Ultra performance liquid chromatography – mass spectrometry studies of formalin‐induced alterations of human brain lipidome

The development of ‘omics’ sciences offers new opportunities for the study of neurodegenerative diseases but increases at the same time the sample demand on brain banks that collect and store valuable human post‐mortem tissue. Our study aims to evaluate in lipidomics the potential of formalin‐fixed tissue compared with the cryopreservation method, considered as the gold standard for biochemical research. Two complementary liquid chromatography/mass spectrometry analytical platforms were used on the basis of hybrid quadrupole time‐of‐flight and triple quadrupole mass spectrometers. Untargeted fingerprinting, semitargeted profiling of specific lipid classes and targeted monitoring of lipid species were performed in formalin‐fixed and cryopreserved samples to provide detailed information at the molecular level on the formalin‐induced alterations of the brain tissue. In vitro incubations of lipid standards were also performed to further describe the degradation processes induced by formaldehyde. Phospholipid compounds were found to be extensively hydrolysed, whilst the sphingolipid ones were preserved. N‐methylation and N‐formylation of amine‐containing phospholipids have also been evidenced. These findings show that the potential detrimental effect of formalin on the analytes of interest must be taken into account when analysing formalin‐fixed samples. Copyright © 2014 John Wiley & Sons, Ltd.     

A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross‐links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.     

Reaction of cytisine with formalin

The reaction of cytisine with formalin in various solvents was studied. Methylene-bis-cytisine was produced in addition to the expected N-methylolcytisine in all solvents except acetone, in which N-(3-oxobutyl)cytisine was produced. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 579–580, November–December, 2006.     

Antinociceptive Synergism of Pomegranate Peel Extract and Acetylsalicylic Acid in an Animal Pain Model

Several modern drugs, which are derived from traditional herbal medicine are used in contemporary pharmacotherapy. Currently, the study of drug–plant interactions in pain has increased in recent years, looking for greater efficacy of the drug and reduce side effects. The antinociception induced by intragastric co-administration of the combination of pomegranate peel extract (PoPEx) and acetylsalicylic acid (ASA) was assessed using the isobolographic analysis in formalin test (nociceptive and inflammatory pain). The effective dose that produced 30% of antinociception (ED₃₀) was calculated for both drugs from the logarithmic dose–response curves, subsequently generating a curve with the combination on fixed proportions (1:1) of PoPEx and ASA. Through isobolographic analysis, this experimental ED₃₀ was compared with the calculated theoretical additive ED₃₀. The result was a synergistic interaction, the experimental ED₃₀ was significantly smaller (p < 0.05) than the theoretical ED₃₀. The antinociceptive mechanism of the PoPEx-ASA combination involves the l-Arginine/NO/cGMP pathway, antioxidant capacity, and high content of total phenols. These findings suggest that an interaction between PoPEx and ASA could be a novel treatment for inflammatory and nociceptive pain, also diminish the secondary reactions of ASA.     

Extracellular matrix alterations in low‐grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry

Lung adenocarcinoma (LUAD) is the second most common cancer, affecting both men and women. Fibrosis is a hallmark of LUAD occurring throughout progression with excess production of extracellular matrix (ECM) components that lead to metastatic cell processes. Understanding the ECM cues that drive LUAD progression has been limited due to a lack of tools that can access and report on ECM components within the complex tumor microenvironment. Here, we test whether low‐grade LUAD can be distinguished from normal lung tissue using a novel ECM imaging mass spectrometry (ECM IMS) approach. ECM IMS analysis of a tissue microarray with 20 low‐grade LUAD tissues and 20 normal lung samples from 10 patients revealed 25 peptides that could discriminate between normal and low‐grade LUAD using area under the receiver‐operating curve (AUC) ≥0.7, P value ≤.001. Principal component analysis demonstrated that 62.4% of the variance could be explained by sample origin from normal or low‐grade tumor tissue. Additional work performed on a wedge resection with moderately differentiated LUAD demonstrated that the ECM IMS analytical approach could distinguish LUAD spectral features from spectral features of normal adjacent lung tissue. Conventional liquid chromatography with tandem mass spectrometry (LC‐MS/MS) proteomics demonstrated that specific sites of hydroxylation of proline (HYP) were a main collagen post translational modification that was readily detected in LUAD. A distinct peptide from collagen 3A1 modified by HYP was increased 3.5 fold in low‐grade LUAD compared with normal lung tissue (AUC 0.914, P value <.001). This suggests that regulation of collagen proline hydroxylation could be an important process during early LUAD fibrotic deposition. ECM IMS is a useful tool that may be used to define fibrotic deposition in low‐grade LUAD.     

Do formalin fixation and freeze‐thaw affect near‐infrared Raman spectroscopy of cartilaginous tissue? A preliminary ex vivo analysis of native human articular cartilage

Near‐infrared (NIR) Raman microprobe spectroscopy has been applied to the non‐invasive characterization of the biochemical structure of extracellular matrix in articular cartilage, a step forward along the path of in vivo diagnostic application of chondropathy. In most studies handling ex vivo cartilage specimens, formalin fixation or freeze‐thaw treatments have been applied in order to stabilize tissue and cell constituents prior to spectroscopic measurements. However, these pre‐processing manipulations might significantly affect certain target bands of the cartilage spectra, thus introducing biases in the characterizations, and potentially leading to data misinterpretation. In this study, we evaluated how formalin fixing and freeze‐thaw processes affect Raman spectra from human femur cartilage. Healthy cartilage specimens were fixed/stored either in a 10% neutral buffered formalin solution or in a deep freezer set at −80 °C. The results of this study show that formalin fixation significantly affects the NIR Raman spectra of cartilage specimens due to concurrent formalin absorption and water dehydration within both collagen and glycosaminoglycan macromolecules. Water dehydration was also confirmed in the amide I structure in the frozen‐thawed specimen, but to a much lesser extent. Furthermore, soaking the tissues in phosphate‐buffered saline solution minimized the storage‐induced Raman artifacts, but its immersion had limited effectiveness in formalin‐fixed specimens, predominantly due to an overlap of signals from the formalin liquid (i.e. emitting at 1046 and 1492 cm⁻¹). Therefore, to provide a highly accurate biochemical evaluation of extracellular matrix using NIR Raman spectroscopy, freeze‐thaw processes are more suitable for ex vivo samples of human cartilage than formalin fixation. Copyright © 2015 John Wiley & Sons, Ltd.     

A technique for relative quantitation of cancer biomarkers in formalin‐fixed,paraffin‐embedded (FFPE) tissue using stable‐isotope‐label based mass spectrometry imaging (SILMSI)

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin‐fixed paraffin‐embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix‐free laser desorption ionization time‐of‐flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.     

Acicular Nickel on Carbon Nanofiber as Electrochemical Sensor Electrode for the Rapid Detection of Aqueous and Gaseous Formaldehyde at Room Temperature

The rapid and straightforward detection of formaldehyde (FA) in the environment is crucial for preventing the accidental inhalation of FA and limiting skin exposure to FA. In this study, we developed a simple nickel-based electrocatalytic electrode on carbon nanofibers (CNFs−Ni), which is suitable for rapidly detecting FA at room temperature. Centrifugal electrospinning was used to obtain polyacrylonitrile (PAN) nanofibers, which was subsequently stabilized and carbonized to fabricate the CNFs. Carbonization of the CNFs occurred at various temperatures (T_c=1200, 1300, 1400, and 1500 °C). PAN CNFs served as a highly conductive template for electroless plating under a magnetic field of 500 G to grow acicular nickel. The amperometric responses of the CNFs−Ni to aqueous FA were then measured. A lab-built amperometric gas sensor (CNFs−Ni 1–8), which comprised CNFs with a reduced Ni loading, was used as the electrode for detecting gaseous FA. Scanning electron microscopy (SEM), linear sweep voltammetry (LSV), cyclic voltammetry (CV), and chronoamperometry were used to evaluate the sensitivities of the electrodes. Within the linear range of 0.05–91.5 mM, the CNFs1400-Ni electrode was highly sensitive for detecting aqueous FA (2592 μA mM⁻¹ cm⁻²), as evidenced by the fast response time (6 s). At a low concentration of gaseous FA (0.5 ppm), the laboratory-built FA gas sensor was stable (98.3 %) and had a fast response time (5 s) after 9 h of continuous operation.