Search

Controlled-release formulations, in the form of micro- or nanoparticles, are increasingly attractive to the pharmaceutical industry for drug delivery. For respiratory illnesses, controlled-release microparticle formulations provide an opportunity to deliver a higher percentage of an inhaled medicament dose to the lung, thus potentially reducing the therapeutic dose, frequency of dosing, and minimising side-effects. We describe the use of a multimodal approach consisting of MALDI MS imaging, 3D depth profiling TOF-SIMS analysis, and histopathology to monitor the distribution of drug and excipients in sections taken from excised rat lungs following an inhaled administration of drug-laden microparticles. Following a single dose, the administered drug was detected in the lung via both MALDI MS and TOF-SIMS over a range of time points. Both imaging techniques enabled the characterisation of the distribution and retention of drug particles and identified differences in the capabilities of both imaging modalities. Histochemical staining of consecutive sections was used to provide biological context to the findings and will also be discussed in this presentation. We demonstrate how this multimodal approach could be used to help increase our understanding of the use of controlled release microparticles.

BackgroundCurrently the only available therapies for fibrotic Interstitial Lung Disease are administered systemically, often causing significant side effects. Inhaled therapy could avoid these but to date there is no evidence that drug can be effectively delivered to distal, fibrosed lung. We set out to combine mass spectrometry and histopathology with rapid sample acquisition using transbronchial cryobiopsy to determine whether an inhaled drug can be delivered to fibrotic, distal lung parenchyma in participants with Interstitial Lung Disease.MethodsPatients with radiologically and multidisciplinary team confirmed fibrotic Interstitial Lung Disease were eligible for this study. Transbronchial cryobiopsies and endobronchial biopsies were taken from five participants, with Interstitial Lung Disease, within 70 minutes of administration of a single dose of nebulised ipratropium bromide. Thin tissue cryosections were analysed by Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging and correlated with histopathology. The remainder of the cryobiopsies were homogenised and analysed by Liquid Chromatography – tandem Mass Spectrometry. ResultsDrug was detected in proximal and distal lung samples from all participants. Fibrotic regions were identified in research samples of four of the five participants. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry imaging showed co-location of ipratropium with fibrotic regions in samples from three participants.ConclusionsIn this proof of concept study, using mass spectrometry, we demonstrate for the first-time that an inhaled drug can deposit in distal fibrotic lung parenchyma in patients with Interstitial Lung Disease. This suggests that drugs to treat pulmonary fibrosis could potentially be administered by the inhaled route.Trial RegistrationA prospective clinical study approved by London Camden and Kings Cross Research Ethics Committee and registered on clinicaltrials.gov (NCT03136120)

Post-extubation dysphagia is associated with an increased incidence of nosocomial pneumonias, longer hospitalizations, and higher re-intubation rates. The purpose of this study was to determine if it is necessary to delay swallow evaluation for 24 hours post-extubation.A prospective investigation of swallowing was conducted at 1, 4, and 24 hours post-extubation to determine if it is necessary to delay swallow evaluation following intubation. Participants were 202 adults from 5 different intensive care units (ICU).A total of 166 of 202 (82.2%) passed the Yale Swallow Protocol at 1 hour post-extubation, with an additional 11 (177/202; 87.6%) at 4 hours, and 8 more (185/202; 91.6%) at 24 hours. Only intubation duration ≥4 days was significantly associated with nonfunctional swallowing.We found it is not necessary to delay assessment of swallowing in individuals who are post-extubation. Specifically, the majority of patients in our study (82.2%) passed a swallow screening at 1 hour post-extubation.

Rationale Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is routinely employed to monitor the distribution of compounds in tissue sections and generate two-dimensional (2D) images. Whilst informative the images do not represent the distribution of the analyte of interest through the entire organ. The generation of 3D images is an exciting field that can provide a deeper view of the analyte of interest throughout an entire organ.Methods Serial sections of mouse and rat lung tissue were obtained at 120 mu m depth intervals and imaged individually. Homogenate registration markers were incorporated in order to aid the final 3D image construction. Using freely available software packages, the images were stacked together to generate a 3D image that showed the distribution of endogenous species throughout the lungs.Results Preliminary tests were performed on 16 serial tissue sections of mouse lungs. A 3D model showing the distribution of phosphocholine at m/z 184.09 was constructed, which defined the external structure of the lungs and trachea. Later, a second experiment was performed using 24 serial tissue sections of the left lung of a rat. Two molecular markers, identified as [PC (32:1) + K](+) at m/z 770.51 and [PC (36:4) + K](+) at m/z 820.52, were used to generate 3D models of the parenchyma and airways, respectively.Conclusions A straightforward method to generate 3D MALDI-MS images of selected molecules in lung tissue has been presented. Using freely available imaging software, the 3D distributions of molecules related to different anatomical features were determined.

The benefits of Tele-ICU are only achieved with proper implementation. In this article, we discuss Tele-ICU outcomes (including the effect on inter-hospital transfers and financial considerations) and detail how these are achieved. A key aspect of the Tele-ICU concept is acuity scoring and alert systems. These allow the Tele-ICU to efficiently identify patients who require closer attention. Sepsis is presented as an example of critical illness where acuity scoring and alert systems associated with Tele-ICU improve outcomes. Areas requiring further investigation are discussed.

Contemporary trials and meta-analyses have evaluated the role of thrombolytics in hemodynamically stable patients with acute pulmonary embolism (PE) and evidence of right ventricular dysfunction and myocardial injury (intermediate-risk PE). The most important findings of these studies are that thrombolytic therapy may prevent hemodynamic deterioration and even all-cause mortality but increases major (including intracranial and fatal) bleeding. These benefits and harms are finely balanced, with no convincing net benefit from thrombolytic therapy among unselected patients. Among patients with intermediate-high risk PE, additional prognostic factors (e.g., syncope, elevated lactate, concomitant deep vein thrombosis, severe respiratory insufficiency) or subtle hemodynamic changes (e.g. increasing heart rate or persistent downtrend of systolic blood pressure) might alter the risk-benefit assessment in favor of thrombolytic therapy before the development of frank hemodynamic instability.

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) offers a label-free alternative for the screening of biochemical targets in both 1536- and 6144-assay formats, as well as potentially providing increased sensitivity, reproducibility, and the simultaneous detection of multiple assay components within a specified m/z range. Ion suppression effects are one of the principal limitations reported for MS analysis. Within MALDI-MS screening, it has been identified that certain biochemical components incorporated into the assay (e.g., the buffers used to preserve the physiological conditions of the enzyme, salts, and other additives) induce suppression of the analyte ion signals monitored. This poorly understood phenomenon of ion suppression is a key reason the screening community has been reluctant to shift their investigations toward MS methods with reduced sample cleanup. Using acetylcholine as an assay substrate mimic, we have generated robust data to quantify the degree to which the most highly used components (base buffers, additional components, detergents, cell culture media, and other additives) within current screening assays are compatible with MALDI-MS. Here, the most suitable buffers and components, along with their identified optimal concentrations in terms of limiting ion suppression effects, are proposed for use in screening assays measured by MALDI-MS.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters - gamma-aminobutyric acid, dopamine and serotonin - with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

There is an increasing need in the pharmaceutical industry to reduce drug failure at late stage and thus reduce the cost of developing a new medicine. Since most drug targets are intracellular, this requires a better understanding of the drug disposition within a cell. Secondary ion mass spectrometry has been identified as a potentially important technique to do this, as it is label-free and allows imaging in 3D with subcellular resolution and recent studies have shown promise for amiodarone. An important analytical parameter is sensitivity, and we measure this in a bovine liver homogenate reference sample for 20 drugs representing important class types relevant to the pharmaceutical industry. We also measure the sensitivity for pure drug and show, for the first time, that the secondary ion mass spectrometry (SIMS) positive ionization efficiency for small molecules is a simple power-law relationship to the log P value. This discovery will be important for advancing the understanding of the SIMS ionization process in small molecules that has, until now, been elusive. This simple relationship is found to hold true for drug doped in the bovine liver homogenate reference sample, except for fluticasone, nicardipine, and sorafenib which suffer from severe matrix suppression. This relationship provides a simple semiempirical method to determine drug sensitivity for positive secondary ions. Furthermore, we show, on chosen models, how the use of different solvents during sample preparation can affect the ionization of analytes.

Introduction: Systemic side effects of oral treatments for fibrotic ILD might be avoided by inhaled drug delivery. Here we report the first prospective study demonstrating the distal deposition of inhaled drug in ILD patients by combining transbronchial cryobiopsy (TBCB) and MALDI-MS Imaging analysis. Methods: ILD patients requiring histological diagnostic confirmation were eligible for enrolment. Ipratropium Bromide (500mcg) was administered via Phillips Portaneb nebulizer. After clinical TBCB biopsies, research samples (1 to 2 TBCB & up to 3 proximal endobronchial controls), were taken. Samples were analysed on Bruker MALDI TOF/TOF instrument. Positive MS/MS detections were compared to predefined limits of acceptance for an ipratropium standard. Adjacent histology sections were stained to correlate MS/MS detection with anatomy and pathological findings. Results: 7 subjects were enrolled in the study, 5 had research TBCBs samples taken. In 4 subjects ipratropium could be positively detected within TBCB samples. Correlation with histopathology demonstrated drug co-localisation with fibrotic regions in 3 subjects (Fig 1). Conclusion: By combining MALDI MS Imaging & TBCB we show that inhaled medication can be delivered to distal fibrotic lung regions. This paves the way for inhaled drug therapy in fibrotic ILD. GSK funded. NCT03136120

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence\ud in the forensic investigation of serious offences. Because many of the current enhancement\ud methods used by crime scene investigators are presumptive, the visualisation of blood is not always\ud reliable nor does it bear additional information. In the work presented here, two methods employing a\ud shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols\ud employ both an in solution digestion method and a recently proposed procedure involving immobilization\ud of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes).\ud Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate.

Patients with life-threatening pulmonary embolism (PE) offer clinicians a unique opportunity to intervene effectively on the patient's behalf. Hemodynamic status remains the most important short-term prognostic factor for patients with acute PE. Although the evidence is limited, the use of thrombolytic therapy is recommended for patients with acute symptomatic PE and associated hypotension or shock (i.e., high-risk PE) because these patients have a high short-term mortality risk (i.e., >15%) even when receiving anticoagulant treatment. In this setting, the hemodynamic benefits of thrombolytic treatment far outweigh its bleeding risk. For hemodynamically stable patients with PE, the identification of a subgroup of patients with a risk of PE-related complications similar to patients with PE and cardiovascular instability (i.e., intermediate-risk group) may assist with decision making regarding therapy. Given the lack of clear mortality benefit and increased bleeding risk, guidelines do not recommend routine use of systemic thrombolysis for this subgroup of patients. Careful monitoring and rescue fibrinolysis for intermediate-risk PE patients who experience hemodynamic compromise or deterioration while receiving standard anticoagulant therapy can minimize deaths from PE. For patients with life-threatening PE at high risk of bleeding, clinicians might consider the use of low-dose thrombolytic therapy, catheter-directed thrombolysis, or surgical embolectomy, if they have access to the required expertise and resources. The evidence does not support the use of inferior vena cava filters in patients with life-threatening PE unless there is a contraindication to anticoagulation. Since various medical and surgical specialties offer different perspectives and expertise, a multidisciplinary approach to patients with intermediate- and high-risk PE might improve patient outcomes.

Overdiagnosis of venous thromboembolism is associated with increasing numbers of patient complications and health care burden. Multiple clinical tools exist to estimate the probability of pulmonary embolism and deep venous thrombosis. When used with d-dimer testing, these can further stratify venous thromboembolism risk to help inform the use of additional diagnostic testing. Although there are similar tools to estimate bleeding risk, these are not as well-validated and lack reliability.

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual’s fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1140-z) contains supplementary material, which is available to authorized users.

RATIONALE The signal intensity of a given molecule across a tissue section when measured using mass spectrometry imaging (MSI) is prone to changes caused by the molecular heterogeneity across the surface of the tissue. Here we propose a strategy to investigate these effects using electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) on a single high-resolution mass spectrometry (HRMS) platform. METHODS A rat was administered with a single inhaled dose of a compound and sacrificed 1 h after dosing. Sections were prepared from the excised frozen lung and analysed using MALDI, liquid extraction surface analysis (LESA) nano-ESI-MS and nano-ESI liquid chromatography (LC)/MS. The ESI and MALDI ion sources were mounted either side of the ion transfer system of the same Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. RESULTS MALDI MSI clearly demonstrated widespread distribution of the dosed molecule throughout the lung, with the exception of a non-lung section of tissue on the same sample surface. Comparison of the lipid signals across the sample indicated a change in signal between the lung and the adipose tissue present on the same section. Use of ESI and MALDI, with and without an internal standard, supported the evaluation of changes in the signal of the dosed molecule across the tissue section. CONCLUSIONS The results demonstrate the successful application of a dual ion source HRMS system to the systematic evaluation of data from MALDI MSI, used to determine the distribution of an inhaled drug in the lung. The system discussed is of great utility in investigating the effects of ion suppression and evaluating the quantitative and qualitative nature of the MSI data. Copyright © 2014 John Wiley & Sons, Ltd.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease with high mortality and few treatment options. This article reviews the epidemiology of CTEPH and identifies risk factors for its development. The pathobiology and the progression from thromboembolic events to chronically increased right-sided pressures are discussed. The diagnosis and assessment of CTEPH requires several modalities and the role of these is detailed. The pre-operative evaluation assesses peri-operative risk and determines the likelihood of benefit from PTE. Pulmonary thromboendarterectomy (PTE) remains the treatment of choice in appropriate patients. Nonsurgical therapies for CTEPH may provide benefit in patients who cannot be offered surgery.

The purpose of this paper is to describe how HealthPartners health system in Minneapolis, MN, has translated a clinical guideline for obesity among adults into an efficient care delivery practice operations system. Based on a foundation provided by the physician-led Institute of Clinical Systems Improvement (ICSI)-developed Prevention and Management of Obesity for Adults Health Care Guideline, HealthPartners adapted the guideline into an electronic health record-based “Smart Set” that provides frontline physicians with the information, treatment options, and referral steps necessary to care for their patients with obesity. Additional context is provided in terms of insurance coverage and systems-based resources designed to prevent and treat obesity for adults.

Upper extremity deep vein thrombosis (UEDVT) is associated with significant morbidity and mortality. The susceptible populations and risk factors for UEDVT are well-known. The presenting symptoms can be subtle, and therefore objective testing is necessary for diagnosis. The optimal diagnostic strategy has not been determined, and more than one test may be required to exclude the diagnosis. Proper treatment reduces the occurrence of complications, and treatment should include long-term anticoagulation if the patient has no contraindications. This article discusses the risk factors, pathogenesis, diagnosis, complications, and management of UEDVT.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA) with nanoelectrospray ionization mass spectrometry (nESI-MS) have both been successfully employed to determine the degree of percutaneous absorption of three novel nonsteroid glucocorticoid receptor (GR) agonists in porcine ear sections. Historically, the ability of a glucocorticoid to elicit a skin blanching response when applied at low dose in ethanol solution to the forearms of healthy human volunteers has been a reliable predictor of their topical anti-inflammatory activity. While all three nonsteroidal GR agonists under investigation caused a skin blanching effect, the responses did not correlate with in vitro GR agonist potencies and different time courses were also observed for the skin blanching responses. MALDI MSI and LESA with nESI-MS were used to investigate and understand these different responses. The findings of the investigation was that the depth of porcine skin penetration correlates to the degree of skin blanching obtained for the same three compounds in human volunteers.

Progression of an acute asthma attack to a severe asthma exacerbation can occur rapidly and lead to devastating consequences, such as hospitalization and respiratory failure. Physiologic processes leading to severe asthma exacerbation may include inflammation, inappropriate hyperresponsiveness of bronchial cells, and excessive mucus production. Specific treatments for acute severe asthma consist of oxygen, intermittent or continuous bronchodilators, systemic corticosteroids, and magnesium sulfate. Close monitoring of patient progress is critical to identify patients most likely to have a severe course of illness. Prior to discharge, patient education and identification of obstacles impeding successful management and control of asthma must be addressed.

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.

Bradykinin and substance P have been derivatised with cyclic diethylenetriaminepentaacetic anhydride (cDTPA) and subsequently labelled with natural and isotopically enriched Eu(3+). This enabled the detection and relative quantitation of the peptides using element-selective detection by high-performance liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS). Relative quantitation was achieved by differentially labelling two peptide sources, after derivatisation with cDTPA, using natural and enriched (151)Eu respectively. The (151)Eu:(153)Eu isotope ratio was measured and used to calculate the original peptide ratio. The measured ratios came within 5.2% of the known ratio. Derivatisation and chelation reactions were additionally confirmed using LC-ESI-MS.

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The FWHM lateral resolution measured across an intracellular interface in a high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.

Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in

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.

Laser ablation–inductively coupled plasma–mass spectrometry (LA–ICP–MS) has been developed as a new strategy for detection and imaging of β-amyloid protein in immunohistochemical sections from the brains of a transgenic mouse model of Alzheimer’s disease. The distribution of β-amyloid deposits in tissue was based on measurement of Eu- and Ni-coupled antibodies. The laser-based methodologies (spot ablation, single line raster, and two-dimensional imaging) were also used to detect and map trace element distributions and thus provide a novel probe for both elemental and protein data. We also report the combination of laser capture microdissection with LA–ICP–MS as an alternative strategy for microanalysis of immunohistochemical sections.

A simple method for the analysis of peptides based on mass spectrometry has been applied to the study of interactions between human bleomycin hydrolase (hBH) or neprilysin (NEP; EC 3.4.24.11) and peptides Ac-HHQKLVFFAG-NH2, Ac-SEVNLDAEFG-NH2 and Ac-GGVVIATVIG-NH2, three substrates cleaved by α, β and γ-secretase, respectively. These proteases are involved in the catabolism of amyloid protein precursor (APP), a protein implicated in Alzheimer's disease. The results indicate that hBH does not cleave the three substrates tested. Conversely, NEP cleaves Ac-HHQKLVFFAG-NH2 at multiple sites and Ac-SEVNLDAEFG-NH2 at only one position, whereas no cleavage was observed with Ac-GGVVIATVIG-NH2. The fragments generated by cleavage with NEP were clearly identified without any further analysis by tandem mass spectrometry. The implication of the role of NEP in the Alzheimer detoxification process is discussed. This technique provides useful information on peptide cleavage points and could be applied easily to a mixture of peptides for the determination of amino acid sequences preferred by any protease.

A method is described whereby stable isotopic signatures were partially incorporated into both termini of a peptide sequence giving rise to a characteristic cluster of four peaks in the mass spectral analysis. Cleavage of this peptide by a protease between the labeled positions generates two fragments both displaying their own individual signature peaks. The event of protease cleavage of the peptide was monitored by the changes in clusters within the spectrum. We believe that this technique could be used to aid the discovery of new cleavage substrates for proteases. Additionally, the analysis can be automated with dedicated software designed to select and interpret the data since all peaks of interest contain predefined signatures and can be easily distinguished from background noise. Copyright © 2002 John Wiley & Sons, Ltd.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.

Bradykinin is a small peptide that acts mainly as a hormone by activating specific receptors that confer protection against the development of hypertension. The efficacy of bradykinin is influenced by the activities of various kininases present in plasma and blood. In this study, both human and rat plasma were incubated with a labelled form of bradykinin (at 4 and 12.5 µM), that will be referred to as bromobradykinin. The metabolic fate of bromobradykinin was monitored by liquid chromatography coupled to an orthogonal acceleration time-of-flight mass spectrometer (oaTOF). Quantification measurements of the bromine-containing metabolites were performed on-line, via flow splitting, by inductively coupled plasma mass spectrometry (ICPMS). The data obtained highlighted that the mechanism(s) of bradykinin metabolism in human and rat plasma are different, with the metabolism of bradykinin in rat plasma being much more aggressive than that observed in human plasma. In addition to the known bradykinin metabolites, e.g. [1,5], [1,7] from ACE, [1,8] from carboxypeptidase and [2,9] from aminopeptidase activity, we have identified the presence of new bradykinin metabolites in both human and rat plasma. These have been identified as fragment [5], the amino acid phenylalanine, which was present in both the human and rat plasma and the fragments [2,8] and [4,8] in rat plasma. To our knowledge it is the first time that these fragments have been recorded in human and rat plasma. The occurrence of these new fragments provides evidence for the presence of potentially new enzymes and mechanisms of bradykinin metabolism. The method described here provides a powerful technique for monitoring the activity of the many kininases involved in bradykinin metabolism such as ACE (angiotensin I converting enzyme), carboxypeptidase N and aminopeptidase P. In addition, this procedure could be used as a screening assay for selecting and monitoring the actions of inhibitors of the enzymes implicated in bradykinin metabolism directly in plasma or serum. Copyright © 2002 John Wiley & Sons, Ltd.

[reaction: see text] An analytical construct resin, designed to aid the analysis of solid-phase chemistry, has been mixed in a small proportion with a conventional resin. The analytical construct ("reporter resin") contains two orthogonal linkers that allow cleavage of either the synthetic intermediates (at linker 2) or their analytically enhanced derivatives (at linker 1). The convenient and rapid monitoring of each step in the syntheses of representative library compounds was possible using small resin aliquots.

The signal intensity of a given molecule across a tissue section when measured using mass spectrometry imaging (MSI) is prone to changes caused by the molecular heterogeneity across the surface of the tissue. Here we propose a strategy to investigate these effects using electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) on a single high-resolution mass spectrometry (HRMS) platform.A rat was administered with a single inhaled dose of a compound and sacrificed 1 h after dosing. Sections were prepared from the excised frozen lung and analysed using MALDI, liquid extraction surface analysis (LESA) nano-ESI-MS and nano-ESI liquid chromatography (LC)/MS. The ESI and MALDI ion sources were mounted either side of the ion transfer system of the same Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer.MALDI MSI clearly demonstrated widespread distribution of the dosed molecule throughout the lung, with the exception of a non-lung section of tissue on the same sample surface. Comparison of the lipid signals across the sample indicated a change in signal between the lung and the adipose tissue present on the same section. Use of ESI and MALDI, with and without an internal standard, supported the evaluation of changes in the signal of the dosed molecule across the tissue section.The results demonstrate the successful application of a dual ion source HRMS system to the systematic evaluation of data from MALDI MSI, used to determine the distribution of an inhaled drug in the lung. The system discussed is of great utility in investigating the effects of ion suppression and evaluating the quantitative and qualitative nature of the MSI data.

A dual linker analytical construct based on a photolabile carbamate is described. Photochemical cleavage from the solid support can be effected to afford an analytical fragment, containing the substrate, which is sensitised to electrospray mass spectrometry. We believe this simple construct now renders all substrates visible to high throughput mass specroscopic analysis.

The structure of a chloroform-extractable siderophore from the supernatant of a nonpathogenic mycobacteria, Mycobacterium smegmatis, has been determined and it closely resembles the structure of mycobactin, the intracellular siderophore found in all mycobacteria. The difference in structures is that the extracellular siderophore has a family of short carboxylic chains attached to the mycobactin nucleus instead of a long alkyl chain and hence the name 'carboxymycobactin' is proposed to distinguish it from mycobactin itself as well as from the major siderophore that is produced - exochelin MS. © Rapid Science 1998.

Drug interactions involving cyclosporine following transplantation are a challenging issue for the transplant clinician. This is especially true when ketoconazole is the second agent used in conjunction with cyclosporine. Because both agents are metabolized by the cytochrome P-450 IIIA4 enzyme system, cyclosporine levels rise dramatically in the presence of ketoconazole. Many other agents interact with ketoconazole, either by competitive enzyme inhibition in the liver and gastrointestinal tract, or by reducing the absorption of ketoconazole by agents that increase the pH of the gastrointestinal tract. Despite the potential cost savings when using ketoconazole to reduce cyclosporine doses, adverse effects associated with ketoconazole put patients at risk when using this combination. Close monitoring of cyclosporine levels is imperative when adding ketoconazole to cyclosporine, and once the dosage adjustments are complete, the addition of a third drug that interacts with either cyclosporine or ketoconazole could result in an unexpected rejection episode or toxic cyclosporine side effect.

A series of novel extracellular siderophores, termed carboxymycobactins, have been isolated from M. avium as iron complexes and their structures determined. They are the first extracellular siderophores to be characterised from pathogenic mycobacteria and are analogous to their intracellular mycobactin counterparts, except that their R 1 alkyl sidechain is shorter and terminates in a carboxylic acid, resulting in much greater hydrophilicity and explaining their occurrence in the aqueous extracellular medium.

Objective. Many patients with chronic disease use alternative therapies. Our objective was to investigate complications resulting from the use of Chinese herbal medications containing undeclared prescription drugs, and to analyze these pills. Methods. Medical records of 5 patients with complications were reviewed. Pills from symptomatic and asymptomatic individuals were analyzed for possible content of undeclared prescription drugs. Results. All pills analyzed contained mefenamic acid and diazepam. Complications related to the presence of these substances included, among others, massive gastrointestinal bleeding. Conclusion. Chinese herbal medications may contain undeclared prescription drugs including nonsteroidal antiinflammatory drugs and benzodiazepines.