Lipid changes within the epidermis of living skin equivalents observed across a time-course by MALDI-MS imaging and profiling

© 2015 Mitchell et al. Abstract Background: Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. Here, its application to the study of the distribution of lipids in sections of reconstructed living skin equivalents during their development and maturation is described. Methods: Living skin equivalent (LSE) samples were obtained at 14 days development, re-suspended in maintenance medium and incubated for 24 h after delivery. The medium was then changed, the LSE re-incubated and samples taken at 4, 6 and 24 h time points. Mass spectra and mass spectral images were recorded from 12 μm sections of the LSE taken at each time point for comparison using matrix assisted laser desorption ionisation mass spectrometry. Results: A large number of lipid species were identified in the LSE via accurate mass-measurement MS and MSMS experiments carried out directly on the tissue sections. MS images acquired at a spatial resolution of 50 μm × 50 μm showed the distribution of identified lipids within the developing LSE and changes in their distribution with time. In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species. Conclusions: MSI can be used to study changes in lipid composition in LSE. Determination of the changes in lipid distribution during the maturation of the LSE will assist in the identification of treatment responses in future investigations

Mass spectrometry imaging of 3D tissue models

A 3D cell culture is an artificially created environment in which cells are permitted to grow/interact with their surroundings in all three dimensions. Derived from 3D cell culture, organoids are generally small‐scale constructs of cells that are fabricated in the laboratory to serve as 3D representations of in vivo tissues and organs. Due to regulatory, economic and societal issues concerning the use of animals in scientific research it seems clear that the use of 3D cell culture and organoids in for example early stage studies of drug efficacy and toxicity will increase. The combination of such 3D tissue models with mass spectrometry imaging provides a label free methodology for the study of drug absorption/penetration, drug efficacy/toxicity and drug biotransformation. In this article, some of the successes achieved to date and challenges to be overcome before this methodology is more widely adopted are discussed

Matrix-assisted ionisation in vacuum mass spectrometry and imaging on a modified quadrupole-quadrupole-time-of-flight mass spectrometer

Matrix-Assisted Ionisation in Vacuum (MAIV) is a new ionisation technique which ionises non-volatile compounds producing electrospray ionisation-like spectra. Its simple, matrix-assisted laser desorption/ionisation-like sample preparation allows for rapid analysis, with no requirement for external energy in the form of a laser or high voltage to produce ions. Ionisation occurs when the matrix (often 3-nitrobenzonitrile) is exposed to sub-ambient pressure. Here, the first use of this revolutionary new ionisation technique to image biological samples is reported. A commercial quadrupole-quadrupole-time-of-flight mass spectrometer was modified to incorporate control of the ion source pressure and a reduced sampling cone orifice diameter. In initial experiments, optimisation of source pressure and matrix composition was carried out to increase the longevity of ion formation. It was noted during these experiments that ion production was only observed when the sample was directly under the sampling cone. Optimisation of sample extraction into the MAIV matrix by the addition of 5 % chloroform enabled MAIV mass spectrometry imaging of lipids in rat brain sections to be carried out in raster imaging mode. Modification of the size and position of the sampling cone improved the selectivity obtainable in these images. Although the quality of these initial images is relatively poor, work is underway to improve the spatial resolution by further modification of the ion source and progress is reported

The investigation of unexpected arsenic compounds observed in routine biological monitoring urinary speciation analysis

This study investigates the identity of two unexpected arsenic species found separately in a number of urine samples sent to the Health and Safety Executive's Health and Safety Laboratory for arsenic speciation (arsenobetaine, AB; arsenite, As3+; arsenate, As5+; monomethylarsonic acid, MMA5+; and dimethylarsinic acid, DMA5+). Micro liquid chromatography coupled to inductively coupled plasma mass spectrometry (μLC-ICP-MS) and electrospray time of flight tandem mass spectrometry (ESI-QqTOF-MS/MS) were used to identify the two arsenic peaks by comparison to several characterized arsenicals: arsenocholine, AC; trimethyl arsine oxide, TMAO; dimethylarsenoacetate, DMAA; dimethylarsenoethanol, DMAE; thio-dimethylarsinate, thio-DMA; thio-dimethylarsenoacetate, thio-DMAA and thio-dimethylarsenoethanol, thio-DMAE. The results from both the ICP-MS and ESI-QqTOF-MS/MS investigations indicate that the unexpected arsenic species termed peak 1 was thio-DMA. While the unexpected arsenic species termed peak 2 has yet to be identified, this investigation shows that it was not AC, TMAO, DMAA, DMAE, thio-DMA, thio-DMAA or thio-DMAE. This study demonstrates the incidence of unexpected arsenic species in both routine and non-routine urine samples from both workers and hospital patients

Quantitative investigation of terbinafine hydrochloride absorption into a living skin equivalent model by MALDI-MSI

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods

The use of hydrazine-based derivatization reagents for improved sensitivity and detection of carbonyl containing compounds using MALDI-MSI

Hydrazine-based derivatization reagents have been used to detect the presence of the carbonyl containing glucocorticoid fluticasone proprionate in rat lung tissue by MALDI-MSI. Such reagents also act as a matrix for analysis by MALDI-MS and have been termed “reactive matrices”. Cryosections of rat lung tissue (12 μm), spotted with a range of concentrations of fluticasone proprionate, were derivatized in situ with 2,4-dinitrophenylhydrazine (DNPH) and 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) by the use of an acoustic reagent spotter. It has been demonstrated that DMNTH gave superior results compared to DNPH and that analysis of samples immediately after application of DMNTH resulted in the detection of the protonated hydrazone derivative ([MD + H]+) of fluticasone propionate at a concentration of 500 ng/μL. It has been further shown that a prolonged reaction time (~48 h) improves the detection limit of the protonated hydrazone derivative to 50 ng/μL and that improvements in sensitivity and limits of detection are obtained when a conventional MALDI matrix CHCA is employed in conjunction with the DNPH/DMNTH reactive matrix

Communication of multi-modal imaging: MRI, MSI, and histology

In an environment where patients are expecting increasingly more information about their condition and care pathway, this tool offers the potential for visual multi-model confirmation of findings. Both normal and abnormal tissue are clearly identified, confirmed by multiple tests, enabling the healthcare professional to easily demonstrate to the patient the effects of treatment

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues

Pre-validation of a MALDI MS proteomics-based method for the reliable detection of blood and blood provenance

Abstract: The reliable identification of blood, as well as the determination of its origin (human or animal) is of great importance in a forensic investigation. Whilst presumptive tests are rapid and deployed in situ, their very nature requires confirmatory tests to be performed remotely. However, only serological tests can determine blood provenance. The present study improves on a previously devised Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS)—proteomics based method for the reliable detection of blood by enabling the determination of blood provenance. The overall protocol was developed to be more specific than presumptive tests and faster/easier than the gold standard liquid chromatography (LC) MS/MS analysis. This is considered a pre-validation study that has investigated stains and fingermarks made in blood, other biofluids and substances that can elicit a false-positive response to colorimetric or presumptive tests, in a blind fashion. Stains and marks were either untreated or enhanced with a range of presumptive tests. Human and animal blood were correctly discriminated from other biofluids and non-biofluid related matrices; animal species determination was also possible within the system investigated. The procedure is compatible with the prior application of presumptive tests. The refined strategy resulting from iterative improvements through a trial and error study of 56 samples was applied to a final set of 13 blind samples. This final study yielded 12/13 correct identifications with the 13th sample being correctly identified as animal blood but with no species attribution. This body of work will contribute towards the validation of MALDI MS based methods and deployment in violent crimes involving bloodshed

Adaptation of the kirkstall QV600 LLI microfluidics system for the study of gastrointestinal absorption by mass spectrometry imaging and LC-MS/MS

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature