Compounds In Biological Tissues Research Articles

4-Nitrocatechol as a novel matrix for low-molecular-weight compounds in situ detection and imaging in biological tissues by MALDI-MSI

Low-molecular-weight (LMW) compounds are ubiquitous in living organisms and play essential roles in biological processes. The direct analysis of LMW compounds in biological tissues by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) could provide a more comprehensive understanding of their essential functions. Here, we evaluated 4-nitrocatechol (4-NC) as a novel positive-ion matrix for enhancing in situ detection and imaging of LMW compounds from the rat liver, brain, and germinating Chinese-yew seed by MALDI-MS. Our results showed that the 4-NC possessed remarkable features, including strong ultraviolet absorption, uniform matrix crystal, excellent chemical stability, and fewer matrix-related background peaks. The use of 4-NC led to the successful detection of 232, 218, and 193 LMW compounds from the three abovementioned tissue sections, respectively. Also, the use of 4-NC improved the imaging quality of LMW compounds in tissue sections through MALDI-MSI and has the potential as a matrix for MALDI tissue imaging of LMW compounds.

Effects of dopants in the imaging of mouse brain by desorption electrospray ionization/post-photoionization mass spectrometry.

Desorption electrospray ionization/post-photoionization (DESI/PI) is a newly developed ionization method by the combination of DESI and post-photoionization for the simultaneous imaging of polar and nonpolar compounds in biological tissues. Dopants are of great importance in DESI/PI for the enhancement of signal intensities through ion-molecule reactions. In this work, to evaluate the performance of dopants in DESI/PI, an efficient homogenate model was developed, and four kinds of dopants (toluene, chlorobenzene, bromobenzene, and anisole) were tested using homogenate of mouse brain tissue as target sample. The influences of the dopants on the signal enhancements of different compounds were explained reasonably by the ionization mechanism. Then, the dopants with their optimum volume contents were applied to the mass spectrometry imaging (MSI). For a comprehensive imaging of various compounds with different polarities, methanol/toluene/formic acid (7:3:0.1) was chosen as the best choice. Finally, the stronger quantitative ability of DESI/PI with toluene as dopant for a few compounds in mouse brain tissue was demonstrated.

3,4-Dimethoxycinnamic Acid as a Novel Matrix for Enhanced In Situ Detection and Imaging of Low-Molecular-Weight Compounds in Biological Tissues by MALDI-MSI.

Low-molecular-weight (low-MW) compounds have many essential functions in biological processes, and the molecular imaging of as many low-MW compounds as possible is critical for understanding complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging molecular-imaging technology that enables determination of the spatial distributions and the relative abundances of diverse endogenous compounds in tissues. New matrices suitable for the imaging of low-MW compounds by MALDI-MSI are important for the technological advancement of tissue imaging. In this study, 3,4-dimethoxycinnamic acid (DMCA) was evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI because of its strong ultraviolet absorption, low matrix-ion related interferences below m/ z 500, and high ionization efficiency for the analysis of low-MW compounds. DMCA was successfully used for improved in situ detection of low-molecular-weight metabolites ( m/ z < 500) and lipids in rat liver, rat brain, and germinating Chinese-yew seed tissue sections. The use of DMCA led to the successful in situ detection of 303, 200, and 248 low-MW compound ion signals from these three tissues, respectively. Both MALDI-MS/MS and LC-MS/MS were used to identify these ion signals, leading to the identification of 115 low-MW compounds from rat liver (including 53 lipids, 29 oligopeptides, and 33 metabolites), 130 low-MW compounds from rat brain (including 104 lipids, 5 oligopeptides, and 21 metabolites), and 111 low-MW compounds from germinating Chinese-yew seeds (including 77 lipids, 22 oligopeptides, 8 flavonoids, and 4 alkaloids). A larger number of low-MW compounds could be detected and imaged when DMCA was used as the MALDI matrix than with other commonly used MALDI matrices such as 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2-mercaptobenzothiazole, graphene oxide, and silver nanoparticles. Our work provides a new and powerful matrix for enhanced MALDI-MS profiling of low-MW compounds in both animal and plant tissues.

In vivo imaging-guided microsurgery based on femtosecond laser produced new fluorescent compounds in biological tissues.

Femtosecond laser microsurgery has become an advanced method for clinical procedures and biological research. The tissue treated by femtosecond laser can become highly fluorescent, indicating the formation of new fluorescent compounds that can naturally label the treated tissue site. We systematically characterized the fluorescence signals produced by femtosecond laser ablation in biological tissues in vivo. Our findings showed that they possess unique fluorescence properties and can be clearly differentiated from endogenous signals and major fluorescent proteins. We further demonstrated that the new fluorescent compounds can be used as in vivo labelling agent for biological imaging and guided laser microsurgery.

High resolution metabolite imaging in the hippocampus following neonatal exposure to the environmental toxin BMAA using ToF-SIMS.

The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is suggested to be linked with neurodegenerative disease. In a rat model, neonatal exposure to BMAA induced selective uptake in the hippocampus and caused cell loss, mineralization and astrogliosis as well as learning and memory impairments in adulthood. Moreover, neonatal exposure resulted in increased protein ubiquitination in the cornus ammonis 1 (CA1) region of the adult hippocampus indicating that BMAA may induce protein aggregation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) based imaging is a powerful technology for spatial profiling of small molecular weight compounds in biological tissues with high chemical specificity and high spatial resolution. The aim of this study was to characterize neurochemical changes in the hippocampus of six month-old rats treated neonatally (postnatal days 9-10) with BMAA. Multivariate data analysis of whole section ToF-SIMS scans was performed to delineate anatomical regions of interest based on their chemical distribution pattern. Further analysis of spectral data obtained from the outlined anatomical regions, including CA1 and dentate gyrus (DG) revealed BMAA-induced long-term changes. Increased levels of phospholipids and protein fragments in the histopathologically altered CA1 region as well as phosphate depletion in the DG were observed. Moreover, high resolution SIMS imaging revealed a specific localization of phosphatidylcholine lipids, protein signals and potassium in the histopathologically altered CA1. These findings demonstrate that ToF-SIMS based imaging is a powerful approach for probing biochemical changes in situ and might serve as promising technique for investigating neurotoxin-induced brain pathology.

Biomaterial imaging with MeV-energy heavy ion beams

The spatial distribution of several chemical compounds in biological tissues and cells can be obtained with mass spectrometry imaging (MSI). In conventional secondary ion mass spectrometry (SIMS) with keV-energy ion beams, elastic collisions occur between projectiles and atoms of constituent molecules. The collisions produce fragments, making the acquisition of molecular information difficult. In contrast, ion beams with MeV-energy excite near-surface electrons and enhance the ionization of high-mass molecules; hence, SIMS spectra of fragment-suppressed ionized molecules can be obtained with MeV-SIMS. To compare between MeV and conventional SIMS, we used the two methods based on MeV and Bi3-keV ions, respectively, to obtain molecular images of rat cerebellum. Conventional SIMS images of m/z 184 were clearly observed, but with the Bi3 ion, the distribution of the molecule with m/z 772.5 could be observed with much difficulty. This effect was attributed to the low secondary ion yields and we could not get many signal counts with keV-energy beam. On the other hand, intact molecular ion distributions of lipids were clearly observed with MeV-SIMS, although the mass of all lipid molecules was higher than 500Da. The peaks of intact molecular ions in MeV-SIMS spectra allowed us to assign the mass. The high secondary ion sensitivity with MeV-energy heavy ions is very useful in biomaterial analysis.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury, butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt4 and NaBPr4 was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing (201)Hg-enriched methylmercury (MeHg), (199)Hg-enriched inorganic mercury (iHg), (119)Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade (116)Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12% for all analytes, and LOQs of 0.06-1.45 pg for GC-MS and 0.02-0.27 pg for GC-ICP/MS.

Mass spectrometry imaging: linking molecule profiles to tissue spatial distribution

Advances in Bioanalytical Mass Spectrometry: 5th Symposium of the Göttingen Proteomics ForumMass Spectrometry with Spatial Resolution: MALDI-Imaging and Laser Capture MicroscopyGöttingen, Germany, 22 November 2012MALDI mass spectrometry imaging (MSI) combines the speed and molecular specificity of MALDI-MS detection with information on spatial organization. In the last years, MSI found large application in proteomics research for determining the spatial distribution of compounds in biological tissues and started to draw increasing interest in clinical research. To shed light on the new developments in the field of MSI, the Göttingen Proteomics Forum organized a symposium that was held in Göttingen as part of the series of regular symposia organized by the members of the Göttingen Proteomics Forum. The symposium was on 22 November 2012, with more than 80 delegates that attended the event entitled ‘Mass spectrometry with spatial resolution: MALDI-imaging and laser capture microscopy’. The one-day agenda consisted of nine oral presentations from renowned experts in the field with subsequent discussion sessions. As usual, the meeting was fruitful and offered a good platform for discussion between the delegates and proteomics specialists.

A candidate reference material ‘Mya arenaria’ for the monitoring of butyltin compounds in mollusks in the marine environment

Most analytical techniques used for the speciation analysis of butyltin compounds in biological tissues usually contain multiple procedures (e. g., extraction, derivatization, separation, preconcentration, determination) which may increase the risk of systematic errors. The use of reference materials (RM) makes it possible to correct most of these errors and improve the validity of quality control. In this study, a new candidate RM for butyltin compounds in mollusks was developed. It was prepared with a mollusk 'Mya arenaria' directly bought from the market. The preparation of this candidate RM, the homogeneity and stability studies, the value assignment and the evaluation of uncertainty were described. The certified values of 0.33 +/- 0.08, 0.62 +/- 0.09, 2.64 +/- 0.20 mu g Sn g(-1) (based on dry mass) for monobutyltin (MBT, [Sn(C4H9)(3+)]), dibutyltin (DBT, [Sn(C4H9)(2)(2+)]) and tributyltin (TBT, [Sn(C4H9)(3)(+)]) respectively have been assigned by inter-laboratory comparison. This work will be of great support to the monitoring of butyltin compounds in biota, as well as meeting the need for implementing international treaties in China.

In Vivo Solid-Phase Microextraction For Tissue Bioanalysis

Conventional in vitro or ex vivo bioanalytical quantitative sample preparation methods for the determination of compounds in biological tissues are often coupled with challenges in obtaining an assay representative of the system of interest. The rising interest in in vivo microsampling bioanalytical methods is due to the unique advantages they offer over their in vitro counterparts. In vivo solid-phase microextraction (SPME), a diffusion-based microsampling tool, has been successfully applied in recent studies to various biological systems. This review presents recent trends in tissue bioanalysis using in vivo SPME as a sample preparation tool. Efforts were made to discuss the various bioapplications of the method while highlighting possible strategies for improved sensitivity where needed. In vivo SPME devices currently employed for the various applications have also been described. In addition, we highlight selectivity of a new class of biocompatible coatings that can potentially improve the coverage of metabolites for untargeted metabolomics.

Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis

Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index). The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination. Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.

Validated Comprehensive Analytical Method for Quantification of Coenzyme A Activated Compounds in Biological Tissues by Online Solid-Phase Extraction LC/MS/MS

We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.

Physicochemical prediction of a brain–blood distribution profile in polycyclic amines

Recent investigation into the pharmacological character of the pentacyclo[5.4.0.0 2,6.0 3,10.0 5,9]undecyl and related polycyclic amines has revealed interesting facts regarding their possible use as neuroprotective agents. At this stage however, a clear shortcoming in the quest for further development of this novel class of compounds is the lack of concrete data on their ability to cross the blood-brain barrier (BBB). Working towards the aim of predicting BBB permeability, a series of related N-substituted 8-amino-8,11-oxapentacyclo[5.4.0.0 2,6.0 3,10.0 5,9]undecanes were synthesised. Compounds were characterised by both experimental and calculative methods, followed by biological assessment and statistical manipulation of the results obtained. In doing so, a simple biological model was established for the comparative evaluation of brain–blood distribution properties within the class. A highly sensitive ESI-MS.MS analytical procedure was developed for the detection of these compounds in biological tissues, indicating significant drug concentrations in the brain after intraperitoneal administration to C57Bl/6 mice. Stepwise multiple linear regression analysis of all data yielded two meaningful models ( R 2=0.9996 and R 2=0.7749) depicting lipophilicity (log P oct), solvent accessible molecular volume (SV), molar refractivity (MR) and system energy as the prime determinants of the brain–blood profile for these amines. The inherently high lipophilicity potential within the series is attributed to strong hydrophobic influences dominating hydrogen bonding effects. A possible conformational and energy dependent preference at the site of permeation is also suggested. The proposed estimations allow for the expedient and reliable prediction of brain partitioning behaviour for related polycyclic amines, facilitating the early rejection of unsuitable candidates and enabling research to focus on neuroprotective activity.

Radiowave dielectric investigation of boron compounds distribution in cultured tumour cells: relevance to boron neutron capture therapy

The distribution of two main Boron neutron capture therapy (BNCT) agents, borocaptate sodium ( BSH) and borono-phenylalanine ( BPA), in C6 rat glioma cells has been investigated by means of radiowave dielectric spectroscopy measurements. Significant differences between cells treated with the two different boron carriers were found in the magnitude of passive electrical cell parameters. This technique offers a new procedure for the measurement of boron compounds interactions with different biological environments at cellular level and is suggested to have the potentiality for becoming an attractive tool for biodistribution studies of BNCT compounds in biological tissues.

Determination of tamoxifen and metabolites in mouse fetal tissue using nonaqueous capillary electrophoresis.

Tamoxifen (TAM), an antiestrogen, has been approved for use by women at risk for developing hormone-dependent breast cancer. Administration of TAM to pregnant CD-1 mice apparently results in reproductive tract toxicity in female offspring. However, there is little or no data describing potential TAM-induced fetal toxicity to women who may become pregnant while receiving prophylactic TAM treatment. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, the present work describes a capillary electrophoresis (CE)-based analytical technique used for detection of TAM and two major metabolites, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in CD-1 mouse fetal tissue. TAM-derived material was extracted from CD-1 mouse fetuses 2-12 h following TAM administration (100 mg/kg) to dams on gestation day 16. The presence of TAM, DMT, and 4-HT was confirmed in the solvent extracts by nonaqueous CE. The limit of detection of TAM by UV absorption was approximately 675 amol at a signal-to-noise ratio of 2:1. This work demonstrates both transplacental transport of TAM in CD-1 mice and a sensitive analytical technique for detecting low concentrations of TAM and similar compounds in biological tissues.

A new mussel certified reference material (CRM 477) for the quality control of butyltin determination in the marine environment.

The analytical techniques used for the determination of butyltin compounds in biological tissues are generally based on a succession of steps (e.g., extraction, derivatization, separation, detection) which are all prone to systematic errors. An interlaboratory programme performed at the EU level and based on a stepwise approach has enabled one to identify and remove most of these errors and to improve considerably the state of the art so that certification of a mussel reference material has been made possible. This paper describes the preparation of this mussel reference material containing monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT), the homogeneity and stability studies and the analytical work performed for the certification of the contents of MBT [Sn(C4H9)3+], DBT [Sn(C4H9)2(2+)] and TBT [Sn(C4H9)3+]. The results obtained by a group of expert EU laboratories are discussed along with the methods used to certify the mass fractions (based on dry mass) of monobutyltin (1.50 +/- 0.28 mg kg-1 as MBT cations), dibutyltin (1.54 +/- 0.12 mg kg-1 as DBT cations) and tributyltin (2.20 +/- 0.19 mg kg-1 as TBT cations). The paper also describes an attempt to certify the content of triphenyltin in this material which, although reasonable agreement was found among the participants' results, failed owing to the instability of this compound. This new material will be of great support to ensure the quality control of butyltin determination in shellfish, as carried out in environmental monitoring programmes.

Isolation and structural elucidation of the predominant geometrical isomers of alpha-carotene.

The recent development and application of a polymeric C30 stationary phase have given unique separations of cis-trans carotenoid isomers in reversed-phase (RP) liquid chromatography (LC) owing to the exceptional shape selectivity of this stationary phase. In the present research, several geometrical isomers of alpha-carotene were at least partially resolved from a photo-isomerized mixture when chromatographed on a 3-microns polymeric C30 column. Double bond configurations of the five predominant alpha-carotene peaks, as isolated on a semi-preparative C30 column, were unambiguously assigned using 1H nuclear magnetic resonance (NMR) spectroscopy, giving the following order of elution: 13-cis, 13'-cis, all-trans, 9-cis, and 9'-cis geometrical forms. Electronic absorption spectra for these isomers were in agreement with the identification of peaks. The alpha-carotene isomers separated and identified herein had not been previously resolved in RPLC. Confirmation of the structures of geometrical alpha-carotene isomers will aid further studies on the possible physiological roles of these compounds in biological tissues.

Validation of the analysis of organotin compounds in biological tissues using alkylation and gas chromatography

AbstractSeveral critical steps in the analytical procedure for organotin compounds in the environment are identified in this paper and solutions are suggested. In particular, an improved procedure for quenching excess Grignard reagent is described. After insight into the nature of losses was obtained, the use of internal standards made it possible to reduce further the method variability. The systematic optimization of the analytical procedure resulted in a well investigated and robust method for analysis of organotin compounds in zebra mussel samples. Organotin compounds (OTs) which enter the environment as a result of their use as biocides and their degradation products are regularly found in environmental samples. Many different analytical techniques are currently being used,1 but little is known about their accuracy and precision.

Extraction procedure for the measurement of butyltin compounds in biological tissues using toluene, HBr, and tropolone.

Extraction procedure for the measurement of butyltin compounds in biological tissues using toluene, HBr, and tropolone.