On-tissue chemical derivatization in mass spectrometry imaging.

Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix-assisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on-tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD-MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.

Spatial localization of ß-unsaturated aldehyde markers in murine diabetic kidney tissue by mass spectrometry imaging.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Limitations in current diagnosis and screening methods have sparked a search for more specific and conclusive biomarkers. Hyperglycemic conditions generate a plethora of harmful molecules in circulation and within tissues. Oxidative stress generates reactive α-dicarbonyls and ß-unsaturated hydroxyhexenals, which react with proteins to form advanced glycation end products. Mass spectrometry imaging (MSI) enables the detection and spatial localization of molecules in biological tissue sections. Here, for the first time, the localization and semiquantitative analysis of "reactive aldehydes" (RAs) 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) in the kidney tissues of a diabetic mouse model is presented. Ionization efficiency was enhanced through on-tissue chemical derivatization (OTCD) using Girard's reagent T (GT), forming positively charged hydrazone derivatives. MSI analysis was performed using matrix-assisted laser desorption ionization (MALDI) coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR). RA levels were elevated in diabetic kidney tissues compared to lean controls and localized throughout the kidney sections at a spatial resolution of 100 µm. This was confirmed by liquid extraction surface analysis-MSI (LESA-MSI) and liquid chromatography-mass spectrometry (LC-MS). This method identified ß-unsaturated aldehydes as "potential" biomarkers of DN and demonstrated the capability of OTCD-MSI for detection and localization of poorly ionizable molecules by adapting existing chemical derivatization methods. Untargeted exploratory distribution analysis of some precursor lipids was also assessed using MALDI-FT-ICR-MSI.

Non-photopic and photopic visual cycles differentially regulate immediate, early, and late phases of cone photoreceptor-mediated vision.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

Maternal and child fatty acid desaturase genotype as determinants of cord blood long-chain PUFA (LCPUFA) concentrations in the Seychelles Child Development Study.

Optimal maternal long-chain PUFA (LCPUFA) status is essential for the developing fetus. The fatty acid desaturase (FADS) genes are involved in the endogenous synthesis of LCPUFA. The minor allele of various FADS SNP have been associated with increased maternal concentrations of the precursors linoleic acid (LA) and α-linolenic acid (ALA), and lower concentrations of arachidonic acid (AA) and DHA. There is limited research on the influence of FADS genotype on cord PUFA status. The current study investigated the influence of maternal and child genetic variation in FADS genotype on cord blood PUFA status in a high fish-eating cohort. Cord blood samples (n 1088) collected from the Seychelles Child Development Study (SCDS) Nutrition Cohort 2 (NC2) were analysed for total serum PUFA. Of those with cord PUFA data available, maternal (n 1062) and child (n 916), FADS1 (rs174537 and rs174561), FADS2 (rs174575), and FADS1-FADS2 (rs3834458) were determined. Regression analysis determined that maternal minor allele homozygosity was associated with lower cord blood concentrations of DHA and the sum of EPA + DHA. Lower cord blood AA concentrations were observed in children who were minor allele homozygous for rs3834458 (ß = 0·075; P = 0·037). Children who were minor allele carriers for rs174537, rs174561, rs174575 and rs3834458 had a lower cord blood AA:LA ratio (P < 0·05 for all). Both maternal and child FADS genotype were associated with cord LCPUFA concentrations, and therefore, the influence of FADS genotype was observed despite the high intake of preformed dietary LCPUFA from fish in this population.

The influence of fish consumption on serum n-3 polyunsaturated fatty acid (PUFA) concentrations in women of childbearing age: a randomised controlled trial (the iFish Study).

PURPOSE: Long-chain polyunsaturated fatty acids (LCPUFA) can be synthesised endogenously from linoleic acid (LA) and α-linolenic acid (ALA) in a pathway involving the fatty acid desaturase (FADS) genes. Endogenous synthesis is inefficient; therefore, dietary intake of preformed LCPUFA from their richest source of fish is preferred. This study investigated the effect of fish consumption on PUFA concentrations in women of childbearing age while stratifying by FADS genotype. The influence of fish consumption on lipid profile, and markers of inflammation and oxidative stress was also examined. METHODS: Healthy women (n = 49) provided a buccal swab which was analysed for FADS2 genotype (rs3834458; T/deletion). Participants were stratified according to genotype and randomised to an intervention group to receive either no fish (n = 18), 1 portion (n = 14) or 2 portions (n = 17) (140 g per portion) of fish per week for a period of 8 weeks. Serum PUFA was analysed at baseline and post-intervention. Lipid profile, and markers of inflammation and oxidative stress were also analysed. RESULTS: Participants consuming 2 portions of fish per week had significantly higher concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 PUFA, and a lower n-6:n-3 ratio compared to those in the no fish or 1 portion per week group (all p < 0.05). Fish consumption did not have a significant effect on biomarkers of oxidative stress, inflammation and lipid profile in the current study. CONCLUSION: Consumption of 2 portions of fish per week has beneficial effects on biological n-3 PUFA concentrations in women of childbearing age; however, no effects on oxidative stress, inflammation or lipid profile were observed. This trial was registered at www.clinicaltrials.gov (NCT03765580), registered December 2018.

Effects of vitamin D as a regulator of androgen intracrinology in LNCAP prostate cancer cells.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumor regression, but commonly restricted by the eventual emergence of a more lethal 'castrate resistant' form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids, are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). In this study, we explored the capacity of the active vitamin D hormone to interact and elicit changes upon this prostatic intracrine pathway at a metabolic level. We used androgen dependent LNCaP cells cultured under steroid-depleted conditions and assessed the impact of vitamin D-based compounds upon intracrine pathways that convert exogenously added DHEA to relevant metabolites, through Mass Spectrometry (MS). Changes in relevant metabolism-related gene targets were also assessed. Our findings confirm that exposure to vitamin D based compounds, within LNCaP cells, elicits measurable and significant reduction in the intracrine conversion of DHEA to T, DHT and other intermediate metabolites within the androgenic pathway. The aassessment and validation of the biological model and analytical platforms were performed by pharmacological manipulations of the SRD5α and HSD-17ß enzymes. The data provides further confirmation for how a vitamin D-based regime may be used to counter intracrine mechanisms contributing to the emergence of castrate-resistant tumors.

Spatial Localization and Quantitation of Androgens in Mouse Testis by Mass Spectrometry Imaging.

Androgens are essential for male development and reproductive function. They are transported to their site of action as blood-borne endocrine hormones but can also be produced within tissues to act in intracrine and paracrine fashions. Because of this, circulating concentrations may not accurately reflect the androgenic influence within specific tissue microenvironments. Mass spectrometry imaging permits regional analysis of small molecular species directly from tissue surfaces. However, due to poor ionization and localized ion suppression, steroid hormones are difficult to detect. Here, derivatization with Girard T reagent was used to charge-tag testosterone and 5α-dihydrotestosterone allowing direct detection of these steroids in mouse testes, in both basal and maximally stimulated states, and in rat prostate. Limits of detection were ∼0.1 pg for testosterone. Exemplary detection of endogenous steroids was achieved by matrix-assisted laser desorption ionization and either Fourier transform ion cyclotron resonance detection (at 150 µm spatial resolution) or quadrupole-time-of-flight detection (at 50 µm spatial resolution). Structural confirmation was achieved by collision induced fragmentation following liquid extraction surface analysis and electrospray ionization. This application broadens the scope for derivatization strategies on tissue surfaces to elucidate local endocrine signaling in health and disease.

Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging.

The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in non-renal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these two sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in non-renal tissues (unlike global Cyp27b1-KO), but no expression within kidney. Here, utilizing on-tissue chemical derivatization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in non-renal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate non-renal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and non-renal 1,25(OH)2D3 production in cytokine changes.

Mass spectrometry imaging for dissecting steroid intracrinology within target tissues.

Steroid concentrations within tissues are modulated by intracellular enzymes. Such "steroid intracrinology" influences hormone-dependent cancers and obesity and provides targets for pharmacological inhibition. However, no high resolution methods exist to quantify steroids within target tissues. We developed mass spectrometry imaging (MSI), combining matrix assisted laser desorption ionization with on-tissue derivatization with Girard T and Fourier transform ion cyclotron resonance mass spectrometry, to quantify substrate and product (11-dehydrocorticosterone and corticosterone) of the glucocorticoid-amplifying enzyme 11ß-HSD1. Regional steroid distribution was imaged at 150-200 µm resolution in rat adrenal gland and mouse brain sections and confirmed with collision induced dissociation/liquid extraction surface analysis. In brains of mice with 11ß-HSD1 deficiency or inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and accumulation of the alternative 11ß-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues.

Vitamin D and Bone Health of Older Adults within Care Homes: An Observational Study.

Limited studies have reported vitamin D status and health outcomes in care home residents, a group at risk of vitamin D deficiency. This study investigated serum 25-hydroxyvitamin D (25-OHD) concentrations in older adults within care homes in Northern Ireland (NI) and its association with musculoskeletal health (ultrasound T-score, muscle strength, Timed Up & Go test (TUG)), bone turnover markers (BTMs), and immune function markers. A total of 87 participants were recruited with mean ± SD age 83.2 ± 7.9 years. Mean ± SD serum 25-OHD concentration (n 69) was 49.52 ± 35.58 nmol/L. Vitamin D deficiency (25-OHD <25 nmol/L) was observed in 34.8% (n 24) of participants with 17.4% (n 12) classified as insufficient (25-OHD 25−50 nmol/L) and 47.8% (n 33) as sufficient (25-OHD >50 nmol/L). 25-OHD concentration was not an independent predictor of T-score, muscle strength, TUG, or inflammatory cytokines. After adjusting for covariates, a significant negative association was observed between 25-OHD concentration and the BTMs; osteocalcin (ß = −0.395; p = 0.001), procollagen type 1 N propeptide (P1NP) (ß = −0.320; p = 0.012), and C-terminal telopeptide of type 1 collagen (CTX) (ß = −0.377; p = 0.003). Higher 25-OHD concentration was positively associated with use of vitamin D ± calcium supplementation (ß = 0.610; p < 0.001). Vitamin D deficiency and insufficiency were highly prevalent in this sample of care home residents in NI. Higher 25-OHD concentration was associated with greater supplement use and with reduced bone turnover, which in this population is linked with reduced bone loss. These findings emphasize the need for a mandatory vitamin D ± calcium supplementation policy specific for care home residents.

Vitamin D Status and Health Outcomes in School Children in Northern Ireland: Year One Results from the D-VinCHI Study.

(1) Background: Vitamin D status has never been investigated in children in Northern Ireland (UK). (2) Methods: Children (4-11 years) (n = 47) were recruited from November 2019 to March 2020 onto the cross-sectional study. Anthropometry was assessed. Plasma 25-hydroxyvitamin D (25(OH)D) was analysed. Vitamin D intake, parental knowledge and perceptions, participant habits, physical activity and sedentary behaviour were established via questionnaire. Muscle strength was assessed via isometric grip strength dynamometry and balance via dominant single-leg and tandem stance. Parathyroid hormone, bone turnover markers (OC, CTX and P1NP), glycated haemoglobin and inflammatory markers (CRP, IFN-γ, IL-10, IL-12p70, IL-13, IL-1ß, IL-2, IL-4, IL-6, IL-8 and TNF-α) were analysed. (3) Results: Mean (SD) 25(OH)D was 49.17 (17.04) nmol/L (n = 47); 44.7% of the children were vitamin D sufficient (25(OH)D >50 nmol/L), 48.9% were insufficient (25-50 nmol/L) and 6.4% were deficient (<25 nmol/L). 25(OH)D was positively correlated with vitamin D intake (µg/day) (p = 0.012, r = 0.374), spring/summer outdoor hours (p = 0.006, r = 0.402) and dominant grip strength (kg) (p = 0.044, r = 0.317). Vitamin D sufficient participants had higher dietary vitamin D intake (µg/day) (p = 0.021), supplement intake (µg/day) (p = 0.028) and spring/summer outdoor hours (p = 0.015). (4) Conclusion: Over half of the children were vitamin D deficient or insufficient. Wintertime supplementation, the consumption of vitamin D rich foods and spring/summer outdoor activities should be encouraged to minimise the risk of vitamin D inadequacy.

Derivatization with 2-hydrazino-1-methylpyridine enhances sensitivity of analysis of 5α-dihydrotestosterone in human plasma by liquid chromatography tandem mass spectrometry.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.

Assessing the Oxidative Degradation of N-Methylpyrrolidone (NMP) in Microelectronic Fabrication Processes by Using a Multiplatform Analytical Approach.

During the construction of recording head devices, corrosion of metal features and subsequent deposition of corrosion by-products have been observed. Previous studies have determined that the use of N-methylpyrrolidone (NMP) may be a contributing factor. In this study, we report the use of a novel multiplatform analytical approach comprising of pH, liquid chromatography/UV detection (LC/UV), inductively coupled plasma optical emission spectroscopy (ICP-OES), and LC/mass spectrometry (LC/MS) to demonstrate that reaction conditions mimicking those of general photoresist removal processes can invoke the oxidation of NMP during the photolithography lift-off process. For the first time, we have confirmed that the oxidation of NMP lowers the pH, facilitating the dissolution of transition metals deposited on wafer substrates during post-mask and pre-lift-off processes in microelectronic fabrication. This negatively impacts upon the performance of the microelectronic device. Furthermore, it was shown that, by performing the process in an inert atmosphere, the oxidation of NMP was suppressed and the pH was stabilized, suggesting an affordable modification of the photolithography lift-off stage to enhance the quality of recording heads. This novel study has provided key data that may have a significant impact on current and future fabrication process design, optimization, and control. Results here suggest the inclusion of pH as a key process input variable (KPIV) during the design of new photoresist removal processes.

Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging.

Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods.

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.

Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry.

Estrogens circulate at concentrations less than 20pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the "reagent" group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought. Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify "FMP" derivatives of estrogens, following LC separation. Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2pg on-column and the method was linear from 1-400pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24h at 10°C (7-9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5mL) 135-473, 193-722pmol/L; male plasma (1mL) 25-111, 60-180pmol/L and post-menopausal female plasma (2mL), 22-78, 29-50pmol/L. Thus FMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.

Measurement of tamsulosin in human serum by liquid chromatography-tandem mass spectrometry.

A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100µL) via liquid-liquid extraction with methyl tert-butyl ether (2mL) following dilution with 0.1M ammonium hydroxide (100µL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis(®) Express C18 (100mm×3mm, 2.7µm) column using a gradient elution with mobile phases methanol and 2mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409→228 and d9-finasteride m/z 382→318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10ppm). The limit of quantitation was 0.2ng/mL, and the method was validated in the linear range 0.2-50ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia.

11ß-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo.

11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7ß-hydroxy- and 7-keto-cholesterol (7ßOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11ßHSD1 to the balance of circulating levels of 7KC and 7ßOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7ßOHC in mice were 91.3±22.3 and 22.6±5.7 nM respectively, increasing to 1240±220 and 406±39 nM in ApoE(-/-) mice receiving atherogenic western diet. Disruption of 11ßHSD1 in mice increased (p<0.05) the 7KC/7ßOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7ßOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11ßHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7ßOHC was a competitive inhibitor of oxidation of corticosterone (Ki=0.9 µM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11ßHSD1 influenced the abundance and balance of circulating and tissue levels of 7ßOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors.