A metabolomic study of the urine of rats with Alzheimer's disease and the efficacy of Ding‐Zhi‐Xiao‐Wan on the afflicted rats

As a well‐known traditional Chinese medicine formula, Ding‐Zhi‐Xiao‐Wan has long been used for the routine treatment of Alzheimer's disease. However, the mechanism of Ding‐Zhi‐Xiao‐Wan in treating Alzheimer's disease is unclear. Therefore, a nontargeted metabolomics method based on ultrahigh performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry has been established to explore the metabolic variations in the urine of Alzheimer's disease rats and investigate the therapeutic mechanism of Ding‐Zhi‐Xiao‐Wan on Alzheimer's disease. To develop a better rat model of Alzheimer's disease, amyloid β25‐35 was injected into the bilateral hippocampus of Sprague–Dawley rats. Multivariate analysis approaches were applied to differentiate the urine components between the four groups. Thereafter, a targeted metabolomics method was used to verify the identified endogenous metabolites and determine the mechanism of action of Ding‐Zhi‐Xiao‐Wan. Altogether, 26 potential biomarkers were found, of which 15 biomarkers (10 of which are potential biomarkers found in nontargeted metabolomics) were identified. The results show that Ding‐Zhi‐Xiao‐Wan mainly affects the pathways of taurine and hypotaurine metabolism, tryptophan metabolism, and phenylalanine metabolism. Ding‐Zhi‐Xiao‐Wan might play a role in the treatment of Alzheimer's disease by mediating antioxidative stress, regulation of energy metabolism, improvement of intestinal microbes, and protection of nerve cells.     

Incubation with Cu(II) and Zn(II) salts enhances MALDI‐TOF mass spectra of amyloid‐beta and α‐synuclein toward in vivo analysis

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build‐up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix‐assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid‐beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix‐assisted laser desorption/ionization mass spectrometry. In addition, α‐synuclein also fragments predictably by in‐source decay. This provides information that may be applied to the identification and localization of amyloid‐beta and α‐synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition‐metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to “clean” the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid‐beta, synthetic α‐synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human‐derived samples.     

Brain metabolomics study for the protective effects of Rhodiola crenulata extract on Alzheimer's disease by HPLC coupled with Fourier transform‐ion cyclotron resonance mass spectrometry

In order to investigate the protective effects of Rhodiola crenulata extract on Alzheimer's disease, a brain metabolomics study in rats was conducted by high performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry. Rat model was constructed by bilateral hippocampal injection of amyloid‐β peptide and immunohistochemistry was performed to evaluate the pharmacological effect of Rhodiola crenulata extract. Multivariate statistical analysis was used to discover potential biomarkers in rat brain and related metabolic pathways analysis was conducted to elucidate the action mechanism of Rhodiola crenulata extract. As a result, a total of 19 metabolites contributing to Alzheimer's disease progress were identified and nine of them were restored to the normal levels after drug administration. Pathway analysis revealed that the protective effects of Rhodiola crenulata extract are related to the regulation of glutathione metabolism and arachidonic acid metabolism in rat brain. In conclusion, this work demonstrates that the developed metabolomics method is useful to investigate the protective effects of Rhodiola crenulata extract against Alzheimer's disease. These outcomes may further provide reliable evidence to illuminate the intervention mechanism of other traditional Chinese medicines on Alzheimer's disease.     

Applications of multifunctional magnetic nanoparticles for the enrichment of proteins for PAGE separation

This paper describes the applications of multifunctional magnetic nanoparticles (MNPs) for the enrichment of low‐abundance proteins for polyacrylamide gel electrophoresis (PAGE) separation. The hemoglobin‐functionalized MNPs, named Hb–MNPs, were obtained based on electrostatic interactions and covalent binding between the hemoglobin (Hb) and the MNPs. It was demonstrated that the proteins in human serum were selectively conjugated to Hb‐MNPs, which can be used for the selective enrichment of low‐abundance proteins. Three and seven kinds of proteins were identified by MS after 1‐D and 2‐D PAGE, respectively. Comparing with native PAGE without the treatment of MNPs, some proteins were observed, such as human serum amyloid P component (SAP), vitamin D‐binding protein, and serine peptidase inhibitor. Because the high concentration of SAP can be considered as a signal for the neurodegeneration of Alzheimer's disease, the present Hb‐MNPs‐based method was applied to investigate the serum level of SAP for the diagnosis of Alzheimer's disease, and the results are satisfying.     

The internal link of serum steroid hormones levels in insomnia,depression, and Alzheimer's disease rats: Is there an effective way to distinguish among these three diseases based on potential biomarkers?

Insomnia, depression, and Alzheimer's disease are all neurodegenerative diseases and are associated with the levels of steroid hormones. To investigate the internal connection and difference of steroid hormones among these three diseases and distinguish them from the perspective of biomarkers, an easy, quick, and efficient high‐performance liquid chromatography with tandem mass spectrometry method was established and validated to determine six steroid hormones simultaneously in rat serum. The separation was accomplished on a SHIM‐PACK XR‐ODS chromatographic column with 0.1% v/v formic acid and methanol as the mobile phase and the detection was performed with electrospray ionization source in the positive ion mode. Based on the concentrations of steroid hormones, all the groups could be distinguished obviously from each other by using partial least square discriminant analysis. Meanwhile, 11‐deoxycortisol, corticosterone, and cortisol were identified as potential biomarkers and 100% of samples were classified correctly by Bayes’ discriminant function. These biomarkers were further screened by one‐way analysis of variance and cortisol was significantly different among all these groups. Bayes’ discriminant function was also built by cortisol and the classification accuracy was 87.2%. This workflow including determination of steroid hormones and discrimination among three neurological diseases would provide a basis for further clinical studies.     

Simultaneous quantification of fatty acids in serum with dual derivatization by liquid chromatography-tandem mass spectrometry

Fatty acids have multitudinous biological functions and play a crucial role in many biological processes, but due to poor ionization efficiency and lack of appropriate internal standards, the comprehensive quantification of fatty acids by liquid chromatography-tandem mass spectrometry is still challenging. In this study, a new, accurate, and reliable method for quantifying 30 fatty acids in serum using dual derivatization was proposed. Indole-3-acetic acid hydrazide derivants of fatty acids were used as the internal standard and indole-3-carboxylic acid hydrazide derivants of them were used to quantify. The derivatization conditions were systematically optimized and the method validation results showed good linearity with R² > 0.9942, low detection limit (0.03–0.6 nM), precision (1.6%–9.8% for intra-day and 4.6%–14.1% for inter-day), recovery (88.2%–107.2% with relative standard deviation < 10.5%), matrix effect (88.3%–105.2% with the relative standard deviation < 9.9%) and stability (3.4%–13.8% for fatty acids derivants in 24 h at 4°C and 4.2%–13.8% for three freeze-thaw cycles). Finally, this method was successfully applied to quantify fatty acids in serum samples of Alzheimer's patients. In contrast to the healthy control group, nine fatty acids showed a significant increase in the Alzheimer's disease group.     

Developing column material for the separation of serum amyloid P and C reactive protein from biological sources

In this study, we have investigated the isolation of serum amyloid P (SAP) and C‐reactive protein (CRP) from rainbow trout. It has recently been found that SAP is deposited in atherosclerotic lesions or neurofibrillary tangles, which are related to aging process and Alzheimer's disease. Given the importance of CRP, the CRP level in blood is becoming recognized as a potential means of monitoring cardiovascular risk. These two proteins, members of the pentraxin family of oligomeric serum proteins, were isolated from rainbow trout using N‐methacryloyl‐phosphoserine (MA‐pSer) immobilized poly (2‐hydroxy ethylmethacrylate) (PHEMA) cryogels as a column material in a fast protein liquid chromatography system. The separation process was verified in two steps. First, SAP and CRP proteins were isolated together from serum sample of rainbow trout using MA‐pSer/PHEMA cryogel columns. Second, SAP protein was separated chromatographically from CRP protein using the Ca²⁺ ion immobilized PHEMA cryogel column. According to the data, a new and effective technique has been developed for the isolation of SAP and CRP proteins from a biological source, rainbow trout. Finally, purified SAP and CRP were loaded using sodium dodecyl sulfate–polyacrylamide gel and western blot analysis to investigate the purity of chromatographically isolated SAP and CRP compared with commertial SAP and CRP. Copyright © 2014 John Wiley & Sons, Ltd.     

Activity‐Based Probes for 15‐Lipoxygenase‐1

Human 15‐lipoxygenase‐1 (15‐LOX‐1) plays an important role in several inflammatory lung diseases, such as asthma, COPD, and chronic bronchitis, as well as various CNS diseases, such as Alzheimer's disease, Parkinson's disease, and stroke. Activity‐based probes of 15‐LOX‐1 are required to explore the role of this enzyme further and to enable drug discovery. In this study, we developed a 15‐LOX‐1 activity‐based probe for the efficient activity‐based labeling of recombinant 15‐LOX‐1. 15‐LOX‐1‐dependent labeling in cell lysates and tissue samples was also possible. To mimic the natural substrate of the enzyme, we designed activity‐based probes that covalently bind to the active enzyme and include a terminal alkene as a chemical reporter for the bioorthogonal linkage of a detectable functionality through an oxidative Heck reaction. The activity‐based labeling of 15‐LOX‐1 should enable the investigation and identification of this enzyme in complex biological samples, thus opening up completely new opportunities for drug discovery.     

A Long‐Living Bioengineered Neural Tissue Platform to Study Neurodegeneration

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long‐term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk‐based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic‐like indicators of increased pathological β‐amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long‐term stability of the platform is suited to study chronic brain disease including neurodegeneration.     

Treatment of Alzheimer's disease with small-molecule photosensitizers

Alzheimer’s disease is a neurodegenerative disease that signals for excess β-amyloid(Aβ) aggregation.Although people have made great attempts to control the aggregation of Aβ, no effective medications have been produced yet. Due to its excellent temporal and spatial selectivity, photodynamic treatment has been gradually employed and interfered in the aggregation process of Aβ, with some achievement. To enhance the research and application of photodynamic therapy in Alzheimer’s disease, this pape...     

A UPLC‐TOF/MS‐based metabolomics study of rattan stems of Schisandra chinensis effects on Alzheimer's disease rats model

A UPLC‐TOF/MS‐based metabolomics method was established to explore the therapeutic mechanisms of rattan stems of S. chinensis (SCS) in Alzheimer's disease (AD). Experimental AD model was induced by intra‐hippocampal Aβ_1–42 injection in rats. Cognitive function and oxidative stress condition in brain of AD rats were assessed using Morris water maze tests and antioxidant assays [malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px)], respectively. UPLC‐TOF/MS combined with multivariate statistical analysis were conducted to study the changes in metabolic networks in serum of rats. The results indicated that the AD model was established successfully and the inducement of Aβ_1–42 caused a decline in spatial learning and memory of rats. The injection of Aβ_1–42 in rat brains significantly elevated the level of MDA, and reduced SOD and GSH‐Px activities. In addition, SCS showed significant anti‐AD effects on model rats. A total of 30 metabolites were finally identified as potential biomarkers of AD and 14 of them had a significant recovery compared with the AD model after SCS administration. Changes in AD metabolite profiling were restored to different levels through the regulation of 13 pathways. This is first report on the use of the UPLC‐TOF/MS‐based serum metabolomics method to investigate therapeutic effects of SCS on AD, and enrich potential biomarkers and metabolic networks of AD.     

Ganmaidazao decoction alleviated cognitive impairment on Alzheimer's disease rats by regulating gut microbiota and their corresponding metabolites

Traditional Chinese medicine targeted at gut microbiota has good effects in relieving the clinical manifestation of Alzheimer's disease, and intestinal metabolites are considered as a bridge of communication between the brain-gut axis. In order to explore the molecular mechanism of Ganmaidazao decoction treatment, first, the model rats induced by Aβ25-35 and d-gal were used to test the therapy of Ganmaidazao extract using the Morris Water Maze, Western Blot and Elisa. Then the 16S rDNA gene sequencing of the gut microbiota as well as UPLC-QTOF/MS-based metabolomic analysis of feces were carried out. Last, the relationship between Alzheimer's disease, gut microbiota and metabolites was analyzed. Results showed that the abundance and diversity of gut microbiota were rescued and the changes of fecal metabolites in rats with Alzheimer's disease were reversed after Ganmaidazao decoction administration, which were mainly related to lipid metabolism, steroid hormone metabolism, energy metabolism, amino acid metabolism and bile acid metabolism. After associating with Spearman’s correlation analysis, we concluded that gut microbiota and metabolites were closely related and Ganmaidazao decoction could interfere with the balance of gut microbiota and their corresponding metabolites to exert anti- Alzheimer’s disease effect. Combined with PICRUSt2 functional prediction of gut microbiota and metabolomics results, phenylalanine metabolism has been focused as a key metabolic pathway, and Ganmaidazao decoction can reduce the abnormal accumulation of phenylalanine and phenylpyruvate and promote their metabolism by restoring the activity of phenylalanine hydroxylase. This integrated omics approach has potential roles in understanding the complex mechanisms of Ganmaidazao decoction in treating Alzheimer’s disease.     

Voltage‐programming‐based capillary gel electrophoresis for the fast detection of angiotensin‐converting enzyme insertion/deletion polymorphism with high sensitivity

A voltage‐programming‐based capillary gel electrophoresis method with a laser‐induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin‐converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin‐converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin‐converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel electrophoresis. In addition, the capillary gel electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel electrophoresis. Consequently, the developed voltage‐programming capillary gel electrophoresis method with laser‐induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease‐related specific DNA molecules.     

Ultra‐fast liquid chromatography with tandem mass spectrometry determination of eight bioactive components of Kai‐Xin‐San in rat plasma and its application to a comparative pharmacokinetic study in normal and Alzheimer's disease rats

A method of ultra‐fast liquid chromatography with tandem mass spectrometry was developed and validated for the simultaneous quantitation of eight bioactive components, including polygalaxanthone III, sibiricaxanthone B, tenuifolin, sibiricose A5, sibiricose A6, tenuifoliside A, ginsenoside Re and ginsenoside Rb1 in rat plasma after oral administration of Kai‐Xin‐San. The plasma samples were extracted by liquid–liquid extraction using digoxin as an internal standard. Chromatographic separation was performed on a Venusil MP C₁₈ column (100 mm × 2.1 mm, 3 μm) with methanol and 0.05% acetic acid in water as mobile phase. The tandem mass spectrometric detection was performed in the multiple reaction monitoring with turbo ion spray source in the negative ionization. Validation parameters were within acceptable ranges. The established method has been successfully applied to compare the pharmacokinetic profiles of the analytes between normal and Alzheimer's disease rats. The results indicated that there were significant differences in pharmacokinetic parameters of some components between two groups, which may be due to the mechanisms of Alzheimer's disease and pharmacological effects of the analytes. The pharmacokinetic research in the pathological state might provide more useful information to guide the clinical usage of herbal medicine.     

Label‐free Electrochemical Sensor for Ex‐vivo Monitoring of Alzheimer's Disease Biomarker

The beta‐amyloid (Aβ) peptide was used as an important biomarker for Alzheimer's disease (AD) diagnosis. The development of an accurate, selective, rapid, and highly sensitive technique for detecting of Aβ level is an important issue in biology, and medicine to assess human health risks. Here, gold nanoparticles (Au NPs) with different size were electrochemically deposited onto the indium tin oxide (ITO) substrate in the presence of different molecular weights of surfactants. The modified substrates were used as a high sensitive electrochemical sensor of in‐vitro as well as ex‐vivo monitoring of Aβ based on cyclic voltammetry and square wave voltammetry techniques. Our findings revealed that the modification of ITO electrode with Au NPs could enhance its sensor performance with high sensitivity for low concentration levels of Aβ over a wide linear range with a detection limit of about 20.7 ng/g, which is less than the concentration of insoluble Aβ40 (105.4±40.2 μg/g) in brain of AD induced. In addition, Au NPs/ITO modified electrodes have demonstrated ability to monitor Aβ in the brain extracted samples without any potential interference with other components. Raman spectroscopy has been used to confirm the presence of Aβ in the AD‐induced samples. Thus, it is applicable for analyzing ex‐vivo samples.     

High-performance liquid chromatography with fast-scanning fluorescence detection and multivariate curve resolution for the efficient determination of galantamine and its main metabolites in serum

Based on green analytical chemistry principles, an efficient approach was applied for the simultaneous determination of galantamine, a widely used cholinesterase inhibitor for the treatment of Alzheimer's disease, and its major metabolites in serum samples. After a simple serum deproteinization step, second-order data were rapidly obtained (less than 6 min) with a chromatographic system operating in the isocratic regime using ammonium acetate/acetonitrile (94:6) as mobile phase. Detection was made with a fast-scanning spectrofluorimeter, which allowed the efficient collection of data to obtain matrices of fluorescence intensity as a function of retention time and emission wavelength. Successful resolution was achieved in the presence of matrix interferences in serum samples using multivariate curve resolution-alternating least-squares (MCR-ALS). The developed approach allows the quantification of the analytes at levels found in treated patients, without the need of applying either preconcentration or extraction steps. Limits of detection in the range between 8 and 11 ng mL⁻¹, relative prediction errors from 7 to 12% and coefficients of variation from 4 to 7% were achieved.     

Anionic Oligothiophenes Compete for Binding of X‐34 but not PIB to Recombinant Aβ Amyloid Fibrils and Alzheimer's Disease Brain‐Derived Aβ

Deposits comprised of amyloid‐β (Aβ) are one of the pathological hallmarks of Alzheimer's disease (AD) and small hydrophobic ligands targeting these aggregated species are used clinically for the diagnosis of AD. Herein, we observed that anionic oligothiophenes efficiently displaced X‐34, a Congo Red analogue, but not Pittsburgh compound B (PIB) from recombinant Aβ amyloid fibrils and Alzheimer's disease brain‐derived Aβ. Overall, we foresee that the oligothiophene scaffold offers the possibility to develop novel high‐affinity ligands for Aβ pathology only found in human AD brain, targeting a different site than PIB.