Western blot detection of brain material in heated meat products using myelin basic protein and neuron-specific enolase as biomarkers

An assay based on Western blotting and detection of central nervous system (CNS)-specific antigens was developed to detect brain tissue in processed (heated) meat products. Bands of antigen-bound primary antibodies were visualised through secondary anti-antibodies labelled with peroxidase, which generated chemiluminescence documented by a photographic film. Ponceau-S staining before antibody incubation and molecular mass information on detected antigens after immunoreactions added information supporting correct identification of brain tissue in the meat products. In this approach B50/growth-associated protein (B50), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), neurofilament (NF), neuron-specific enolase (NSE) and synaptophysin (Syn) proteins were detected in raw luncheon meat and a liver product enriched with brain tissue at a level of 5% (m/m). Only MBP and NSE were considered suitable biomarkers for detection of 1% (m/m) brain tissue in meat products pasteurised at 70 °C or sterilised at 115 °C. The use of an anti-monkey MBP instead of anti-human MBP enabled speciation of the CNS material whether from bovine and ovine brains or from porcine brain tissue. This immunoblot assay potentiates the analysis of approximately 70 samples within 8 h, including sample preparation and the simultaneous probing of NSE and MBP target antigens.     

Raman spectroscopy in combination with background near-infrared autofluorescence enhances the in vivo assessment of malignant tissues

The diagnostic ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy, NIR autofluorescence spectroscopy and the composite Raman and NIR autofluorescence spectroscopy, for in vivo detection of malignant tumors was evaluated in this study. A murine tumor model, in which BALB/c mice were implanted with Meth-A fibrosarcoma cells into the subcutaneous region of the lower back, was used for this purpose. A rapid-acquisition dispersive-type NIR Raman system was employed for tissue Raman and NIR autofluorescence spectroscopic measurements at 785-nm laser excitation. High-quality in vivo NIR Raman spectra associated with an autofluorescence background from mouse skin and tumor tissue were acquired in 5 s. Multivariate statistical techniques, including principal component analysis (PCA) and linear discriminant analysis (LDA), were used to develop diagnostic algorithms for differentiating tumors from normal tissue based on their spectral features. Spectral classification of tumor tissue was tested using a leave-one-out, cross-validation method, and the receiver operating characteristic (ROC) curves were used to further evaluate the performance of diagnostic algorithms derived. Thirty-two in vivo Raman, NIR fluorescence and composite Raman and NIR fluorescence spectra were analyzed (16 normal, 16 tumors). Classification results obtained from cross-validation of the LDA model based on the three spectral data sets showed diagnostic sensitivities of 81.3%, 93.8% and 93.8%; specificities of 100%, 87.5% and 100%; and overall diagnostic accuracies of 90.6%, 90.6% and 96.9% respectively, for tumor identification. ROC curves showed that the most effective diagnostic algorithms were from the composite Raman and NIR autofluorescence techniques.     

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme¶

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time‐resolved, laser‐induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time‐resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time‐resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral‐ (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high‐grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively.     

Comprehensive analysis of nine monoamines and metabolites in small amounts of peripheral murine (C57Bl/6 J) tissues

Monoamines, acting as hormones and neurotransmitters, play a critical role in multiple physiological processes ranging from cognitive function and mood to sympathetic nervous system activity, fight‐or‐flight response and glucose homeostasis. In addition to brain and blood, monoamines are abundant in several tissues, and dysfunction in their synthesis or signaling is associated with various pathological conditions. It was our goal to develop a method to detect these compounds in peripheral murine tissues. In this study, we employed a high‐performance liquid chromatography method using electrochemical detection that allows not only detection of catecholamines but also a detailed analysis of nine monoamines and metabolites in murine tissues. Simple tissue extraction procedures were optimized for muscle (gastrocnemius, extensor digitorum longus and soleus), liver, pancreas and white adipose tissue in the range of weight 10–200 mg. The system allowed a limit of detection between 0.625 and 2.5 pg μL⁻¹ for monoamine analytes and their metabolites, including dopamine, 3,4‐dihydroxyphenylacetic acid, 3‐methoxytyramine, homovanillic acid, norepinephrine, epinephrine, 3‐methoxy‐4‐hydroxyphenylglycol, serotonin and 5‐hydroxyindoleacetic acid. Typical concentrations for different monoamines and their metabolization products in these tissues are presented for C57Bl/6 J mice fed a high‐fat diet.     

Nonphotochemical hole-burning study of selectively stained normal and cancerous human ovarian tissues

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.     

Imaging and differentiation of mouse embryo tissues by ToF-SIMS

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) equipped with a gold ion gun was used to image mouse embryo sections and differentiate tissue types (brain, spinal cord, skull, rib, heart and liver). Embryos were paraffin-embedded and then deparaffinized. The robustness and repeatability of the method was determined by analyzing ten tissue slices from three different embryos over a period of several weeks. Using principal component analysis (PCA) to reduce the spectral data generated by ToF-SIMS, histopathologically identified tissue types of the mouse embryos can be differentiated based on the characteristic differences in their mass spectra. These results demonstrate the ability of ToF-SIMS to determine subtle chemical differences even in fixed histological specimens.     

Analysis of human brain tissue, brain tumors and tumor cells by infrared spectroscopic mapping

This study uses infrared (IR) spectroscopic, point detection, mapping procedures to examine tissue samples from normal brain specimens and from astrocytic gliomas, the most frequent human brain tumors. Model systems were derived from cultured glioma cell lines. IR spectra of normal tissue sections distinguished white matter from gray matter by increased spectral contributions from lipids and cholesterol. Qualitatively the same differences were found in IR spectra of low and high grade glioma tissue sections pointing to a significant reduction of brain lipids with increasing malignancy. Whereas spectral contributions of proteins and lipids were similar in IR spectra of glioma cells and tissues, nucleic acid bands were more intense for cells suggesting higher proliferative activities. For statistical analyses of IR spectroscopic maps from 71 samples, a parameter for the lipid to protein ratio was introduced involving the CH(2) symmetric stretch band with lipids as main contributors and the amide I band of proteins. As this parameter correlated with the grade of gliomas obtained from standard histopathological examination, it was applied to classify brain tissue sections based on IR spectroscopic mapping.     

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.     

Photodynamic therapy of vertebral metastases: evaluating tumor-to-neural tissue uptake of BPD-MA and ALA-PpIX in a murine model of metastatic human breast carcinoma

Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin-derivative monoacid ring A (BPD-MA) and by 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor-bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD-MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD-MA, ALA-PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor-bearing vertebrae compared to spinal cord. The highest ratio for BPD-MA concentration was found 15 min after injection, which can be recommended for therapy in this model.     

Determination of clenbuterol in bovine plasma and tissues by gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay was developed for the determination of clenbuterol in bovine plasma and tissues. Clenbuterol and the internal standard [2H9]clenbuterol were measured by gas chromatography-negative-ion chemical ionization mass spectrometry with methane as the reagent gas. Bovine tissues including muscle, liver, heart, kidney, lung, suet, brain, spinal cord and thymus were ground in a buffer of pH 7 and then extracted using ethyl acetate. After two subsequent purification steps, the cleaned-up organic extract was derivatized with pentafluoropropionic anhydride. The mass spectrometer was set to monitor the abundant ions m/z 368 and 377 of the perfluoroacyl derivatives. This assay was performed with 1 ml of plasma or 0.2 g of tissue. The feasibility of this method was demonstrated by the determination of clenbuterol residues as the femtomole level in a variety of tissues.     

GC-MS detection of central nervous tissues as TSE risk material in meat products: analytical quality and strategy

The detection of central nervous system (CNS) tissue (i.e. brain and spinal cord) by the use of GC-MS and certain fatty acids (FAs) as their methyl esters (FAMEs) was previously shown to be a very promising approach towards identification of CNS tissue as a specified risk material (SRM) in meat products, contrasting available immunochemical methods. This GC-MS method promised to allow quantification of CNS material as low as 0.01%. Here, we show that the CNS-relevant FAMEs C22:6, C24:19, C24:17, C24:0 and C24-OH are present in pure muscle and adipose tissue samples in detectable amounts. Thus, limits of detection are not feasible as quality parameters in this analytical GC-MS approach. Instead, cut-off values have to be applied as calculated from the baseline content of the respective FAME in CNS-free samples and its variation for a given statistical security. Furthermore, the FAs used for quantification of the CNS showed distinct differences depending on species and age. This finding is in accordance with previous studies where it had been concluded that species and age differentiation of CNS might be possible with GC-MS. However, it was not taken into account that it also necessitates a strict analytical strategy for quantification of the CNS content: identification of the presence of CNS (step 1); identification of species and age (step 2); and quantification by use of a species- and age-specific CNS calibration (step 3). Differences between the FA content of the CNS used for calibrating and the CNS in the sample will cause up to fivefold deviation from the true CNS content. Our results show that the FA best suited for identification (step 1) and quantification (step 3) purposes is cerebronic acid C24-OH after silylation. Further in-depth studies are needed in order to elucidate variability of brain FA content and to determine analytical limits. However, the present GC-MS approach is already a highly promising supplement to existing immunochemical methods for the detection of traces of CNS material in meat products.     

Classification of normal and malignant human gastric mucosa tissue with confocal Raman microspectroscopy and wavelet analysis

Thirty-two samples from the human gastric mucosa tissue, including 13 normal and 19 malignant tissue samples were measured by confocal Raman microspectroscopy. The low signal-to-background ratio spectra from human gastric mucosa tissues were obtained by this technique without any sample preparation. Raman spectral interferences include a broad featureless sloping background due to fluorescence and noise. They mask most Raman spectral feature and lead to problems with precision and quantitation of the original spectral information. A preprocessed algorithm based on wavelet analysis was used to reduce noise and eliminate background/baseline of Raman spectra. Comparing preprocessed spectra of malignant gastric mucosa tissues with those of counterpart normal ones, there were obvious spectral changes, including intensity increase at approximately 1156cm(-1) and intensity decrease at approximately 1587cm(-1). The quantitative criterion based upon the intensity ratio of the approximately 1156 and approximately 1587cm(-1) was extracted for classification of the normal and malignant gastric mucosa tissue samples. This could result in a new diagnostic method, which would assist the early diagnosis of gastric cancer.     

Pharmacokinetics of Photogem using fluorescence monitoring in Wistar rats

In this study we investigated the pharmacokinetics of a hematoporphyrin derivative (Photogem) in Wistar rats using the fluorescence spectroscopy to evaluate the drug distribution in liver, kidney and skin tissues. The detection system is composed of a 532 nm exciting laser, a Y-type catheter for light delivery and collection, a monochromator and a computer for data acquisition. The analysis of the fluorescence spectra was based on the intensity of porphyrin emission bands from specific tissues of the investigated organ. A simple transport model is proposed to determine the accumulation and elimination times for each type of investigated tissue. The obtained results show the viability of the fluorescence spectroscopic technique for the drug concentration monitoring in different target tissues and related pharmacokinetics. These effects should be considered before any in vivo study of Photodynamic Therapy using Photogem.     

Structural characterisation of some fatty acids from the brain as biomarkers of BSE risk material

Identification of bovine and ovine tissue from the central nervous system (CNS: brain and spinal cord) in meat products is possible by using certain CNS fatty acids as biomarkers in GC–MS analysis. Furthermore, the relationship between the isomers of the tetracosenic acid (C24:1) is important for differentiation of the species and age of the CNS in view of the legal definition of specified risk material (SRM). This has so far been referred to as the cis/trans ratio of the isomers of nervonic acid; however, structural analysis was not performed. Here we present results from GC–MS structural analysis by retention time and DMDS adduct profiling of the even numbered monoenoic fatty acids from C18:1 to C26:1. Retention times and mass spectra of the FAME standards indicated that the so far designated trans-nervonic acid has a different isomeric structure in the tetracosenic acid from brain-sample extracts. By performing GC–MS analysis of DMDS adducts we have shown that this isomer was actually cis-17-tetracosenic acid in all species so far tested, not trans-15-tetracosenic acid (trans-nervonic acid). The tetracosenic acid isomer ratio proved to be species-specific in accordance with previous results. Thus, instead of the ratio of cis/trans isomers of nervonic acid, the ratio of 9/7-tetracosenic acid (15c-C24:1/17c-C24:1) will have to be used as a correct reference in future publications. Although trans isomers were not detectable in sheep and cattle brain, porcine brain contained, in addition to cis-17-tetracosenic acid, small amounts of the trans isomers of the C18:1, C20:1, C24:1, and C26:1 fatty acids, in decreasing quantities. In future, this might be useful as another means of differentiation between porcine CNS (non-SRM) and ovine or bovine CNS (SRM). Extensive follow-up studies must be performed to elucidate the extent to which this GC–MS approach will facilitate the detection of CNS according to the legal SRM definition.     

Monitoring the accumulation of lipofuscin in aging murine eyes by fluorescence spectroscopy

The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.     

Diagnosis of inflammatory lesions by high-wavenumber FT-Raman spectroscopy

The Raman spectroscopy technique has been extensively used for biological sample characterization. In particular, the fingerprint spectral region (800?C1,800?cm^?1) has been shown to be very promising for optical biopsy purposes. However, limitations for the widespread use of Raman-based optical biopsy technique still persist. For example, fluorescence when one uses visible light (400?C700?nm) spectral sources is often present and appears to affect the mid-IR/Raman region more than the high-wavenumber region (2,800?C3,600?cm^?1). But, both the higher wavenumber spectral region and the mid-IR/Raman region can be fluorescence-free when one uses lasers sources, which do not cause fluorescence, for example, 1,064, 830 or 785?nm sources. In addition, the Raman spectral signal of inflammatory infiltrates can influence the biopsy diagnoses and is one important source of misdiagnosis of normal versus pathological tissues. The present work seeks to evaluate whether the Raman spectra in the high-wavenumber spectral region can be used to distinguish between oral inflammatory fibrous hyperplasia (IFH) lesions and normal (NM) tissues and hence be used as a new diagnostic tool. Thirty spectra of oral IFH lesions and NM tissues from biopsies of 12 patients were analyzed using both principal components analysis (PCA) and a binary logistic regression (BLR) model. It was found that the high-wavenumber region Raman spectra can be used to discriminate between NM tissue and oral IFH tissues by analyzing the 2,800?C3,050?cm^?1 (CH₂ and CH₃ vibrations of lipids and proteins) and 3,050?C3,600?cm^?1 (CH, OH, and NH vibrations of proteins and water) spectral intensities. A simple classification model based on the relative areas of the above cited regions resulted in concordant pairs of 95.3%. Considering the standard errors in the model parameters, it was found that the sensitivity (Se) and specificity (Sp) fall in the interval 87%?<?Se?<?100% and 73%?<?Sp?<?93%, respectively. In addition, it has been found that the Raman scattering cross-sections in the NH, OH, and CH stretching region are more intense than in the mid-IR/Raman (fingerprint) region.     

High-performance liquid chromatographic strategies for the determination and confirmation of anticoagulant rodenticide residues in animal tissues

A comprehensive approach to the analysis of anticoagulant rodenticide residues in animal tissues based on high-performance liquid chromatography (HPLC) has been developed. Residues of warfarin, coumatetralyl, difenacoum, brodifacoum, bromadiolone, diphacinone and chlorophacinone were extracted with chloroformacetone (1:1, v/v). Extracts were cleaned-up by an integrated gel permeation and adsorption chromatographic procedure which divided the rodenticides into two groups. Residues were then determined and confirmed using normal-phase, ion-pair and weak ion-exchange HPLC techniques. Ion-pair gradient separation resolved all seven rodenticides in a single chromatographic analysis. UV detection methods were employed for all seven rodenticides. Use of a diode array detection system permitted additional confirmation of residues down to 0.1 mg kg-1 by matching UV spectra and derivatives of spectra. Sensitive fluorescence detection was possible for the coumarin-based rodenticides but not for diphacinone and chlorophacinone. Post-column pH-switching fluorescence detection methods were shown to be superior to other methods of fluorescence detection of coumarin-based rodenticides. Recoveries from spiked liver tissue were around 90% at levels from 0.05 to 1 mg kg-1. Detection limits of around 0.002 mg kg-1 for most rodenticides and of 0.01 mg kg-1 for warfarin could be achieved with animal tissue extracts.     

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.     

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).