基于1H-NMR技术的肝癌尿液样品代谢组学研究

目的:探索区分健康对照组与肝癌患者的潜在标志代谢物。方法:运用SIMAC 13.0软件对对数据进行多元分析方法 PCA、PLS及OPLS的方法分析。结果:应用SPSS 21.0统计软件对其数据进行ROC曲线分析,发现肝癌患者在α-葡萄糖、β-葡萄糖和肌酸酐的代谢路径上都发生了一定的异常变化。这3个代谢物拟合综合变量的ROC曲线的AUC值为0.972(灵敏度为90.6%,特异性为92.9%),说明此代谢标记物组具有较高的诊断准确度。结论:α-葡萄糖、β-葡萄糖和肌酸酐可以作为区分健康对照人群与肝癌患者的潜在标志代谢物。     

Metabonomic Profiling of Serum and Urine by 1H NMR-Based Spectroscopy Discriminates Patients with Chronic Obstructive Pulmonary Disease and Healthy Individuals

Chronic obstructive pulmonary disease (COPD) has seriously impacted the health of individuals and populations. In this study, proton nuclear magnetic resonance (¹H NMR)-based metabonomics combined with multivariate pattern recognition analysis was applied to investigate the metabolic signatures of patients with COPD. Serum and urine samples were collected from COPD patients (n = 32) and healthy controls (n = 21), respectively. Samples were analyzed by high resolution ¹H NMR (600 MHz), and the obtained spectral profiles were then subjected to multivariate data analysis. Consistent metabolic differences have been found in serum as well as in urine samples from COPD patients and healthy controls. Compared to healthy controls, COPD patients displayed decreased lipoprotein and amino acids, including branched-chain amino acids (BCAAs), and increased glycerolphosphocholine in serum. Moreover, metabolic differences in urine were more significant than in serum. Decreased urinary 1-methylnicotinamide, creatinine and lactate have been discovered in COPD patients in comparison with healthy controls. Conversely, acetate, ketone bodies, carnosine, m-hydroxyphenylacetate, phenylacetyglycine, pyruvate and α-ketoglutarate exhibited enhanced expression levels in COPD patients relative to healthy subjects. Our results illustrate the potential application of NMR-based metabonomics in early diagnosis and understanding the mechanisms of COPD.     

1H NMR-Based Metabolite Profiling of Plasma in a Rat Model of Chronic Kidney Disease

Chronic kidney disease (CKD) is characterized by the gradual loss of the kidney function to excrete wastes and fluids from the blood. ¹H NMR-based metabolomics was exploited to investigate the altered metabolic pattern in rats with CKD induced by surgical reduction of the renal mass (i.e., 5/6 nephrectomy (5/6 Nx)), particularly for identifying specific metabolic biomarkers associated with early of CKD. Plasma metabolite profiling was performed in CKD rats (at 4- or 8-weeks after 5/6 Nx) compared to sham-operated rats. Principle components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots showed a significant separation between the groups. The resulting metabolic profiles demonstrated significantly increased plasma levels of organic anions, including citrate, β-hydroxybutyrate, lactate, acetate, acetoacetate, and formate in CKD. Moreover, levels of alanine, glutamine, and glutamate were significantly higher. These changes were likely to be associated with complicated metabolic acidosis in CKD for counteracting systemic metabolic acidosis or increased protein catabolism from muscle. In contrast, levels of VLDL/LDL (CH₂)_n and N-acetylglycoproteins were decreased. Taken together, the observed changes of plasma metabolite profiles in CKD rats provide insights into the disturbed metabolism in early phase of CKD, in particular for the altered metabolism of acid-base and/or amino acids.     

Pea Fiber and Wheat Bran Fiber Show Distinct Metabolic Profiles in Rats as Investigated by a 1H NMR-Based Metabolomic Approach

This study aimed to examine the effect of pea fiber (PF) and wheat bran fiber (WF) supplementation in rat metabolism. Rats were assigned randomly to one of three dietary groups and were given a basal diet containing 15% PF, 15% WF, or no supplemental fiber. Urine and plasma samples were analyzed by NMR-based metabolomics. PF significantly increased the plasma levels of 3-hydroxybutyrate, and myo-inositol as well as the urine levels of alanine, hydroxyphenylacetate, phenylacetyglycine, and α-ketoglutarate. However, PF significantly decreased the plasma levels of isoleucine, leucine, lactate, and pyruvate as well as the urine levels of allantoin, bile acids, and trigonelline. WF significantly increased the plasma levels of acetone, isobutyrate, lactate, myo-inositol, and lipids as well as the urine levels of alanine, lactate, dimethylglycine, N-methylniconamide, and α-ketoglutarate. However, WF significantly decreased the plasma levels of amino acids, and glucose as well as the urine levels of acetate, allantoin, citrate, creatine, hippurate, hydroxyphenylacetate, and trigonelline. Results suggest that PF and WF exposure can promote antioxidant activity and can exhibit common systemic metabolic changes, including lipid metabolism, energy metabolism, glycogenolysis and glycolysis metabolism, protein biosynthesis, and gut microbiota metabolism. PF can also decrease bile acid metabolism. These findings indicate that different fiber diet may cause differences in the biofluid profile in rats.     

Study of the Cardiotoxicity of Venenum Bufonis in Rats using an 1H NMR-Based Metabolomics Approach

Venenum Bufonis, a well-known traditional Chinese medicine, has been widely used in Asia and has gained popularity in Western countries over the last decade. Venenum Bufonis has obvious side effects that have been observed in clinical settings, but few studies have reported on its cardiotoxicity. In this work, the cardiotoxicity of Venenum Bufonis was investigated using a ¹¹H NMR-based metabolomics approach. The ¹H NMR profiles of the serum, myocardial extracts and liver extracts of specific-pathogen-free rats showed that Venenum Bufonis produced significant metabolic perturbations dose-dependently with a distinct time effect, peaking at 2 hr after dosing and attenuating gradually. Clinical chemistry, electrocardiographic recordings, and histopathological evaluation provided additional evidence of Venenum Bufonis-induced cardiac damage that complemented and supported the metabolomics findings. The combined results demonstrated that oxidative stress, mitochondrial dysfunction, and energy metabolism perturbations were associated with the cardiac damage that results from Venenum Bufonis.     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

1H NMR-Based Metabolite Profiling of Planktonic and Biofilm Cells in Acinetobacter baumannii 1656-2

Acinetobacter baumannii is an aerobic and gram-negative pathogenic bacterium that is resistant to most antibiotics. Recently, A. baumannii 1656-2 exhibited the ability to form biofilms under clinical conditions. In this study, global metabolite profiling of both planktonic and biofilm forms of A. baumannii 1656-2 was performed using high-resolution nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analysis to investigate the metabolic patterns leading to biofilm formation. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed a distinct separation between planktonic and biofilm cells. Metabolites including acetates, pyruvate, succinate, UDP-glucose, AMP, glutamate, and lysine were increasingly involved in the energy metabolism of biofilm formation. In particular, the ratio of N-acetyl-D-glucosamine (GlcNAc) to D-glucosamine (GlcNH₂) was significantly higher during biofilm formation than under the planktonic condition. This study demonstrates that NMR-based global metabolite profiling of bacterial cells can provide valuable insight into the metabolic changes in multidrug resistant and biofilm-forming bacteria such as A. baumannii 1656-2.     

1H-MRS评估慢性肝病脑部代谢异常与肝硬化的相关性

目的:利用~1H-MRS研究慢性肝病脑部代谢改变,并探讨~1H-MRS评估慢性肝病脑部代谢异常与肝硬化Child-Pugh分级的相关性。方法:选取经临床确诊为慢性肝炎肝硬化患者42例(child A 19例,child B14例,child C 9例)及健康志愿者15例(对照组),行磁共振平扫及磁共振单体素~1H-MRS检查,计算相关代谢物N-乙酰天门冬氨酸(NAA)、谷氨酰胺复合物(Glx)、胆碱(Cho)、肌醇(mI)和肌酸(Cr)的峰下面积及前四项指标与Cr的比值(NAA/Cr、Glx/Cr、Cho/Cr、mI/Cr),并进行统计学分析,同时对相关代谢物的变化与肝硬化Child-Pugh分级及肝硬化Child-Pugh分级与肝性脑病的关系进行相关性分析。结果:~1HMRS分析显示与正常对照组相比,慢性肝炎肝硬化组Glx/Cr值升高,Cho/Cr与mI/Cr值降低,且差异均有统计学意义(P0.05);不同程度肝硬化病例组对比显示,Glx/Cr值均随着肝硬化程度加重而增大,且Glx/Cr值的差异在child A、child B、child C组中均有统计学意义(P0.05);肝性脑病(HE)组与非肝性脑病组脑代谢物峰下面积比值Glx/Cr、Cho/Cr、mI/Cr比较,差异有统计学意义(P0.05);Child-Pugh分级与Glx/Cr呈正相关,与Cho/Cr、mI/Cr呈负相关;随着肝硬化程度加重,肝性脑病出现概率越高,差异有统计学意义(P0.05)。结论:~1H-MRS作为一种无创性的评价手段,能够反映慢性肝硬化及肝性脑病患者存在脑代谢物浓度异常改变,可作为早期诊断肝硬化、肝性脑病及评价肝硬化、肝性脑病严重程度的一项指标,在一定程度上评估肝性脑病与肝硬化分级具有相关性。     

A Four-Compartment Metabolomics Analysis of the Liver,Muscle, Serum,and Urine Response to Polytrauma with Hemorrhagic Shock following Carbohydrate Prefeed

Objective

Hemorrhagic shock accompanied by injury represents a major physiologic stress. Fasted animals are often used to study hemorrhagic shock (with injury). A fasted state is not guaranteed in the general human population. The objective of this study was to determine if fed animals would exhibit a different metabolic profile in response to hemorrhagic shock with trauma when compared to fasted animals.

Methods

Proton (1H) NMR spectroscopy was used to determine concentrations of metabolites from four different compartments (liver, muscle, serum, urine) taken at defined time points throughout shock/injury and resuscitation. PLS-DA was performed and VIP lists established for baseline, shock and resuscitation (10 metabolites for each compartment at each time interval) on metabolomics data from surviving animals.

Results

Fed status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The death rate for CPF animals is higher than FS animals (47 vs 28%). The majority of deaths occur post-resuscitation suggesting reperfusion injury. The metabolomics response to shock reflects priorities evident at baseline. FS animals raise the baseline degree of proteolysis to provide additional amino acids for energy production while CPF animals rely on both glucose and, to a lesser extent, amino acids. During early resuscitation levels of metabolites associated with energy production drop, suggesting diminished demand.

Conclusions

Feeding status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The response to shock reflects metabolic priorities at baseline.     

Diagnosing Fatty Liver Disease: A Comparative Evaluation of Metabolic Markers,Phenotypes, Genotypes and Established Biomarkers

Background

To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted.

Aim

We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes.

Methods

The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV).

Results

Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD.

Conclusion

We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.     

Metabolic Profiles are Principally Different between Cancers of the Liver,Pancreas and Breast

Molecular profiling of primary tumors may facilitate the classification of patients with cancer into more homogenous biological groups to aid clinical management. Metabolomic profiling has been shown to be a powerful tool in characterizing the biological mechanisms underlying a disease but has not been evaluated for its ability to classify cancers by their tissue of origin. Thus, we assessed metabolomic profiling as a novel tool for multiclass cancer characterization. Global metabolic profiling was employed to identify metabolites in paired tumor and non-tumor liver (n=60), breast (n=130) and pancreatic (n=76) tissue specimens. Unsupervised principal component analysis showed that metabolites are principally unique to each tissue and cancer type. Such a difference can also be observed even among early stage cancers, suggesting a significant and unique alteration of global metabolic pathways associated with each cancer type. Our global high-throughput metabolomic profiling study shows that specific biochemical alterations distinguish liver, pancreatic and breast cancer and could be applied as cancer classification tools to differentiate tumors based on tissue of origin.     

Exchange of histones H1, H2A, and H2B in vivo

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.     

Investigation of flexibility of neuraminidase 150-loop using tamiflu derivatives in influenza A viruses H1N1 and H5N1

This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite (“150-cavity”) adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported K_i values determined at non-physiological pH should be carefully scrutinized.     

A Guide to the Metabolic Pathways and Function of Metabolites Observed in Human Brain 1H Magnetic Resonance Spectra

The current knowledge of the normal biochemistry of compounds that give rise to resonances in human brain proton magnetic resonance spectra measureable at readily available field strengths (i.e. ≤3 T) is reviewed. Molecules covered include myo- and scyllo-inositol, glycerophospho- and phospho-choline and choline, creatine and phosphocreatine, N-acetylaspartate, N-acetylaspartylglutamate, glutamate, glutamine, γ-aminobutyrate, glucose, glutathione and lactate. The factors which influence changes in the levels of these compounds are discussed. As most proton resonances in the brain at low field are derived from a combination of moieties whose biochemistry is complex and interrelated, an understanding of the mechanisms underlying why these species change is crucial to meaningful interpretation of human brain spectra.     

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium using 1H NMR spectroscopy

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.     

Collagen Peptides in Urine: A New Promising Biomarker for the Detection of Colorectal Liver Metastases

Introduction

For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls.

Materials and Methods

Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM''s) were developed on a UPLC-MS/MS system.

Results

Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%.

Conclusion

Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.     

血清及尿中Kim-1 及CysC 联合监测对急性肾衰竭的诊断意义

目的:探讨血清及尿中Kim-1及CysC联合监测在临床诊断急性肾衰竭中的意义及价值。方法:选取我院重症监护病房的泌尿系统疾病患者108例根据急性肾衰竭诊断标准将其分为两组:58例符合急性肾衰竭诊断标准为ARF组,50例非急性肾衰竭为非ARF组,另取正常体检的健康人5 0例为正常对照组,检测三组患者的血清肌酐浓度、血清尿素氮、血清和尿KIM-1及Cys-C浓度,进而对血清和尿KIM-1及Cys-C水平进行分析。结果:与对照组和非ARF组相比,ARF组血清肌酐浓度、血清尿素氮、血清和尿KIM-1及Cys-C水平较高(P0.05)。与血清和尿KIM-1及Cys-C水平的单独检测相比,两者联合检测敏感性以及特异性较高(P0.05)。结论:血清及尿中Kim-1及CysC水平升高可作为急性肾衰竭的评估指标,两者联合监测对急性肾衰竭患者的诊断具有重要意义,对疾病预后有重要指导价值。     

Association of the Common Catalase Gene Polymorphism rs1001179 With Glycated Hemoglobin and Plasma Lipids in Hyperlipidemic Patients

Catalase represents perhaps the most effective antioxidant defense in the body under conditions of increased oxidative stress, and rs1001179 (CAT-262C >T) is its most extensively studied gene polymorphism. Using an established PCR–RFLP method for genotyping, we examined the association of rs1001179 with glycated hemoglobin (HbA1c) and plasma lipids using univariate analyses with age, sex, body mass index (BMI), smoking, and alcohol abuse as covariates, in a group of dyslipidemic patients from northern Greece. Our results suggest that the TT genotype is a risk factor for increased HbA1c and plasma triglycerides, and that this association is modulated by the BMI and/or age of the patients.