The study of mammalian metabolism through NMR-based metabolomics

High-resolution NMR spectroscopy has been widely used to monitor metabolism almost since the technique's development. It is now one of the principle technologies used in metabolomics, to profile the metabolite compliment of a cell, tissue, organism, or biofluid. This chapter describes how tissue extracts are prepared for NMR spectroscopy and, in particular, focuses on two approaches based on perchloric acid and methanol/chloroform extractions. This is followed by a description of key NMR experiments that can be used to profile tissue extracts, biofluids, or intact tissues. While these NMR techniques should be optimized for a particular sample set, we provide some tried and tested starting parameters for these experiments which should allow the user to acquire good quality spectra

Metabolomic study of the LDL receptor null mouse fed a high-fat diet reveals profound perturbations in choline metabolism that are shared with ApoE null mice

Failure to express or expression of dysfunctional low-density lipoprotein receptors (LDLR) causes familial hypercholesterolemia in humans, a disease characterized by elevated blood cholesterol concentrations, xanthomas, and coronary heart disease, providing compelling evidence that high blood cholesterol concentrations cause atherosclerosis. In this study, we used 1H nuclear magnetic resonance spectroscopy to examine the metabolic profiles of plasma and urine from the LDLR knockout mice. Consistent with previous studies, these mice developed hypercholesterolemia and atherosclerosis when fed a high-fat/cholesterol/cholate-containing diet. In addition, multivariate statistical analysis of the metabolomic data highlighted significant differences in tricarboxylic acid cycle and fatty acid metabolism, as a result of high-fat/cholesterol diet feeding. Our metabolomic study also demonstrates that the effect of high-fat/cholesterol/cholate diet, LDLR gene deficiency, and the diet-genotype interaction caused a significant perturbation in choline metabolism, notably the choline oxidation pathway. Specifically, the loss in the LDLR caused a marked reduction in the urinary excretion of betaine and dimethylglycine, especially when the mice are fed a high-fat/cholesterol/cholate diet. Furthermore, as we demonstrate that these metabolic changes are comparable with those detected in ApoE knockout mice fed the same high-fat/cholesterol/cholate diet they may be useful for monitoring the onset of atherosclerosis across animal models

Kinetic and reactor modelling of lipases catalyzed (R,S)-1-phenylethanol resolution

This study was focused on the development of a kinetic model and a reactor model for the enzymatic resolution of (R,S)-1-phenylethanol. The reaction progress curves catalyzed by immobilized lipases, ChiroCLEC-PC in batch stirred tank reactor were used to develop the kinetic model. The resolution followed Ping-Pong Bi-Bi mechanism with the inhibition of lauric acid, (R,S)-1-phenylethanol and water. The validity of the model was verified by fitting it to another experimental data catalyzed by immobilized lipases, Chirazyme L2, c.-f., C3, lyo at the same reaction conditions. The rate equation was then applied for the development of reactor model in a recirculated packed bed reactor system. The overall effectiveness factor and Peclet number were used to determine the mass transfer and axial dispersion limitation in the reactor performance. The reactor model was verified by fitting it to the larger scale reactor data with the correlation coefficient value more than 0.99

Momordica charantia suppresses inflammation and glycolysis in lipopolysaccharide-activated RAW264.7 macrophages

Macrophage activation is a key event that triggers inflammatory response. The activation is accompanied by metabolic shift such as upregulated glucose metabolism. There are accumulating evidences showing the anti-inflammatory activity of Momordica charantia. However, the effects of M. charantia on inflammatory response and glucose metabolism in activated macrophages have not been fully established. The present study aimed to examine the effect of M. charantia in modulating lipopolysaccharide (LPS)-induced inflammation and perturbed glucose metabolism in RAW264.7 murine macrophages. The results showed that LPS-induced NF-?B (p65) nuclear translocation was inhibited by M. charantia treatment. In addition, M. charantia was found to reduce the expression of inflammatory genes including IL6, TNF-a, IL1ß, COX2, iNOS, and IL10 in LPS-treated macrophages. Furthermore, the data showed that M. charantia reduced the expression of GLUT1 and HK2 genes and lactate production (-28%), resulting in suppression of glycolysis. Notably, its effect on GLUT1 gene expression was found to be independent of LPS-induced inflammation. A further experiment also indicated that the bioactivities of M. charantia may be attributed to its key bioactive compound, charantin. Taken together, the study provided supporting evidences showing the potential of M. charantia for the treatment of inflammatory disorders

Molecular Docking and Pharmacokinetics Analysis of Phytochemicals from Piper caninum as Dengue NS2B-NS3 Protease Inhibitors

Dengue fever affects 390 million people each year. Currently, there is no specific medicine to treat this disease. Thus, the search for potential NS2B-NS3 protease inhibitors has attracted increasing research interest. The dengue protease NS2B-NS3 was used as a molecular target because of its vital function in viral replication. Piper caninum belongs to the Piperaceae family, found inhabited in Malaysia and Indonesia. This plant possesses various phytochemicals with various health benefits. However, the anti-dengue activity of this plant is yet to be discovered. Therefore, the objective of this research is to evaluate the inhibitory activity of phytochemicals from P. caninum against NS2B-NS3 using in silico experimentation. Molecular docking using AutoDock Vina was utilized to identify the binding interaction of phytochemicals on NS2B-NS3. SwissADME and ProTox-II web servers were used to analyse the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of the phytochemicals. Results showed that cepharadione A, bornyl caffeate, and (+)-bornyl p-coumarate had comparable molecular interaction with the reference compound, curcumin. Analysis of in silico pharmacokinetics properties revealed that these phytochemicals have good pharmacokinetics profiles and excellent drug-ability, which obeyed Lipinski’s Rule of Five. This study shows the potential inhibitory activity of the phytochemicals against NS2B-NS3 for a lead in the development of dengue inhibitors

Effect of extraction solvents on the phytochemical content and bioactivity of Momordica charantia Linn. fruits

Momordica charantia (M. charantia) is a herbaceous climber commonly found in Southeast Asia with therapeutic importance for various illnesses. This study focused on the effect of extraction solvents on saponins-containing compounds from M. charantia and their bioactivities. Different organic solvents including water, ethanol, ethyl acetate, a mixture of methanol-water and methanol-n-butanol were used in the extraction process. The total saponin content, total flavonoid and phenolic content for each extract were examined. In addition, the antioxidant capacity of these extracts were evaluated using both 1,1-diphenyl-2-picrylhydrazyl (DPPH) Free Radical Scavenging Activity and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay. Furthermore, a-amylase and lipase inhibition assay were carried out using an in vitro model. The result showed that methanol-n-butanol extracts exhibited the highest total saponin, flavonoid, phenolic content, and ABTS antioxidant activity compared to the other extracts. The a-amylase inhibition assay revealed that water extract and methanol-n-butanol extract from M. charantia contained potent a-amylase inhibitor. On the other hand, the ethyl acetate extract was found to have the most antioxidant capacities based on DPPH radical scavenging assay. The ethyl acetate extract also exhibited the highest inhibition of lipase activities. In conclusion, the methanol-n-butanol solvent was found to be the most effective in extracting saponin from M. charantia. The M. charantia extracts showed inhibition of a-amylase and lipase activities which may suggest the therapeutic potential of M. charantia for obesity and diabetes

Metabolomics of the interaction between PPAR-α and age in the PPAR-α-null mouse

Regulation between the fed and fasted states in mammals is partially controlled by peroxisome proliferator-activated receptor-α (PPAR-α). Expression of the receptor is high in the liver, heart and skeletal muscle, but decreases with age. A combined 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry metabolomic approach has been used to examine metabolism in the liver, heart, skeletal muscle and adipose tissue in PPAR-α-null mice and wild-type controls during ageing between 3 and 13 months. For the PPAR-α-null mouse, multivariate statistics highlighted hepatic steatosis, reductions in the concentrations of glucose and glycogen in both the liver and muscle tissue, and profound changes in lipid metabolism in each tissue, reflecting known expression targets of the PPAR-α receptor. Hepatic glycogen and glucose also decreased with age for both genotypes. These findings indicate the development of age-related hepatic steatosis in the PPAR-α-null mouse, with the normal metabolic changes associated with ageing exacerbating changes associated with genotype. Furthermore, the combined metabolomic and multivariate statistics approach provides a robust method for examining the interaction between age and genotype

Deciphering the metabolic perturbation in hepatic alveolar echinococcosis: a 1 H NMR-based metabolomics study

Background: Hepatic alveolar echinococcosis (HAE) is caused by the growth of Echinococcus multilocularis larvae in the liver. It is a chronic and potentially lethal parasitic disease. Early stage diagnosis for this disease is currently not available due to its long asymptomatic incubation period. In this study, a proton nuclear magnetic resonance (1H NMR)-based metabolomics approach was applied in conjunction with multivariate statistical analysis to investigate the altered metabolic profiles in blood serum and urine samples obtained from HAE patients. The aim of the study was to identify the metabolic signatures associated with HAE. Results: A total of 21 distinct metabolic differences between HAE patients and healthy individuals were identified, and they are associated with perturbations in amino acid metabolism, energy metabolism, glyoxylate and dicarboxylate metabolism. Furthermore, the present results showed that the Fischer ratio, which is the molar ratio of branched-chain amino acids to aromatic amino acids, was significantly lower (P < 0.001) in the blood serum obtained from the HAE patients than it was in the healthy patient group. Conclusions: The altered Fischer ratio, together with perturbations in metabolic pathways identified in the present study, may provide new insights into the mechanistic understanding of HAE pathogenesis and potential therapeutic interventions

Metabolomics analysis of herb-partitioned moxibustion treatment on rats with diarrhea-predominant irritable bowel syndrome

Background: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, which is commonly treated with antidiarrhoeal, antispasmodics, serotonergic agents or laxative agents. These treatments provide relief for IBS symptoms but may also lead to undesired side effects. Previously, herb-partitioned moxibustion (HPM) treatment has been demonstrated to be effective in ameliorating symptoms of IBS. However, the underlying mechanism of this beneficial treatment is yet to be established. The aim of the current study was to systematically assess the metabolic alterations in response to diarrhea-predominant IBS (IBS-D) and therapeutic effect of HPM. Methods: Proton nuclear magnetic resonance spectroscopy (1H NMR)-based metabolomics approach was used to investigate fecal and serum metabolome of rat model of IBS-D with and without HPM treatment. Results: The current results showed that IBS-induced metabolic alterations in fecal and serum sample include higher level of threonine and UDP-glucose together with lower levels of aspartate, ornithine, leucine, isoleucine, proline, 2-hydroxy butyrate, valine, lactate, ethanol, arginine, 2-oxoisovalerate and bile acids. These altered metabolites potentially involve in impaired gut secretory immune system and intestinal inflammation, malabsorption of nutrients, and disordered metabolism of bile acids. Notably, the HPM treatment was found able to normalize the Bristol stool forms scale scores, fecal water content, plasma endotoxin level, and a number of IBS-induced metabolic changes. Conclusions: These findings may provide useful insight into the molecular basis of IBS and mechanism of the HPM intervention