Altered plasma acylcarnitine and amino acid profiles in type 2 diabetic kidney disease

Introduction

Dysregulation of acylcarnitines (AcylCNs) and amino acids metabolism have implicated in abnormality of fatty acid oxidation in type 2 diabetes (T2D). However, it is not well known whether altered plasma AcylCN, and amino acid profiles are associated with albuminuria or diabetic nephropathy (DN) in T2D.

Objective

The aim of this study was to elucidate alterations in plasma levels of AcylCNs and amino acids with respect to the T2D patients with various stages of albuminuria.

Methods

We recruited 52 healthy subjects as control, and 156 T2D patients which were divided into 52 normoalbuminuria, 52 microalbuminuria, and 52 macroalbuminuria. Plasma 37 AcylCNs and 12 amino acids were analyzed by tandem mass spectrometry.

Results

We found that T2D with normoalbuminuria and microalbuminuria had lower shot-, medium-, and long-chain AcylCNs, whereas T2D with macroalbuminuria had higher short-and medium-chain AcylCNs and lower long-chain AcylCNs than healthy subjects. Moreover, estimated glomerular filtration rate (eGFR) was a negative, independent and significant predictor of albumin to creatinine ratio (ACR) levels (β = ?0.376, P < 0.001), whereas plasma Low-density lipoprotein cholesterol (LDL-C) was significantly and positively associated with ACR levels (β = 0.169, P = 0.049). Furthermore, multivariate ordinal logistic regression analysis revealed that isobutyrylcarnitine (C4) was a positive, independent, and significant predictor of ACR levels with higher odds of having T2D patients with progression normoalbuminuria to microalbuminuria [OR = 9.93, 95 % CI (3.51–28.05), P < 0.001].

Conclusions

The findings suggest that plasma C4 may serve as a potential biomarker for the early stages of DN.

     

Metabolomics meets functional assays: coupling LC–MS and microfluidic cell-based receptor-ligand analyses

Introduction

Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.

Objective

We aimed to combine LC–MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.

Methods

We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity.

Results

Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel.

Conclusion

Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC–MS.

     

Clinical relevance of glycerophospholipid,sphingomyelin and glutathione metabolism in the pathogenesis of pharyngolaryngeal cancer in smokers: the Korean Cancer Prevention Study-II

Introduction

Although smoking is a major risk factor for pharyngolaryngeal cancer, most smokers do not develop pharyngolaryngeal cancer.

Objectives

In the prospective Korean Cancer Prevention Study-II (KCPS-II), we investigated the application of metabolomics to differentiate smokers with incident pharyngolaryngeal cancer (pharyngolaryngeal cancer group) from smokers who remained free from cancer (controls) during a mean follow-up period of 7 years and aimed to discover valuable early biomarkers of pharyngolaryngeal cancer.

Methods

We used baseline serum samples from 30 smoking men with incident pharyngolaryngeal cancer and 59 age-matched cancer-free smoking men. Metabolic alterations associated with the incidence of pharyngolaryngeal cancer were investigated by performing metabolomics on baseline serum samples using ultra-performance liquid chromatography-linear-trap quadrupole-Orbitrap mass spectrometry.

Results

Compared to the control group, the pharyngolaryngeal cancer group showed significantly higher oxidized LDL levels. Seventeen metabolites were differentially abundant between the two groups. At baseline, compared to controls, smokers with incident pharyngolaryngeal cancer during follow-up showed significantly higher levels of pyroglutamic acid (glutathione metabolism) but lower levels of lysophosphatidylcholines (lysoPCs) C14:0, C15:0, C16:0, C17:0, C18:0, and C20:5; glycerophosphocholine; PC C36:5; lysoPEs C16:0, C20:1, and C22:0 (glycerophospholipid metabolism); SM (d18:0/16:1); and SM (d18:1/18:1) (sphingomyelin metabolism). Furthermore, smokers with incident pharyngolaryngeal cancer showed significantly higher levels of oleamide and lower levels of tryptophan and linoleyl carnitine at baseline than cancer-free smokers.

Conclusion

This prospective study showed the clinical relevance of dysregulated metabolism of glutathione, glycerophospholipids and sphingolipids to the pathogenesis of pharyngolaryngeal cancer among smokers. These data suggest that the dysregulation of these metabolic processes may be a key mechanism underlying pharyngolaryngeal cancer progression and development.

     

Real-time metabolomic analysis of lactic acid bacteria as monitored by in vitro NMR and chemometrics

Introduction

Lactic acid bacteria (LAB) play an important role in the food industry as starter cultures to manufacture fermented food, and as probiotics. In recent years, there has been an increasing interest in using LAB cultures for biopreservation of food products. It is therefore of great interest to study the detailed metabolism of these bacteria.

Objectives

This study aimed at developing an efficient analytical protocol for real-time in vitro NMR measurements of LAB fermentations, from sample preparation, over data acquisition and preprocessing, to the extraction of the kinetic metabolic profiles.

Method

The developed analytical protocol is applied to an experimental design with two LAB strains (Lactobacillus rhamnosus DSM 20021 and Lactobacillus plantarum subsp. plantarum DSM 20174), two initial pH levels (pH_i 6.5 and 5.5), two levels of glucose concentration (2.5 and 0.25 g/l), and two batch fermentation replicates.

Results

The design factors proved to be strongly significant and led to interesting biological information. The protocol allowed for detailed real-time kinetic analysis of 11 major metabolites involved in the glycolysis, pyruvate catabolism, amino acid catabolism and cell energy metabolism. New biological knowledge was obtained about the different patterns of glutamine and aspartic acid consumption by the two strains. It was observed that L. plantarum consumes more glutamine at low pH (pH 5.5) whereas the opposite applies to L. rhamnosus. Regarding aspartic acid, both of the strains consume it higher at low pH, and overall L. plantarum consumes it more. L. rhamnosus did not consume aspartic acid at pH 6.5.

Conclusion

The developed analytical protocol for real-time in vitro NMR measurements of bacterial metabolism allows a relatively easy investigation of different fermentation factors such as new strains, new substrates, cohabitations, temperature, and pH and has a great potential in biopreservation studies to discover new efficient bioprotective cultures.

     

Coupling solid phase microextraction to complementary separation platforms for metabotyping of <Emphasis Type="Italic">E. coli</Emphasis> metabolome in response to natural antibacterial agents

Introduction

Essential oils are known to possess antimicrobial activity; thus, their use has played an important role over the years in medicine and for food preservation purposes.

Objective

The effect of clove oil and its major constituents as bactericidal agents on the global metabolic profiling of E. coli bacteria was assessed by means of metabolic alterations, using solid phase microextraction (SPME) as a sample preparation method coupled to complementary analytical platforms.

Method

E. Coli cultures treated with clove oil and its major individual components were sampled by HS-SPME-GCxGC-ToF/MS and SPME-UPLC–MS. Full factorial design was applied in order to estimate the most effective antibacterial agent towards E. coli. Central composite design and factorial design were applied to investigate parameters influencing metabolite coverage and efficiency by SPME.

Results

The metabolic profile, including 500 metabolites identified by LC–MS and 789 components detected by GCxGC-ToF/MS, 125 of which were identified as dysregulated metabolites, revealed changes in the metabolome provoked by the antibacterial activity of clove oil, and in particular its major constituent eugenol. Analyses of individual components selected using orthogonal projections to latent structures discriminant analysis showed a neat differentiation between control samples in comparison to treated samples in various sets of metabolic pathways.

Conclusions

The combination of a sample preparation method capable of providing cleaner extracts coupled to different analytical platforms was successful in uncovering changes in metabolic pathways associated with lipids biodegradation, changes in the TCA cycle, amino acids, and enzyme inhibitors in response to antibacterial treatment.

     

Metabolic fingerprints of human primary endothelial and fibroblast cells

Introduction

Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders.

Objectives

In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles.

Methods

Biolog Phenotype MicroArray? technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF).

Results

Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides.

Conclusion

Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.

     

Comprehensive <Superscript>1</Superscript>H NMR metabolic profiling of body fluids for differentiation of meningitis in adults

Introduction

Meningitis, a morbidly infectious central nervous system pathology is accompanied by acute inflammation of the meninges, causing raised intracranial pressure linked with serious neurological sequelae.

Objective

To observe the variation in the metabolic profile, that may occur in serum and urine along with CSF in adults using ¹H NMR spectroscopy, with an attempt of appropriate and timely treatment regimen.

Methods

The ¹H NMR-based metabolomics has been performed in 115 adult subjects for differentiating bacterial meningitis (BM) and tubercular meningitis (TBM).

Results

The discriminant function analysis (DFA) of the three bio-fluids collectively identified 3-hydroxyisovalerate, lactate, glucose, formate, valine, alanine, ketonic bodies, malonate and choline containing compounds (choline and GPC) as significant metabolites among cases versus control group. The differentiation of bacterial meningitis and tuberculous meningitis (BM vs. TBM) can be done on the basis of identification of 3-hydroxyisovalerate, isobutyrate and formate in case of CSF (with a correct classification of 78 %), alanine in serum (correct classification 60 %), valine and acetone in case of urine (correct classification 89.1 %). The NMR spectral bins based orthogonal signal correction principal component analysis score plots of significant metabolites obtained from DFA also provided group classification among cases versus control group in CSF, serum and urine samples. The variable importance in projection scores also identified similar significant metabolites as obtained from DFA, collectively in CSF, serum and urine samples, responsible for differentiation of meningitis.

Conclusion

The CSF contained metabolites which are formed during infection and inflammation, and these were also found in significant quantity in serum and urine samples.

     

Metabolomic study of the bone trabecula of osteonecrosis femoral head patients based on UPLC–MS/MS

The severity and/or progression of osteonecrosis of the femoral head (ONFH) are commonly assessed by radiography, nuclear magnetic resonance image which aren’t invariably correlated to severity of disease and may be disturbed by other factors. Consequently, exploring the novel biochemical signatures of ONFH may be beneficial for diagnosing and understanding this disease. In this work, a bone trabecula metabolomics was undertaken to determine the expression pattern of low molecular mass metabolites in patients of femoral head necrosis based on the ultra-performance liquid chromatography/time-of-flight tandem mass spectrometry (UPLC/TOF MS/MS). Histological study showed that necrotic bone was characterized by necrosis, fibrosis and lacuna, but adjacent “normal” bone was pathologically normal. Principal component analysis in combination with orthogonal partial least-squares discrimination analysis was used to find out changed metabolites. MS/MS was used to speculate the corresponding molecule. Both osteonecrotic bone trabecula (ONBT) and adjacent “normal” bone trabecula (ANBT) showed higher levels of amino acids, such as proline, arginine, glutamine, dipeptides and lower levels of antioxidants. Most disrupted lipids, such as fatty acid esters, glycerophospholipids, sphingolipids, were found in osteonecrotic zone. The area under the receiver operating characteristic curve of combinational biomarkers (d-arginine, l-proline, l-carnitine, inosine) in ONBT and ANBT was 0.996 and 0.950, respectively. Our findings might provide a significant insight to understand the metabolic mechanism and diagnosis biomarkers of ONFH in the future.