Visualization of PFOA accumulation and its effects on phospholipid in zebrafish liver by MALDI Imaging.

Exposure to poly- and perfluoroalkyl substances (PFASs) can result in bioaccumulation. Initial findings suggested that PFASs could accumulate in tissues rich in both phospholipids and proteins. However, our current understanding is limited to the average concentration of PFASs or phospholipid content across entire tissue matrices, leaving unresolved the spatial variations of lipid metabolism associated with PFOA in zebrafish tissue. To address gap, we developed a novel methodology for concurrent spatial profiling of perfluorooctanoic acid (PFOA) and individual phospholipids within zebrafish hepatic tissue sections, utilizing matrix-assisted laser desorption/ionization time of flight imaging mass spectrometry (MALDI-TOF-MSI). 5-diaminonapthalene (DAN) matrix and laser sensitivity of 50.0 were optimized for PFOA detection in MALDI-TOF-MSI analysis with high spatial resolution (25 µm). PFOA was observed to accumulate within zebrafish liver tissue. H&E staining results corroborating the damage inflicted by PFOA accumulation, consistent with MALDI MSI results. Significant up-regulation of 15 phospholipid species was observed in zebrafish groups exposed to PFOA, with these phospholipid demonstrating varied spatial distribution within the same tissue. Furthermore, co-localized imaging of distinct phospholipids and PFOA within identical tissue sections suggested there could be two distinct potential interactions between PFOA and phospholipids, which required further investigation. The MALDI-TOF-IMS provides a new tool to explore in situ spatial distributions and variations of the endogenous metabolites for the health risk assessment and ecotoxicology of emerging environmental pollutants.

A novel visualization method for the composition analysis of processed garlic by MALDI-TOF imaging mass spectrometry (MSI) and Q-TOF LC-MS/MS.

Laba garlic is a kind of vinegar processed garlic (Allium sativum L.) product with multiple health effects. This study applied matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-TOF MSI) and Q-TOF LC-MS/MS for the first time to investigate the garlic tissue spatial distribution changes of low molecular weight compounds during the Laba garlic processing. The distribution characteristics of the compounds were observed in processed and unprocessed garlic including amino acids and derivatives, organosulfur compounds, pigment precursors, polysaccharides and saponins. During Laba garlic processing, some bioactive compounds such as alliin and saponins were lost because they were transformed into other compounds or leached into the acetic acid solution, and some new compounds including pigments-related compounds occurred. This study provided a basis for the spatial distributions and changes of compounds in garlic tissue during Laba garlic processing, which suggested that the bioactivities of garlic might be changed after processing owing to the transformation and change of the constituents.

Screening of potential chemical marker with interspecific differences in Pterocarpus wood and a spatially-resolved approach to visualize the distribution of the characteristic markers.

Profiling the spatial distributions and tissue changes of characteristic compounds with interspecific differences is critical to elucidate the complex species identification during tree species traceability, wood anti-counterfeiting verification and timber trade control. In this research, in order to visualize the spatial position of characteristic compounds in two species with similar morphology (Pterocarpus santalinus and Pterocarpus tinctorius), a high coverage MALDI-TOF-MS imaging method was used to found the mass spectra fingerprints of different wood species. 2-Mercaptobenzothiazole matrix was used to spray wood tissue section to enhance the detection effect of metabolic molecules, and the mass spectrometry imaging data were obtained. Based on this technology, the spatial location of fifteen potential chemical markers with remarkable interspecific differences in 2 Pterocarpus timber species were successfully obtained. Distinct chemical signatures obtained from this method can promote rapid identification at the wood species level. Thus, matrix-assisted laser desorption/time-of-flight/ionization mass spectrometry imaging (MALDI-TOF-MSI) provides a spatial-resolved way for traditional wood morphological classification and breaking through the limitations of traditional wood identification technology.

Study on the spatial distribution of coumarins in Angelica dahurica root by MALDI-TOF-MSI.

INTRODUCTION: The main chemical components of Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. are coumarins and volatile oils, and coumarins are regarded as the representative constituents with various pharmacological effects. OBJECTIVE: Based on matrix-assisted laser desorption/ionization time of flight mass spectrometry imaging (MALDI-TOF-MSI), a method for spatial distribution analysis of coumarins in primary root and lateral root of A. dahurica was established. Also, spatial visualization of coumarins in the roots of A. dahurica was realized. MATERIALS AND METHODS: α-Cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, and 9-aminoacridine were used as matrices. MALDI-TOF-MSI was employed to analyze the standards of imperatorin, oxypeucedanin, and osthole. Based on the higher sensitivity and repeatability of MALDI-TOF-MSI, the CHCA matrix was selected. The matrix was used for MALDI-TOF-MSI in positive mode to analyze the distribution of coumarins in primary root and lateral root of A. dahurica. RESULTS: In total, 37 coumarins were detected in primary root and 36 coumarins were detected in lateral root by MALDI-TOF-MSI. The results showed that the coumarin content in primary root was higher than that in lateral root. Coumarins in primary root of A. dahurica were concentrated in the periderm, cortex, and phloem, whereas coumarins in lateral roots were concentrated in the phloem. CONCLUSION: The coumarins in primary root and lateral root of A. dahurica were directly analyzed without extraction and isolation, and the spatial distribution of coumarins was comprehensively visualized for the first time by MALDI-TOF-MSI, which provided a basis for distinguishing primary root and lateral root.

A reliable and effective sample preparation protocol of MALDI-TOF-MSI for lipids imaging analysis in hard and dry cereals.

A superior sectioning sample preparation protocol is the basic guarantee for maintaining data reliability in MALDI-TOF-MSI analysis. Despite significant advances in sample preparation, visualization of lipids in hard and dry cereals remains difficult due to their inherent physicochemical properties. Thus, a cryosections preparation method was designed via poly-l-lysine soaking, conductive tape adhesion and embedding medium fixation, which preserved the spatial integrity of lipids in cereals without causing analyte delocalization and obvious background interference. Higher signal intensity and clearer imaging of lipids in rice, wheat and barley (Qingke) were obtained using the established sample preparation method. Moreover, the spatial distribution of lipids was visualized in rice with different storage years, which found that the phosphatidylinositols (PIs) and phosphatidylglycerols (PGs) could be used for evaluation of rice aging degree. Our study provided molecular level guidance for further discussion of rice aging mechanism, rice quality evaluation and safety monitoring.

Distribution and metabolism of daidzein and its benzene sulfonates in vivo (in mice) based on MALDI-TOF MSI.

Daidzein (D1) has been proved to be of great benefit to human health. More and more attention was paid to the metabolic process of D1. Most studies focused on the metabolites of D1 and analogs were determined through the excretion of animals and humans by traditional HPLC-MS, while their in situ distribution and metabolism in organs in vivo has not been reported. In our group, novel daidzein sulfonate derivatives were synthesized and confirmed to have excellent pharmaceutical properties. They exhibited good anti-inflammatory, inhibitory activities on human vascular smooth muscle cell proliferation and other bioactivities. Compared with traditional analytical methods, matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) can directly analyze the distribution of compounds in tissues and organs. In this study, we investigate the in situ distribution and metabolism of D1 and its derivatives (DD2, DD3) in the organs of mice based on MALDI-TOF MSI for the first time. Trace prototype compounds were detected in the plasma 4 h after the intravenous injection of D1, DD2, and DD3. Seven phase I metabolites and seven phase II metabolites were detected. D1 sulfates were found in the plasma and in organs except the heart. The presence of D1 and DD3 monosulfates in the brain indicated that they could penetrate the blood-brain barrier. DD2 and DD3 could be hydrolyzed into D1 and their metabolic pathways were similar to those of D1. In addition, a ligand-receptor docking of D1 and DD2 with mitogen-activated protein kinase 8 (JNK1) was performed because of their significant anti-inflammatory activities through the JNK signaling pathway. It showed that the binding energy of DD2 with JNK1 was obviously lower than that of D1 which was consistent with their anti-inflammatory activities. It provided a theoretical basis for further validation of their anti-inflammatory mechanism at the protein level. In summary, the research will provide beneficial guidance for further pharmacological, toxicological studies and the clinical-use research of these compounds.

Dl-3-n-butylphthalide inhibits neuroinflammation by stimulating foxp3 and Ki-67 in an ischemic stroke model.

Dl-3-n-butylphthalide (NBP) has been widely used to treat ischemic stroke in China. To investigate the mechanisms underlying NBP activity, we established a permanent middle cerebral artery occlusion (pMCAO) rat model and injected the rats with 4 mg/kg/d NBP for nine days. We then assessed neuroinflammation, neovascularization and nerve regeneration within the brain. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) was used to determine the phospholipid distribution, while laser ablation-inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) was used to measure Foxp3, Ki-67 and pCREB levels in the brain. Immunohistochemistry was used to investigate the expression of NLR family pyrin domain containing 3 (NLRP3) and its inflammatory products, caspase-1 and interleukin-1ß, in brain tissues. NBP attenuated ischemic damage and ameliorated neurological deficits in rats with pMCAO. In the ischemic brain region, NBP reduced phosphatidylethanolamine (18:0), NLRP3, caspase-1 and interleukin-1ß levels, but increased levels of Foxp3, Ki-67, pCREB and several phospholipids. In molecular docking analyses, NBP bound to NLRP3, interleukin-1ß, caspase-1, Foxp3, and Ki-67. These results demonstrate that NBP reduces neuroinflammation in brain tissues and promotes nerve and blood vessel regeneration, thus protecting neuromorphology and function.

The distribution and changes of glycoalkaloids in potato tubers under different storage time based on MALDI-TOF mass spectrometry imaging.

Glycoalkaloids (GAs) are toxic secondary metabolites in potatoes, which are harmful to human body. The storage time has a great influence on the biosynthesis and distribution of GAs. In present study, an imaging mass microscope (iMScope) was used to investigate the distribution and changes of GAs in potato tubers under different storage time (0, 10, 15, 20, 30, 40 and 60 days). We established a growth model with logistic equation to evaluate the growth trends of four major GAs in sprout, periderm and medulla. The results showed that the growth rate and relative contents of four GAs in sprout and periderm were significantly higher than that in medulla. In addition, four GAs also presented different change trends. For dehydrosolanine and α-solanine, rapid growth period of these two GAs in sprout (about at the day 23, similar to these in medulla) was later than which period in periderm (about at the day 17), while rapid growth of dehydrochaconine and α-chaconine appeared at almost the same time (about at the day 20). Based on the biosynthesis and metabolism of GAs, we have made possible explanations for these results. This study is useful for comprehending the metabolism of GAs in different parts and monitoring food safety in potatoes.

Novel imaging and related techniques for studies of diseases of the central nervous system: a review.

Imaging technologies for the analysis of the central nervous system are rapidly developing. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging, tracer-based magnetic resonance imaging, CLARITY technology and optogenetics can be used to visualize small molecules in brain tissues, the interstitial system of the brain and neuronal circuits in whole-brain samples. These tools serve as powerful technical means to explore the mechanisms underlying disease models and to evaluate the effects of drugs. Here, we review the constituting principles of these imaging techniques and describe their applications in the field of neuroscience.

Immersion by rotation-based application of the matrix for fast and reproducible sample preparations and robust results in mass spectrometry imaging.

Automated matrix deposition for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is crucial for producing reproducible analyte ion signals. Here we report an innovative method employing an automated immersion apparatus, which enables a robust matrix deposition within 5 minutes and with scalable throughput by using MAPS matrix and non-polar solvents. MSI results received from mouse heart and rat brain tissues were qualitatively similar to those from nozzle sprayed samples with respect to peak number and quality of the ion images. Overall, the immersion-method enables a fast and careful matrix deposition and has the future potential for implementation in clinical tissue diagnostics.

Function of complement factor H and imaging of small molecules by MALDI-MSI in a methamphetamine behavioral sensitization model.

BACKGROUND: At present, the harm of new-type drug, methamphetamine (METH), has gradually exceeded that of the traditional opioid drugs, and METH abuse has become a serious public health and social problem. In our previous study, complement factor H (CFH) was found to be upregulated in the sera of METH-addicted patients and rats and in certain brain regions in the rats. METHODS: We used ELISA and immunofluorescence to confirm the changes in CFH in the serum and hippocampus of a METH behavioral sensitization mouse model, and C1q expression was also detected by immunofluorescence in the hippocampus. We aimed to elucidate the involvement of CFH and C1q in the mechanism of METH addiction. We also detected the distribution of various small molecules by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in select brain regions: the nucleus accumbens, the hippocampus and the ventral tegmental area. RESULTS: The expression of CFH was upregulated in the serum and hippocampus of METH behavioral sensitization model mice, consistent with our previous research on conditioned place preference rats. In contrast, C1q decreased dramatically in the mossy fibers of the hippocampus. The results of small-molecule imaging by MALDI-MSI showed that the levels of K+, antioxidants, neurotransmitters, and ATP metabolism-related molecules were altered in different regions. CONCLUSIONS: These results indicate the involvement of the complement system in the mechanism of METH addiction and validate the presence of oxidative stress, energy metabolism changes during addiction. This suggests the utility of further investigation into the above aspects.

Effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenfu injection is a popular Chinese herbal formula that has been widely used in the treatment of shock in China. AIM OF THE STUDY: To investigate the effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits. MATERIALS AND METHODS: We established a septic shock model in rabbits by administering an intravenous injection of 0.6 mg/kg LPS to anesthetized rabbit, and 15 min after LPS challenge, the rabbits were intravenously administered the Shenfu injection. In these in vivo experiments, the jugular vein of the rabbits was cannulated for LPS and drug administration, and the right common carotid artery was cannulated to record the mean arterial pressure (MAP) over a 6-h period. In addition, various serum biochemical parameters, including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), glutamate transaminase (ALT), creatinine (Cre), and urea nitrogen (Urea), were measured at 0, 3, and 6 h. Serum LPS levels at 6 h were determined by the test kit. And histological changes in the heart, liver and kidney tissues were observed by HE staining. Furthermore, some related small molecules in the heart tissues were detected by MALDI-TOF-MSI. RESULTS: We found that Shenfu injection can increase the MAP, decrease the serum LPS, LDH and AST levels, and improve the tissue morphology of the heart, liver and kidney in rabbits with LPS-induced septic shock. In addition, Shenfu injection can increase the contents of ATP and taurine while reducing the content of AMP in the heart tissue during septic shock. CONCLUSIONS: These results indicate that Shenfu injection exerts a protective effect on LPS-induced septic shock in rabbits.

Visualization of soluble carbohydrate distribution in apple fruit flesh utilizing MALDI-TOF MS imaging.

To confirm availability of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry imaging (MSI) for visualizing distribution of soluble carbohydrates in apple (Malus domestica) fruits a horizontal fruit flesh specimen was cut from a matured 'Fuji' fruit, mounted on a glass slide, lyophilized and then ion intensities of individual soluble carbohydrates were probed around the specimen using a MALDI-TOF MSI apparatus automatically. Contents of soluble carbohydrates in adjacent tissue of the same fruit were also determined using HPLC to compare the distribution of individual carbohydrate based on the ion intensities from MALDI-TOF MSI with those from HPLC. Positive correlation (P < 0.001, R2 > 0.95) was confirmed between the concentration of each standard carbohydrate and the relative ion intensity of MALDI-TOF mass spectrometry (MS), and thus it seems possible to use the ion intensity of MALDI-TOF MS for determining the relative concentration of carbohydrates in a sample. Singly charged ions attached with a potassium ion only were detected from the apple fruit specimen when DHB was used as a matrix for MALDI-TOF MSI. Graded increase of sucrose content from center to cortex side of the fruit flesh was confirmed by both MALDI-TOF MSI and HPLC. When pseudo color images on the distribution of individual carbohydrates based on the results from MALDI-TOF MSI were compared with the content of carbohydrates in the adjacent 16 tissue blocks quantified using HPLC, strong (P < 0.001, R2 = 0.6222) and weak (P < 0.10, R2 = 0.2123) correlation was confirmed between the brightness and the content of sucrose and sorbitol, respectively. These facts indicate that distribution of sucrose and sorbitol in apple fruit tissue can be visualized using MALDI-TOF MSI. Thus, MALDI-TOF MSI will be useful for examining carbohydrate metabolism during the maturing of apple fruit.

Visualizing phosphatidylcholine via mass spectrometry imaging: relevance to human health.

INTRODUCTION: Mass spectrometry imaging (MSI) techniques are nowadays widely used to obtain spatially resolved metabolite information from biological tissues. Since (phospho)lipids occur in all animal tissues and are very sensitively detectable, they are often in the focus of such studies. This particularly applies for phosphatidylcholines (PC) which are very sensitively detectable as positive ions due to the permanent positive charge of their choline headgroup. Areas covered: After a short introduction of lipid species occurring in biological systems and approaches normally used to obtain spatially resolved mass spectra (with the focus on matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) MSI) a survey will be given which diseases have so far been characterized by changes of the PC composition. Expert commentary: Since PC species are very sensitively detectable by MS, sensitivity is not a major issue. However, spatial resolution is still limited and cellular dimensions can be hardly resolved by MALDI-TOF MSI, which is a critical point of the available approaches. Due to lacks of reproducibility and standardization further development is required.

Optimization and evaluation of MALDI TOF mass spectrometric imaging for quantification of orally dosed octreotide in mouse tissues.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) has received considerable attention in recent years since it allows molecular mapping of diverse bimolecular in animal/plant tissue sections, although some barriers still exist in absolute pixel-to-pixel quantification. Octreotide, a synthetic somatostatin analogue, has been widely used to prevent gastrointestine bleeding in the clinic. The aim of the present study is to develop a MALDI-TOF-MSI method for quantitatively visualizing spatial distribution of octreotide in mouse tissues. In this process, a structurally similar internal standard was spotted onto tissue section together with matrix solution to minimize signal variation and give excellent quantitative results. The 2,5-dihydroxybenzoic acid was chosen as the most suitable matrix via comparing the signal/noise generated by MALDI-TOF-MSI after cocrystallization of octreotide with different matrix candidates. The reliability of MALDI-TOF-MSI, with respect to linearity, sensitivity and precision, was tested via measuring octreotide in fresh tissue slices at different concentrations. The validated method was then successfully applied to visualize the distribution of octreotide in mouse tissues after oral administration of octreotide at 20mg/kg. The results demonstrated that MALDI-TOF-MSI could not only clearly visualize the spatial distribution of octreotide, but also make the calculation of the key pharmacokinetic parameters (Tmax and t1/2) possible. More importantly, the tissue concentration-time curves of octreotide determined by MALDI-TOF-MSI agreed well with those measured based on LC-MS/MS.These findings illustrate the potential of MALDI-TOF-MSI in pharmacokinetic profiling during drug development.

A new study of the bacterial lipidome: HPTLC-MALDI-TOF imaging enlightening the presence of phosphatidylcholine in airborne Pseudomonas fluorescens MFAF76a.

Lipids are major functional components of bacterial cells that play fundamental roles in bacterial metabolism and the barrier function between cells and the environment. In an effort to investigate the bacterial lipidome, we adopted a protocol using MALDI-TOF MS imaging coupled to HPTLC to screen a large number of phospholipid classes in a short span of time. With this method, phospholipids of airborne Pseudomonas fluorescens MFAF76a were visualized and identified in sample extracts (measurement accuracy below 0.1 Da, phospholipid identification by means of four characteristic fragment peaks). Via this technique, the P. fluorescens lipidome was shown to comprise three major lipid classes: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The protocol described herein is simple, rapid and effective for screening of bacterial phospholipid classes. The remarkable presence of a eukaryotic phospholipid, phosphatidylcholine, was observed in P. fluorescens MFAF76a. This lipid is known to play a role in bacteria-host interactions and had not been known to be found in P. fluorescens cells.