma. Gene expression profiles and data from the methylation chip with clinical details from TCGA and Chinese Glioma Genome Atlas (CGGA) of glioma samples were examined. The methylation of TXNDC11 in glioma was evaluated by 450K methylation chip data analysis. The pathways involved in TXNDC11 expression were screened by gene set enrichment analysis (GSEA). The correlation between TXNDC11 and immune cells was analyzed. Protein level of TXNDC11 was detected by IHC staining in glioma specimens. RESULTS: TXNDC11 was highly expressed in glioma, and high TXNDC11 expression was associated with poor overall survival (OS) and worse clinical prognostic variables. The methylation of cg04399632 was statistically different between glioma samples and normal samples, and was negatively correlated with TXNDC11 expression in glioma patients. Survival analysis demonstrated a poorer prognosis in glioma patients with cg04399632 hypomethylation. TXNDC11-high phenotype was associated with certain immune-related pathways and other signaling pathways in glioma. The expression of TXNDC11 was correlated positively with M2 macrophage infiltration and negatively with M0 and M1 macrophage infiltration. IHC staining confirmed that TXNDC11 expression increased in higher-grade glioma. CONCLUSIONS: High expression of TXNDC11 may predict unfavorable prognosis of glioma patients.

A highly sensitive LC-MS/MS method for the simultaneous quantitation of serum androstane-3α, 17ß-diol and androstane-3ß, 17ß-diol in post-menopausal women.

Sensitive and accurate measurement of androstane-3ß,17ß-diol and androstane-3α,17ß-diol in the circulation is important for clinical research and accurate clinical diagnosis. This report describes a highly sensitive, specific, precise and reliable assay for the simultaneous accurate measurement of serum androstane-3α,17ß-diol and androstane-3ß,17ß-diol in postmenopausal women. The LLOQ of 1 pg/mL has been achieved with nicotinic acid derivatization, which is superior to picolinic acid by a factor of 5 to 10 in terms of signal to noise ratio. The difference is attributed to the higher acidity of picolinic acid which forms a more stable intermediate, thus decreasing derivatization efficiency. Potential interference from androstane-3α, 17α-diol, androstane-3ß, 17α-diol, and 5-androstenediol has been well separated from the two target diols. The high level of specificity has been determined by well-developed chromatography and ion ratio monitoring. A good linearity in the range of 1 pg/mL to 200 pg/mL (0.03 pg to 6 pg on column) was obtained for both compounds at R > 0.998. The bias and coefficients of variation of all the QC levels are within the range of 10% while the recovery in both charcoal-stripped and unstripped human serum is around 85%. The matrix effect was evaluated and the results well met the acceptance criteria according to the guidelines of bioanalytical method development and validation. Using this newly developed method, the concentrations of both androstane-3α,17ß diol and androstane-3ß,17ß diol were measured in normal postmenopausal serum, where the concentrations range from 2 pg/mL to 32 pg/mL for androstane-3α,17ß diol and from 1 pg/mL to 10 pg/mL for androstane-3ß,17ß diol, respectively.

Concentration range of serum sex steroids in normal postmenopausal women and those with diagnosis of vulvovaginal atrophy.

OBJECTIVE: The aim of the study was to determine the range of serum sex-related steroids in normal postmenopausal women and in women of the same age with a diagnosis of vulvovaginal atrophy (VVA). METHODS: Validated mass spectrometry-based assays coupled to gas or liquid chromatography were used over a 10-year period for steroid measurements. Serum samples were obtained in up to 1,512 women aged 55 to 65 years. RESULTS: Serum estrone sulfate (E1S) and androsterone glucuronide (ADT-G), the main metabolites of estrogens and androgens, respectively, were 16.9% (P = 0.005) and 16.1% (P = 0.001) higher in women not diagnosed with moderate/severe VVA than those diagnosed with VVA. Serum estrone (E1) was 14.5% (P < 0.0001) higher in women with no diagnosis of VVA, whereas the other steroids did not show meaningful differences. The limited biological significance of serum estradiol (E2) and testosterone is supported by the lack of statistical significance in the serum concentrations of these two steroids between the two groups. Most importantly, for the women without a diagnosis of VVA, the normal upper limit (95 centile) of serum E2 was 9.15 pg/mL (n = 364) and 10.7 pg/mL (n = 67) for a weighted average of 9.99 pg E2/mL. A limit of 10 pg E2/mL has recently been found by two other laboratories. When comparing 50- to 59-year-old and 70- to 79-year-old women, serum E2, E1S, ADT-G, and DHEA were, respectively, 24.4%, 22.6%, 27.0%, and 85.9% higher in the younger group. CONCLUSIONS: Somewhat higher values, namely, 16.9% and 16.1%, are observed in the serum concentrations of the estrogen (E1S) and androgen (ADT-G) metabolites in normal compared with women with a diagnosis of VVA. Such data indicating a lower estrogenic and androgenic global exposure in women diagnosed with VVA offers an opportunity for the local intravaginal administration of DHEA to replace the deficiency in endogenous DHEA.

Evaluation of a one-step sample preparation protocol for analysis of total serum testosterone by LC-MS/MS.

In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC-MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

Impact of sample extraction on the accurate measurement of progesterone in human serum by liquid chromatography tandem mass spectrometry.

In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315>97 & 315>109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315>97-315>109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315>109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1×3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70%∼80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1×3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5-2000pg/mL, a good linearity is observed with R>0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74∼91.7pg/mL, which is well below the reported concentrations of 314 pg/mL∼942pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mL∼18.6ng/mL.

A sensitive and accurate LC-MS/MS assay with the derivatization of 1-Amino-4-methylpiperazine applied to serum allopregnanolone, pregnenolone and androsterone in pre- and postmenopausal women.

The concentrations of allopregnanolone (Allopreg), pregnenolone (Preg) and androsterone (ADT) are very low in the circulation, especially in postmenopausal women, resulting in a considerable challenge for their accurate measurements in serum or plasma. In this report, a sensitive and reliable LC-MS/MS assay method has been developed using a simple sample preparation and the 1-Amino-4-methylpiperazine (AMP) derivatization procedure. A 5pg/ml (0.1pg on column) of low limit of quantitation has been achieved for Allopreg, Preg and ADT, with a sensitivity comparable to data obtained with the commercial reagent. The major benefit of this reagent is to limit the matrix effect since the excess amount of reagent can be removed during the reaction. Multiple reaction monitoring (MRM) from the derivatization of AMP not only increases the detection of these compounds but also provides a good resolution for Allopreg, Preg and ADT from interferences, especially for Allopreg from its isomers. Within the calibration range of 5pg/ml to 2000pg/ml, a good linearity was obtained with R>0.99 where the weighing factor is 1/X. Bias and coefficients of variance are within 15% for all QC levels. The matrix effect has been evaluated, well meeting the acceptance criteria according to the FDA guidelines. With this method, the concentrations of Allopreg, Preg and ADT in postmenopausal serum are in the range of 6.4-53.6pg/ml, 16.2-68.0pg/ml and 23.9-114.0pg/ml, respectively, while the ranges in premenopausal serum are 8.2-701.5pg/ml, 31.2-135.2pg/ml and 47.8-310.0pg/ml, respectively.

Precise and accurate assay of pregnenolone and five other neurosteroids in monkey brain tissue by LC-MS/MS.

A series of steroids present in the brain have been named "neurosteroids" following the possibility of their role in the central nervous system impairments such as anxiety disorders, depression, premenstrual dysphoric disorder (PMDD), addiction, or even neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Study of their potential role requires a sensitive and accurate assay of their concentration in the monkey brain, the closest model to the human. We have thus developed a robust, precise and accurate liquid chromatography-tandem mass spectrometry method for the assay of pregnenolone, pregnanolone, epipregnanolone, allopregnanolone, epiallopregnanolone, and androsterone in the cynomolgus monkey brain. The extraction method includes a thorough sample cleanup using protein precipitation and phospholipid removal, followed by hexane liquid-liquid extraction and a Girard T ketone-specific derivatization. This method opens the possibility of investigating the potential implication of these six steroids in the most suitable animal model for neurosteroid-related research.

Serum steroid concentrations remain within normal postmenopausal values in women receiving daily 6.5mg intravaginal prasterone for 12 weeks.

This study integrates all data obtained in women aged 40-80years enrolled with moderate to severe symptoms of vulvovaginal atrophy (VVA) who received daily intravaginal administration of 0.50% (6.5mg) dehydroepiandrosterone (DHEA; prasterone) for 12weeks (n=723; ITT-S population) as compared with placebo (n=266; ITT-S population). To this end, serum steroid levels (DHEA, DHEA-sulfate (DHEA-S), androst-5-ene-3ß, 17ß-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17ß-diol 17-glucuronide (3α-diol-17G)) were measured at Day 1 and Week 12 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following validation performed according to the FDA guidelines [1-6]. In agreement with the mechanisms of intracrinology where DHEA is exclusively transformed intracellularly into active sex steroids which act and are inactivated locally before being released as glucuronided or sulfated metabolites for elimination by the kidneys and liver, all sex steroids remained well within normal postmenopausal values following administration of intravaginal DHEA. Serum estradiol, the most relevant sex steroid, was measured after 12weeks of treatment at 3.36pg/ml (cITT-S population) or 19% below the normal postmenopausal value of 4.17pg/ml. On the other hand, serum E1-S, the best recognized marker of global estrogenic activity, shows an average value of 209pg/ml at 12 weeks compared to 220pg/ml in normal postmenopausal women. Moreover, serum ADT-G, the main metabolite of androgens, also remains well within normal postmenopausal values. The present data shows that a low daily intravaginal dose (6.5mg) of DHEA (prasterone) which is efficacious on the symptoms and signs of VVA, permits to achieve the desired local efficacy without systemic exposure, in agreement with the stringent mechanisms of menopause established after 500 million years of evolution where each cell in each tissue is the master of its sex steroid exposure.

The conversion of 16ß hydroxyldehydroepiandrosterone in human serum.

The circulating levels of 16ß hydroxydehydroepiandrosterone (16ß OH-DHEA) are at the limit of detection (less than 10 pg/mL), unlike the serum concentrations of 16α-hydroxydehydroepiandrosterone (16α OH-DHEA, 10-300 pg/mL) in premenopausal, postmenopausal and male serum. A major reason could be the rapid conversion of 16ß OH-DHEA to 5-androstene-3beta, 17beta-diol 16 one (3ß, 17ß-diol 16-oxo) in serum due to the stereospecific structure of 16ß OH-DHEA. In ultrapure H2O, there is no apparent conversion observed while 16ß OH-DHEA (10 ng/mL) spiked in stripped or unstripped serum is quickly converted to 3ß, 17ß-diol 16-oxo at room temperature. During this conversion, a further converted product was observed with a difference in molecular weight of 16 Da from that of 16ß OH-DHEA and 3ß, 17ß-diol 16-oxo, which could be their hydroxylation product, i.e. triol-ketone. Under basic conditions, further conversion occurs. The present data can explain the practically undetectable concentration of serum 16ß OH-DHEA while 3ß, 17ß-diol 16-oxo is at the level of less than 50 pg/mL. Serum concentrations of (0.0-9.9 pg/mL for 16ß OH-DHEA, 8.9-50.7 pg/mL for 3ß, 17ß-diol 16-oxo and 10.0-285.0 pg/mL for 16α OH-DHEA are measured in sera of premenopausal, postmenopausal women and men over 50 years of age.

A validated LC-MS/MS method for the sensitive quantitation of serum 7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone using a novel derivatization reagent.

7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7ß OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC-MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10 pg/mL (0.2 pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10-2000 pg/mL, a good linearity was obtained with R>0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze-thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7ß OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7ß OH-DHEA in both types of sera.

Serum steroids remain within the same normal postmenopausal values during 12-month intravaginal 0.50% DHEA.

OBJECTIVE: Analyze the serum levels of DHEA (prasterone) and its metabolites after daily intravaginal 0.50% (6.5 mg) DHEA in postmenopausal women with vulvovaginal atrophy (VVA). METHODS: Serum samples were obtained at baseline and after 12, 26 and 52 weeks of treatment. The serum levels of DHEA, DHEA-sulfate (DHEA-S), androstene-3ß, 17ß-diol (5-diol), androstenedione (4-dione), testosterone, dihydrotestosterone (DHT), estrone (E1), estradiol (E2), E1-sulfate (E1-S), androsterone glucuronide (ADT-G) and androstane-3α,17ß-diol 17-glucuronide (3α-diol-17G) were measured by validated liquid chromatography-tandem mass spectrometry. RESULTS: A total of 435 women were exposed for 52 weeks. All serum steroids remained within normal values with no significant differences between lengths of treatment. For the most relevant estrogen-related compounds, namely E1, E2, and E1-S, a reliable marker of total estrogen exposure, the values in the DHEA-treated group at 52 weeks were -3.4%, -9.1% and +1.8%, respectively, compared to the normal postmenopausal values, thus clearly confirming the absence of significant systemic estrogen exposure. CONCLUSION: While confirming that all serum sex steroids originating exclusively from DHEA after menopause are maintained within the normal postmenopausal values, the present data show that the dose of intravaginal DHEA used is free from systemic exposure with no detectable change in metabolism up to 52 weeks of treatment.

Serum levels of sex steroids and metabolites following 12 weeks of intravaginal 0.50% DHEA administration.

The objective of the present phase III, placebo-controlled, double-blind, prospective and randomized study was to confirm the efficacy of daily intravaginal administration of 0.50% dehydroepiandrosterone (DHEA; prasterone) ovules for 12 weeks on moderate to severe dyspareunia (or pain at sexual activity) as most bothersome symptom of vulvovaginal atrophy (VVA) while having serum steroid concentrations within normal postmenopausal values. To this end, serum levels of DHEA, DHEA-sulfate (DHEA-S), Androst-5-ene-diol-3ß, 17ß-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17ß-diol 17-glucuronide (3α-diol-17G) were measured by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). In agreement with the mechanisms of intracrinology, all serum sex steroids and metabolites concentrations after 12 weeks of daily intravaginal administration of 0.50% DHEA remain well within the limits of normal postmenopausal women. More specifically, the 12-week serum E2 concentration was measured at 22% below the average normal postmenopausal value (3.26 versus 4.17 pg/ml), thus eliminating any fear of E2 exposure outside the vagina. In addition, serum E1-S, a particularly reliable indicator of global estrogenic activity, shows serum levels practically superimposable to the value observed in normal postmenopausal women (219 versus 220 pg/ml). Similarly, serum ADT-G, the major metabolite of androgens, remains within normal postmenopausal values. The present data confirm the intracellular transformation of DHEA in the vagina resulting in local efficacy without any systemic exposure to sex steroids, observations which are in agreement with the physiological mechanisms of menopause.

Accurate and sensitive liquid chromatography/tandem mass spectrometry simultaneous assay of seven steroids in monkey brain.

BACKGROUND: Following its secretion mainly by the adrenal glands, dehydroepiandrosterone (DHEA) acts primarily in the cells/tissues which express the enzymes catalyzing its intracellular conversion into sex steroids by the mechanisms of intracrinology. Although reliable assays of endogenous serum steroids are now available using mass spectrometry (MS)-based technology, sample preparation from tissue matrices remains a challenge. This is especially the case with high lipid-containing tissues such as the brain. With the combination of a UPLC system with a sensitive tandem MS, it is now possible to measure endogenous unconjugated steroids in monkey brain tissue. METHODS: A Shimadzu UPLC LC-30AD system coupled to a tandem MS AB Sciex Qtrap 6500 system was used. RESULTS: The lower limits of quantifications are achieved at 250 pg/mL for DHEA, 200 pg/mL for 5-androstenediol (5-diol), 12 pg/mL for androstenedione (4-dione), 50 pg/mL for testosterone (Testo), 10 pg/mL for dihydrotestosterone (DHT), 4 pg/mL for estrone (E1) and 1 pg/mL for estradiol (E2). The linearity and accuracy of quality controls (QCs) and endogenous quality controls (EndoQCs) are according to the guidelines of the regulatory agencies for all seven compounds. CONCLUSION: We describe a highly sensitive, specific and robust LC-MS/MS method for the simultaneous measurement of seven unconjugated steroids in monkey brain tissue. The single and small amount of sample required using a relatively simple preparation method should be useful for steroid assays in various peripheral tissues and thus help analysis of the role of locally-made sex steroids in the regulation of specific physiological functions.

A rapid and sensitive UPLC-MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women.

Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200µL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.