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Metabolic Assessment of Human Liver Transplants from Biopsy Samples at the Donor and Recipient Stages Using High-Resolution Magic Angle Spinning 1H NMR Spectroscopy
Abstract
This work presents the first application of high-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy to human liver biopsy samples, allowing a determination of their metabolic profiles before removal from donors, during cold perfusion, and after implantation into recipients. The assignment of peaks observed in the 1H HR-MAS NMR spectra was aided by the use of two-dimensional J-resolved, TOCSY and 1H-13C HMQC spectra. The spectra were dominated by resonances from triglycerides, phospholipids, and glycogen and from a variety of small molecules including glycerophosphocholine (GPC), glucose, lactate, creatine, acetate, amino acids, and nucleoside-related compounds such as uridine and adenosine. In agreement with histological data obtained on the same biopsies, two of the six livers were found to contain high amounts of triglycerides by NMR spectroscopy, which also indicated that these tissues contained a higher degree of unsaturated lipids and a lower proportion of phospholipids and low molecular weight compounds. Additionally, proton T2 relaxation times indicated two populations of lipids, a higher mobility triglyceride fraction and a lower mobility phospholipid fraction, the proportions of which changed according to the degree of fat content. GPC was found to decrease from the pretransplant to the posttransplant biopsy of all livers except for one with a histologically confirmed high lipid content, and this might represent a biomarker of liver function posttransplantation. NMR signals produced by the liver preservation solution were clearly detected in the cold perfusion stage biopsies of all livers but remained in the posttransplant spectra of only the two livers with a high lipid content and were prominent mainly in the graft that later developed primary graft dysfunction. This study has shown biochemical differences between livers used for transplants that can be related to the degree and type of lipid composition. This technology might therefore provide a novel screening approach for donor organ quality and a means to assess function in the recipient after transplantation.
... Further MRI studies in vivo have demonstrated a close relation between hepatic steatosis and body adiposity, and a close correlation between MRI estimation of adiposity and histological assessment in two of these patients [11] . Although studies have shown a close association between in vivo estimates and biopsies, in vitro MRS assessment of lipid content in liver biopsies by MAS MRS would allow direct comparison with histology, reducing the effect of sampling error [33] . ...
... In a preliminary study, Duarte and colleagues used in vitro 1 H MAS MRS to assess biopsies taken at three time-points from six livers, before removal from donors, during cold perfusion and following implantation into the recipient [33] . The biopsies with the highest concentration of peaks reflecting fatty acyl chain (triglyceride) resonances were also identified as those also estimated to have the highest fat content on histological analysis. ...
... The biopsies with the highest concentration of peaks reflecting fatty acyl chain (triglyceride) resonances were also identified as those also estimated to have the highest fat content on histological analysis. Other metabolites were identified, including glycerophosphocholine (GPC), which were reported to decrease from pre-to post-transplant [33] . However, in further studies such spectral changes need to be correlated with a range of clinical endpoints, including the premorbid clinical history of the liver donor, the pretransplantation clinical history and nutritional status of the recipient, the subsequent post-transplantation liver function tests, pre-and post-transplantation indices of nutrition in the recipient and ultimately, the final clinical outcome. ...
... The spectra were processed by Java based magnetic resonance user interface (jMRUI, V6.0, Leuven, Belgium) using accurate, robust and e cient spectral (AMARES) tting of the MRS data. The chemical shifts of the main metabolites are inferred from the results of ex vivo 1 H-NMRS in liver biopsy specimens [22][23][24], the position of the glycogen peaks were assigned according to previous publication [23,25]. Correction factors for estimating lipid and glycogen content were used as previously described [2]. ...
... The spectra were processed by Java based magnetic resonance user interface (jMRUI, V6.0, Leuven, Belgium) using accurate, robust and e cient spectral (AMARES) tting of the MRS data. The chemical shifts of the main metabolites are inferred from the results of ex vivo 1 H-NMRS in liver biopsy specimens [22][23][24], the position of the glycogen peaks were assigned according to previous publication [23,25]. Correction factors for estimating lipid and glycogen content were used as previously described [2]. ...
Purpose
Glycogenic hepatopathy is associated with significant psychosocial consequences and health costs. Metabolic Dysfunction-Associated Steatotic Liver Disease and glycogenic hepatopathy are frequently confused as “fatty liver” when seen on ultrasonography. We wished to examine liver fat and glycogen content in groups defined based on metabolic and liver disease phenotypes.
Methods
This case-control study undertaken in a tertiary hospital used nuclear proton magnetic resonance spectroscopy to examine liver fat and glycogen content in five clinical groups, each containing five participants: 1. type 1 diabetes with glycogenic hepatopathy, 2. satisfactorily controlled type 1 diabetes with no liver disease, 3. poorly controlled type 1 diabetes without liver disease, 4. a control group of body mass index- and age-matched individuals without diabetes or liver disease, and 5. hepatic steatosis.
Results
Fat content was highest in the hepatic steatosis (median 15.4%, IQR 10.0-19.3) and glycogenic hepatopathy (median 6.5%, IQR 4.5–9.1) groups and compared to both of these groups was lower in the control group (median 1.0%, IQR 0.7–1.1, p 0.002 and 0.022), the T1DM group with satisfactory control (median 0.3%, IQR 0.2–0.6, p < 0.001 and < 0.001), and the T1DM group with poor control without liver disease (median 1.1%, IQR 0.9–1.1, p 0.001 and 0.012).
Conclusions
The hyperechoic ultrasonographic liver changes seen in type 1 diabetes mellitus are often termed “fatty liver”, but may relate to glycogen accumulation and not fat. In cases diagnosed with glycogenic hepatopathy there may be significant concomitant fat accumulation, compounding the already elevated cardiovascular risk in this cohort.
... The first use of NMR spectroscopy on human liver samples was reported in 2005, determining metabolic profiles before organ retrieval, during HMP and after Tx [62]. The revealed variations in donor livers were consistent in most donors. ...
... First, GPC decreased in the majority of livers, suggesting increased cell turnover. Interestingly, in the graft that developed PGD, GPC remained stable, probably reflecting a lower degree of cellular activity, and therefore this substance might be a new biomarker for liver function [62]. ...
In solid organ transplantation (Tx), both survival rates and quality of life have improved dramatically over the last few decades. Each year, the number of people on the wait list continues to increase, widening the gap between organ supply and demand. Therefore, the use of extended criteria donor grafts is growing, despite higher susceptibility to ischemia-reperfusion injury (IRI) and consecutive inferior Tx outcomes. Thus, tools to characterize organ quality prior to Tx are crucial components for Tx success. Innovative techniques of metabolic profiling revealed key pathways and mechanisms involved in IRI occurring during organ preservation. Although large-scale trials are needed, metabolomics appears to be a promising tool to characterize potential biomarkers, for the assessment of graft quality before Tx and evaluate graft-related outcomes. In this comprehensive review, we summarize the currently available literature on the use of metabolomics in solid organ Tx, with a special focus on metabolic profiling during graft preservation to assess organ quality prior to Tx.
... The possibility to correlate metabolic alterations and histopathologic features of a surgical tumor specimen may help decipher tumor heterogeneity and gain better molecular insights into tumor pathogenic mechanisms [5,13]. Indeed, 1 H HR-MAS NMR is now increasingly used for the characterization of tumor lesions in humans, including brain [14,15], breast [16], liver [17], colon [18], and prostate cancers [19]. Notably, 1 H HR-MAS NMR has also been applied to the study of articular cartilage degradation [20], intervertebral disc degeneration [21], gamma-radiated bones of non-cancer mouse models [22], and human bone mineral tissue [23]. ...
... The examination of relatively small amounts of tumor tissues could then be used to assess slight differences in the biochemical profile of the tissue samples prior to histopathology. In this respect, 1 H HR-MAS NMR spectroscopy is increasingly emerging as a highly versatile tool [6,11,[13][14][15]17,18]. ...
Multiple myeloma (MM) is a malignancy of plasma cells characterized by multifocal osteolytic bone lesions. Macroscopic and genetic heterogeneity has been documented within MM lesions. Understanding the bases of such heterogeneity may unveil relevant features of MM pathobiology. To this aim, we deployed unbiased¹H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) metabolomics to analyze multiple biopsy specimens of osteolytic lesions from one case of pathological fracture caused by MM. Multivariate analyses on normalized metabolite peak integrals allowed clusterization of samples in accordance with a posteriori histological findings. We investigated the relationship between morphological and NMR features by merging morphological data and metabolite profiling into a single correlation matrix. Data-merging addressed tissue heterogeneity, and greatly facilitated the mapping of lesions and nearby healthy tissues. Our proof-of-principle study reveals integrated metabolomics and histomorphology as a promising approach for the targeted study of osteolytic lesions.
... The unambiguous assignment of the resonance signals in the 1 H-NMR spectra was achieved by verification of the results through 2D NMR ( 1 H/ 1 H COSY, 1 H/ 1 H TOCSY, 1 H/ 13 C HSQC) spectroscopy. The assignment of the resonance signals of the specific metabolites in 1 H-NMR spectra of the samples studied were in agreement with the data published in the literature [42,43]. ...
... However, the lipid signals could be attenuated at the expense of the enhancement of the resonance signals of low molecular weight metabolites. The NMR spectral data we obtained are in agreement with the data published in the literature [42,43,67]. In the results obtained from the different CM, the differences detected (compared to unconditioned media) were basically a consequence of cell metabolization (as the glucose depletion for example). ...
Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.
... Liver tissue has been well studied by both MALDI imaging and NMR spectroscopy. [114][115][116][117][118][119][120][121] Liver disease, such as cirrhosis or fatty liver disease, as well as liver-based toxicity, profoundly impacts metabolic and lipidomic pathways. 117,118,122,123 Utilizing a combination of ex vivo NMR spectroscopy and MALDI imaging would allow for in-depth region-specific molecular analysis of liver samples. ...
Nuclear magnetic resonance (NMR) spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple‐negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several metabolites phospholipid species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy – MALDI imaging to advance metabolomic and lipidomic studies.
... Using this well-established [31][32][33][34][35] and validated [36] technology, we here compare metabolite profiles of a spectrum of indolent and aggressive TETs and identify enriched metabolic pathways through metabolite set enrichment analysis (MSEA). Since we investigate a rare form of cancer with only a small cohort of tumors available for analysis and no independent cohort of snap frozen tissue samples available for validation-implying that MSEA may be vulnerable to false positive results-we combine metabolic profiling for functional pathway elucidation with analysis of transcriptomic profiles of these pathways in a large, independent cohort of TETs (the TCGA database). ...
Simple Summary
Thymomas and thymic carcinomas (TCs) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. This is the first metabolomics investigation on thymic epithelial tumors employing nuclear magnetic resonance spectroscopy of tissue samples. We could detect and quantify up to 37 metabolites in the major tumor subtypes, including acetylcholine that was not previously detected in other non-endocrine cancers. A metabolite-based cluster analysis distinguished three clinically relevant tumor subgroups, namely indolent and aggressive thymomas, as well as TCs. A metabolite-based metabolic pathway analysis also gave hints to activated metabolic pathways shared between aggressive thymomas and TCs. This finding was largely backed by enrichment of these pathways at the transcriptomic level in a large, publicly available, independent TET dataset. Due to the differential expression of metabolites in thymic epithelial tumors versus normal thymus, pathways related to proline, cysteine, glutathione, lactate and glutamine appear as promising therapeutic targets. From these findings, inhibitors of glutaminolysis and of the downstream TCA cycle are anticipated to be rational therapeutic strategies. If our results can be confirmed in future, sufficiently powered studies, metabolic signatures may contribute to the identification of new therapeutic options for aggressive thymomas and TCs.
Abstract
Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public “The Cancer Genome Atlas” (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other non-endocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong “Warburg effect”, glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.
... Moreover, ischemia-dependent membrane lysis could account for the modulation of O-phosphocholine. Indeed, cell membrane disruption takes place in ischemic livers as a result of ATP depletion and activation of calcium-dependent phospholipases [14,15,17,26,33]. Consistently, different metabolites involved in the phospholipid turnover were found to be modulated in perfused compared to non-perfused grafts. ...
Machine perfusion (MP) allows the maintenance of liver cells in a metabolically active state ex vivo and can potentially revert metabolic perturbations caused by donor warm ischemia, procurement, and static cold storage (SCS). The present preclinical research investigated the metabolic outcome of the MP procedure by analyzing rat liver tissue, bile, and perfusate samples by means of high-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy. An established rat model of normothermic MP (NMP) was used. Experiments were carried out with the addition of an oxygen carrier (OxC) to the perfusion fluid (OxC-NMP, n = 5) or without (h-NMP, n = 5). Bile and perfusate samples were collected throughout the procedure, while biopsies were only taken at the end of NMP. Two additional groups were: (1) Native, in which tissue or bile specimens were collected from rats in resting conditions; and (2) SCS, in which biopsies were taken from cold-stored livers. Generally, NMP groups showed a distinctive metabolomic signature in all the analyzed biological matrices. In particular, many of the differentially expressed metabolites were involved in mitochondrial biochemical pathways. Succinate, acetate, 3-hydroxybutyrate, creatine, and O-phosphocholine were deeply modulated in ex vivo perfused livers compared to both the Native and SCS groups. These novel results demonstrate a broad modulation of mitochondrial metabolism during NMP that exceeds energy production and redox balance maintenance.
... A systematic review on the use of metabolomics to discover liver biomarkers for transplantation outcomes in liver tissue biopsies highlighted promising results [17]. These first studies identified lipid molecules [18][19][20], tryptophan, kynurenine and S-adenosylmethionine as liver biomarkers [21,22]. ...
The current shortage of livers for transplantation has increased the use of marginal organs sourced from donation after circulatory death (DCD). However, these organs have a higher incidence of graft failure, and pre-transplant biomarkers which predict graft function and survival remain limited. Here, we aimed to find biomarkers of liver function before transplantation to allow better clinical evaluation. Matched pre- and post-transplant liver biopsies from DCD (n = 24) and donation after brain death (DBD, n = 70) were collected. Liver biopsies were analysed using mass spectroscopy molecular phenotyping. Discrimination analysis was used to parse metabolites differentiated between the two groups. Five metabolites in the purine pathway were investigated. Of these, the ratios of the levels of four metabolites to those of urate differed between DBD and DCD biopsies at the pre-transplantation stage (q < 0.05). The ratios of Adenosine monophosphate (AMP) and adenine levels to those of urate also differed in biopsies from recipients experiencing early graft function (EGF) (q < 0.05) compared to those of recipients experiencing early allograft dysfunction (EAD). Using random forest, a panel consisting of alanine aminotransferase (ALT) and the ratios of AMP, adenine, and hypoxanthine levels to urate levels predicted EGF with area under the curve (AUC) of 0.84 (95% CI (0.71, 0.97)). Survival analysis revealed that the metabolite classifier could stratify six-year survival outcomes (p = 0.0073). At the pre-transplantation stage, a panel composed of purine metabolites and ALT could improve the prediction of EGF and survival.
... For the lipidomic analysis, the 1 H single-pulse NMR-spectra were used. Due to the broad character of the lipid signals, seven groups of lipid signals (L1-L7) were assessed and designated by the moieties present within 19 : L1: lipid methyls; L2: lipid aliphatic chain; L3: lipid β-methylenes; L4: lipid allylic methylenes; L5: lipid α-methylenes; L6: lipid polyunsaturated allylic methylenes and L7: lipid alkenes ( figure 1A). Due to the broadness of the lipid signals, lipid groups were compared in patients as ratios relative to the lipid methyl group L1. ...
Objectives
The differential diagnosis of seronegative rheumatoid arthritis (negRA) and psoriasis arthritis (PsA) is often difficult due to the similarity of symptoms and the unavailability of reliable clinical markers. Since chronic inflammation induces major changes in the serum metabolome and lipidome, we tested whether differences in serum metabolites and lipids could aid in improving the differential diagnosis of these diseases.
Methods
Sera from negRA and PsA patients with established diagnosis were collected to build a biomarker-discovery cohort and a blinded validation cohort. Samples were analysed by proton nuclear magnetic resonance. Metabolite concentrations were calculated from the spectra and used to select the variables to build a multivariate diagnostic model.
Results
Univariate analysis demonstrated differences in serological concentrations of amino acids: alanine, threonine, leucine, phenylalanine and valine; organic compounds: acetate, creatine, lactate and choline; and lipid ratios L3/L1, L5/L1 and L6/L1, but yielded area under the curve (AUC) values lower than 70%, indicating poor specificity and sensitivity. A multivariate diagnostic model that included age, gender, the concentrations of alanine, succinate and creatine phosphate and the lipid ratios L2/L1, L5/L1 and L6/L1 improved the sensitivity and specificity of the diagnosis with an AUC of 84.5%. Using this biomarker model, 71% of patients from a blinded validation cohort were correctly classified.
Conclusions
PsA and negRA have distinct serum metabolomic and lipidomic signatures that can be used as biomarkers to discriminate between them. After validation in larger multiethnic cohorts this diagnostic model may become a valuable tool for a definite diagnosis of negRA or PsA patients.
... However, for samples that are naturally rich in lipids (tissues, various foods, living, and whole organisms), broad 1 H-NMR signals from the lipids often dominate the aliphatic region of the spectrum. Although these signals contain important information themselves, [22] they overlap with many of the smaller metabolite signals of interest and are problematic in solution-state and HR-MAS NMR. [23] Particularly for environmental metabolomics, it would be highly beneficial if experiments can be performed in vivo, since under the current practice of buffer-based extraction of metabolites, [5] as valuable information could be lost in the sample preparation procedure. ...
Proton NMR spectra of intact biological samples often show strong contributions from lipids which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids verses small metabolites could be exploited to suppress lipid signals, in HR‐MAS NMR spectroscopy. In solution state NMR, suspended samples can exhibit very broad water signals which are challenging to supress. Here, improved water suppression is incorporated into the sequence and the CPMG train is replaced with a low power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR‐MAS as well as static solution‐state samples. Applications are also extended to include in vivo organisms. For solution‐state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR‐MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non‐invasive NMR‐based analysis of biological samples with a high lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.
... L'utilisation de la HR-MAS-a déjà été rapportée dans 2 articles. Le 1 er évaluait l'évolution des profils métaboliques au cours de la conservation de 6 greffons hépatiques et montrait une diminution de la GPC excepté dans le seul greffon ayant présenté une dysfonction.121 Une analyse métabolomique par UPLC-MS sur une large série validait le principe que le profil metabolique de greffon permettait de prédire la survenue d'une dysfonction de greffon définie selon Olthoff. ...
La principale limite en chirurgie hépatobiliaire est représentée par l’insuffisance hépatocellulaire (IHC) posthépatectomie (Hx) ou la dysfonction du greffon (EAD) après transplantation (TH). Peu d’études ont évalué le métabolisme du foie dans son ensemble, du fait du manque de technique utilisable en clinique. La métabolomique HR-MAS-RMN pourrait pallier à ce manque. Le but de cette thèse était d’évaluer l’apport de cette technique en chirurgie hépatobiliaire.En TH (n=42), le profil métabolique (PM) prédisait le risque d’EAD et identifiait le lactate et la phosphocholine comme biomarqueurs permettant d’envisager un matching métabolique. Après Hx majeure (n=45), le PM prédisait la survenue d’un décès par IHC. Ce PM différait du profil cirrhotique en décompensation et était compatible avec celui de système cellulaire prolifératif. Une étude préliminaire montrait que le PM prédisait la récidive à 1 an après hépatectomie. Ce travail montre l’intérêt de la métabolomique par HR MAS RMN pour prédire l’issue d’une Hx ou d’une TH dans un temps compatible avec la clinique. Ces données orientent vers la piste de l’intervention métabolique en chirurgie hépatique.
... The metabolomic platform used was high-resolution magic angle spinning (HR-MAS) 1 H-NMR, a variation of conventional 1 H-NMR that can be performed on solid samples, and it was the first metabolomic application of such spectroscopy used with human liver tissues. A larger amount of triglycerides and unsaturated lipids, and lower levels of phospholipids, were found on the grafts with fatty infiltration (Duarte et al. 2005). Recently, Xu et al. described for the first time different lipid profiles between two types of donors as DBD and DCD. ...
The term biomarker usually refers to the biochemical molecules used in basic and clinical research, and also in the clinical practice, as surrogate markers that offer the advantage of being an objective, quantifiable, and reproducible measure. The most common applications of biomarkers include diagnosis, screening and monitoring of disease, assessment of response during therapy, risk assessment, and prognosis. Metabolomics or metabonomics enables the determination of hundreds of small molecules at the same time, which provides more comprehensive information than the determination of a single biomarker. Using metabolomics as an approach for searching biomarkers is supported by its capabilities to detect subtle metabolic changes triggered by external stimuli or perturbation. Metabolome changes are quite dynamic compared to genomics and transcriptomics, or even proteomics. Therefore, such metabolite alterations are found early in different samples, like tissues, cell lysates, blood, serum, plasma, feces, urine, etc. Application of metabolomics in liver transplantation is still in its early stages and has focused mainly on studying three aspects: post-reperfusion damage and rejection and dysfunction of the organ. In the current era when lack of organs suitable for transplantation is the most important limiting factor, the existence of an accepted functional assessment of grafts before transplantation would help to not only recover initially discarded organs but to also assess the therapies used to improve the quality of these organs. Different metabolic approaches have been used to search for objective markers of graft function and quality, but further analytical and clinical validation in multicentre studies is mandatory before they are incorporated into clinical routines.
... The metabolomic platform used was high-resolution magic angle spinning (HR-MAS) 1 H-NMR, a variation of conventional 1 H-NMR that can be performed on solid samples, and it was the first metabolomic application of such spectroscopy used with human liver tissues. A larger amount of triglycerides and unsaturated lipids, and lower levels of phospholipids, were found on the grafts with fatty infiltration (Duarte et al. 2005). Recently, Xu et al. described for the first time different lipid profiles between two types of donors as DBD and DCD. ...
The term biomarker usually refers to the biochemical molecules used in basic and clinical research, and also in the clinical practice, as surrogate markers that offer the advantage of being an objective, quantifiable, and reproducible measure. The most common applications of biomarkers include diagnosis, screening and monitoring of disease, assessment of response during therapy, risk assessment, and prognosis. Metabolomics or metabonomics enables the determination of hundreds of small molecules at the same time, which provides more comprehensive information than the determination of a single biomarker. Using metabolomics as an approach for searching biomarkers is supported by its capabilities to detect subtle metabolic changes triggered by external stimuli or perturbation. Metabolome changes are quite dynamic compared to genomics and transcriptomics, or even proteomics. Therefore, such metabolite alterations are found early in different samples, like tissues, cell lysates, blood, serum, plasma, feces, urine, etc. Application of metabolomics in liver transplantation is still in its early stages and has focused mainly on studying three aspects: post-reperfusion damage, rejection and dysfunction of the organ. In the current era when lack of organs suitable for transplantation is the most important limiting factor, the existence of an accepted functional assessment of grafts before transplantation would help to not only recover initially discarded organs but to also assess the therapies used to improve the quality of these organs. Different metabolic approaches have been used to search for objective markers of graft function and quality, but further analytical and clinical validation in multicentre studies is mandatory before they are incorporated into clinical routines.
... In all publications, classical NMR experiments (one-dimensional nuclear Overhauser effect spectroscopy and/or Car-Purcell-Meiboom-Gill) and 2D experiments (total correlation spectroscopy and J-resolved) were recorded. A study by Duarte et al (28) using heteronuclear multiple quantum coherence 1H-13C spectra proposed to easily identify metabolites from chemical shifts. Notably, this study also measured T1 and T2 relaxation times for CH 2 -CH 2 -CO lipid protons, which are correlated with lipid mobility. ...
... Since NMR/MRI based approaches allow for the quantification of metabolite concentrations of intact tissue, either in vivo [57] or ex vivo [58], the majority of metabolomics studies use one-dimensional (1D) proton ( 1 H) NMR spectroscopy of cell or tissue extracts as the primary method of analysis. Because the 1 H is abundant in biological molecules, and is also the second most NMR sensitive nucleus (after tritium), this method will be discussed here. ...
Although significant advances have been made in recent years, the structural elucidation of small molecules continues to remain a challenging issue for metabolite profiling. Many metabolomic studies feature unknown compounds; sometimes even in the list of features identified as " statistically significant " in the study. Such metabolic " dark matter " means that much of the potential information collected by metabolomics studies is lost. Accurate structure elucidation allows researchers to identify these compounds. This in turn, facilitates downstream metabolite pathway analysis, and a better understanding of the underlying biology of the system under investigation. This review covers a range of methods for the structural elucidation of individual compounds, including those based on gas and liquid chromatography hyphenated to mass spectrometry, single and multi-dimensional nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry and includes discussion of data standardization. Future perspectives in structure elucidation are also discussed; with a focus on the potential development of instruments and techniques, in both nuclear magnetic resonance spectroscopy and mass spectrometry that, may help solve some of the current issues that are hampering the complete identification of metabolite structure and function.
... Providing a more realistic insight into metabolic profiles, this original technique has been successfully applied to address a variety of questions related to organ transplantation. Indeed, metabolomics is a useful tool to assess the quality of transplants from biopsy samples at the donor stage [11], to improve the preservation and storage of the graft before transplantation [12], and to monitor organ function after transplantation by following metabolic perturbations in biofluids such as blood and urine [13]. The literature presents some examples of metabolomic applications mainly with NMR in the specific field of islet transplantation, from the preservation of the donor pancreas to the detection of islet rejection. ...
Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. ¹H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with ¹H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.
... The hepatic metabolic profile ( Fig. 1a) was characterised by high levels of betaine, lactate and glucose. This was the only biological matrix where it was possible to detect glutathione (in its oxidised form since the total pool of glutathione becomes oxidised during tissue extraction), in very small quantities, in contrast to what is commonly found in mammalian hepatic metabolic profiles (Martin et al. 2007;Waters et al. 2002;Duarte et al. 2005;Claus et al. 2008). Table 1 Similarly, kidney metabolic profiles (Fig. 1b) were rich in lactate, which is consistent with the important role of the kidney in energy metabolism. ...
IntroductionPoultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments. Objectives
As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus). Methods
Metabolic extractions were performed prior to 1H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized. ResultsNearly 80 metabolites were identified. A cross-comparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice. Conclusion
This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.
... Gross examination and intraoperative extemporaneous microscopic examination are unreliable. In recent years, metabolomics, or global metabolite profiling, has been used for investigating metabolite changes associated with some pathologic conditions (eg, colorectal cancers, 3 breast cancers, 4 liver cancers, 5 and pancreatic cancers 6 ). ...
Background:
Primary hyperparathyroidism (PHPT) may be related to a single gland disease or multiglandular disease, which requires specific treatments. At present, an operation is the only curative treatment for PHPT. Currently, there are no biomarkers available to identify these 2 entities (single vs. multiple gland disease). The aims of the present study were to compare (1) the tissue metabolomics profiles between PHPT and renal hyperparathyroidism (secondary and tertiary) and (2) single gland disease with multiglandular disease in PHPT using metabolomics analysis.
Methods:
The method used was (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Forty-three samples from 32 patients suffering from hyperparathyroidism were included in this study.
Results:
Significant differences in the metabolomics profile were assessed according to PHPT and renal hyperparathyroidism. A bicomponent orthogonal partial least square-discriminant analysis showed a clear distinction between PHPT and renal hyperparathyroidism (R(2)Y = 0.85, Q(2) = 0.63). Interestingly, the model also distinguished single gland disease from multiglandular disease (R(2)Y = 0.96, Q(2) = 0.55). A network analysis was also performed using the Algorithm to Determine Expected Metabolite Level Alterations Using Mutual Information (ADEMA). Single gland disease was accurately predicted by ADEMA and was associated with higher levels of phosphorylcholine, choline, glycerophosphocholine, fumarate, succinate, lactate, glucose, glutamine, and ascorbate compared with multiglandular disease.
Conclusion:
This study shows for the first time that (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy is a reliable and fast technique to distinguish single gland disease from multiglandular disease in patients with PHPT. The potential use of this method as an intraoperative tool requires specific further studies.
... The various factors considered to indicate a prospective donor liver biopsy are: history of alcohol intake, elevated serum ferritin levels and presence of steatosis on imaging. 2 There are many upcoming modalities like NMR spectroscopy for metabolic assessment of the liver. 3 We set out to identify the significance of donor liver biopsy especially in a developing country where liver biopsy is much cheaper compared to developed countries. 4 ...
Liver transplantation is an important modality of treatment for end-stage liver disease. Liver biopsy evaluation has been an important aspect of the donor evaluation protocol. With the advent of newer modalities of donor evaluation such as high resolution CT scan, fibroscan and NMR spectroscopy, the relevance of the liver biopsy appears to be diminishing. We investigated the usefulness of donor liver biopsy evaluation in patients who had been cleared by radiological investigations. We evaluated 184 donor liver biopsies performed over a one-year period and found that 18% showed >5% steatosis and around 40% showed portal inflammation, which was, however, minimal to mild. Fibrosis was detected in 10 cases (5.4%), 7 being in stage 1 and 3 in stage 2. Donors with these findings were not considered for transplantation. We conclude that the liver biopsy still continues to be relevant especially in a developing country and does add additional information to the diagnostic work-up of a liver donor.
... Metabonomics offers a rapid and simultaneous measurement of significant number of metabolites in biological samples (Chen et al. 2007;Clarke and Haselden 2008;Fernie et al. 2004;Nicholson and Wilson 2003). Currently, metabonomic profiling has been performed using various techniques, including high-field nuclear magnetic resonance (NMR) (Nicholls et al. 2003;Duarte et al. 2005), and gas or liquid chromatography/mass spectrometry (GC-MS, LC-MS) (Kushnir et al. 2010;Lutz et al. 2006;Major et al. 2006). Today, ultra performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC/ TOF/MS) has become one of the widely used techniques in metabonomics studies with higher resolutions, higher sensitivities, and more rapid separations (Stolker et al. 2008;Grata et al. 2008;Gika et al. 2008a, b). ...
Metabonomics has become a highly sensitive and powerful tool for screening of biomarkers and elucidation of biochemical processes. Recently, we reported beneficial therapeutic effect of human placenta mesenchymal stem cells (hPMSCs) transplantation on d-galactosamine (GalN)-induced liver injury in Chinese miniature pigs. However, the underlying mechanism remains largely unclear. Here, UPLC/TOF/MS-based metabonomics approach was employed to analyze serum from GalN-treated pigs with the goal of identifying potential biomarkers for acute liver injury. Our results showed that obvious metabolic disturbance occurred during acute liver failure (ALF), which can be ameliorated by the hPMSCs transplantation. The metabolic profiles of the hPMSCs transplantation group returned back to the original state 5 weeks after the hPMSCs transplantation. In addition, the result obtained from metabolic trajectory analysis correlated well with those from biochemical analysis and histological examination. Serum levels of several metabolites including glycoursodeoxycholic acid, glycochenodeoxycholic acid, aromatic amino acids, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, were significantly modified during the process of ALF and cell treatment. Taken together, our study has gained new insight into the molecular mechanism on how hPMSCs administration facilitates the recovery of ALF, and provided strong support for treating liver diseases with stem cell-based therapies.
... Since then a number of research articles and reviews were published regarding the metabolite identification and differentiation of benign and malignant human tissue specimens [44,45] by 1 H NMR HR-MAS spectroscopy. It has already demonstrated real insights into the mechanisms of toxicity and pathology at molecular level [46][47][48][49]. The technique greatly enhances the spectral resolution and provides relevant biochemical information regarding small metabolites present in tissues, which can be mapped and quantitated from a single spectral window. ...
Metabonomics has shown substantial potential as a diagnostic technique in cancer research including cancer recurrence. The minimal acquisition time and maximum information gained justifies its impact in identifying novel cancer biomarkers and developing cancer therapeutics. We discuss the potential application of NMR based metabonomics for biomarker identification in different biofluids and tissue biopsies from patients suffering from oral squamous cell carcinoma. In this review, we broadly emphasise in vitro, ex vivo, in vivo NMR spectroscopy and MRI as diagnostic and prognostic methods.
... The Carr-Purcell-Meiboom-Gill (CPMG) sequence is commonly used as a T2-lter to reduce broad components and therefore also fat contributions from spectra, taking advantage of the short T2 relaxation times of lipids. 2,4 However, long echo times would be required to eliminate most of the lipids, 5 with consequently (A) reduced signal to noise also for small metabolites, (B) a T2 dependency of peak intensities impeding metabolite quantication, (C) heating of the sample, (D) J-evolution of spins, and (E) losing all information on lipids. ...
High Resolution Magic Angle Spinning (HR-MAS) NMR allows to metabolically characterize biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions, overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals of small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both, small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites has potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.
... In the 2D 1 H-13 C NMR spectrum a signal was observed at G 22.87-30.05 ppm, which corresponds to the methylene group of the FA [121]. ...
... In the downfield region, below d H 6 ppm, resonances from various aromatic compounds such as the nucleosides/nucleobases, adenosine, inosine, uridine, uracil and from amino acids, tyrosine, phenylalanine and histidine are observed. As for the muscle extracts, the identification of signals was obtained from 2D NMR spectra and by comparison with the literature (Duarte et al., 2005;Fan, 1996) and the assignment of the different resonances are listed in Table 3. ...
The metabolite profiles from muscles and livers of Atlantic salmon were investigated using high resolution nuclear magnetic resonance (HR-NMR) spectroscopy of aqueous extracts and magic angle spinning (MAS) NMR spectroscopy of intact tissues. Comparison of the data showed that most small metabolites present in the aqueous extracts were also identified in the Carr–Purcell–Meiboom–Gill (CPMG) MAS NMR spectra of the intact tissues. Not only the total omega-3 fatty acid content, but also the EPA and DHA content, in the muscle and liver tissues could be calculated directly from the diffusion-edited MAS NMR spectra without the need for lipophilic extraction. The effect of replacing a normal fish meal during a period of 3months with a diet containing 20% zygomycete was also investigated by multivariate analysis of the NMR spectra. Principal component analysis (PCA) was used to study the small metabolites distribution in the aqueous extracts of liver and muscles, and revealed that the feed containing zygomycete could have an influence on the metabolites profiles of juvenile Atlantic salmon.
Strategies to minimize immune-suppressive (IS) medications after liver transplantation (LT) are limited by allograft rejection. Biopsy of liver is the current standard of care (SOC) in diagnosing rejection. However, it adds to physical and economic burden to the patient, and has diagnostic limitations. In this review we aim to highlight the different biomarkers to predict and diagnose acute rejection. We also aim to explore recent advances in molecular diagnostics to improve the diagnostic yield of liver biopsies.
Liver transplantation has been widely accepted as an effective intervention for end-stage liver diseases and early hepatocellular carcinomas. However, a variety of postoperative complications and adverse reactions have baffled medical staff and patients. Currently, transplantation monitoring relies primarily on nonspecific biochemical tests, whereas diagnosis of multiple complications depends on invasive pathological examination. Therefore, a noninvasive monitoring method with high selectivity and specificity is desperately needed. This review summarized the potential of endogenous small-molecule metabolites as biomarkers for assessing graft function, ischemia-reperfusion injury and liver rejection. Exogenous metabolites, mainly those immunosuppressive agents with high intra- and inter-individual variability, were also discussed for transplantation monitoring.
Liver transplantation is an effective intervention for end-stage liver disease, fulminant hepatic failure, and early hepatocellular carcinoma. Yet, there is marked patient-to-patient variation in liver transplantation outcomes. This calls for novel diagnostics to enable rational deployment of donor livers. Metabolomics is a postgenomic high-throughput systems biology approach to diagnostic innovation in clinical medicine. We report here an original systematic review of the metabolomic studies that have identified putative biomarkers in the context of liver transplantation. Eighteen studies met the inclusion criteria that involved sampling of blood (n = 4), dialysate fluid (n = 4), bile (n = 5), and liver tissue (n = 5). Metabolites of amino acid and nitrogen metabolism, anaerobic glycolysis, lipid breakdown products, and bile acid metabolism were significantly different in transplanted livers with and without graft dysfunction. However, criteria for defining the graft dysfunction varied across studies. This systematic review demonstrates that metabolomics can be deployed in identification of metabolic indicators of graft dysfunction with a view to implicated molecular mechanisms. We conclude the article with a horizon scanning of metabolomics technology in liver transplantation and its future prospects and challenges in research and clinical practice.
Purpose
The aim of this study is to generate a metabolic database for biomedical studies of biopsy specimens by high‐resolution magic angle spinning (HRMAS) nuclear MR (NMR).
Methods
Seventy‐six metabolites, classically found in human biopsy samples, were prepared in aqueous solution at a known concentration and analyzed by HRMAS NMR. The spectra were recorded under the same conditions as the ones used for the analysis of biopsy specimens routinely performed in our hospital.
Results
For each metabolite, a complete set of NMR spectra (1D ¹H, 1D ¹H‐CPMG, 2D J‐Resolved, 2D TOCSY, and 2D ¹H‐¹³C HSQC) was recorded at 500 MHz and 277 K. All spectra were manually assigned using the information contained in the different spectra and existing databases. Experiments to measure the T1 and the T2 of the different protons present in the 76 metabolites were also recorded.
Conclusion
This new HRMAS metabolic database is a useful tool for all scientists working on human biopsy specimens, particularly in the field of oncology. It will make the identification of metabolites in biopsy specimens faster and more reliable. Additionally, the knowledge of the T1 and T2 values will allow to obtain a more accurate quantification of the metabolites present in biopsy specimens.
Background and aims:
The purpose of this study was to evaluate the potential value of high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS-NMR) metabolomic analysis of back-table biopsies for the prediction of early allograft dysfunction and donor-recipient matching. Indeed there is an emerging need to assess the metabolic state of liver allograft especially in the novel setting of machine perfusion preservation and DCD grafts. HR-MAS-NMR could be a useful tool in this setting as it can extemporaneously provide untargeted metabolic profile.
Method:
The metabolic profiles obtained by HR-MAS-NMR of back-table biopsies were compared according to the presence of early allograft dysfunction using PLS-DA. Network analysis was used to identify metabolite with significant changes. The profiles were compared to native livers to identify metabolites for donor-recipient matching.
Results:
metabolic profiles were significantly different between grafts exerting EAD versus no EAD. The constructed model can be used to predict the graft outcome with excellent accuracy. The metabolites showing the most significant differences were lactate level >8.3mmol/g and phosphocholine content >0.646 mmol/g which were significantly associated with graft dysfunction with an excellent accuracy (AUROClactates=0.906; AUROCphosphocholine=0.816). Metabolic profiles from native livers from sarcopenic patients showed opposite content in lactate and glycerophosphocholine. In sarcopenic patients, the risk of EAD was significantly higher when transplanting a graft exerting high risk graft metabolic score.
Conclusion:
This study underlines the cost of metabolic adaptation identifying lactate and choline-derived metabolites as predictors of poor graft function in both native livers and liver grafts. HR-MAS-NMR seems a valid technique to evaluate graft quality and the consequences of cold ischemia on the graft. It could be used to assess the efficiency of graft resuscitation on machine perfusion in future studies.
Lay summary:
Real-time metabolomic profiles of human grafts during back-table can accurately predict graft dysfunction. High lactate and phosphocholine content are highly predictive of graft dysfunction whereas low lactate and phosphocholine content characterize sarcopenic patients. In these patients, the cost of metabolic adaptation may explain poor outcomes.
High-resolution magic angle spinning (HRMAS) MRS is a powerful method for gaining insight into the physiological and pathological processes of cellular metabolism. Given its ability to obtain high-resolution spectra of non-liquid biological samples, while preserving tissue architecture for subsequent histopathological analysis, the technique has become invaluable for biochemical and biomedical studies. Using HRMAS MRS, alterations in measured metabolites, metabolic ratios, and metabolomic profiles present the possibility to improve identification and prognostication of various diseases and decipher the metabolomic impact of drug therapies. In this review, we evaluate HRMAS MRS results on human tissue specimens from malignancies and non-localized diseases reported in the literature since the inception of the technique in 1996. We present the diverse applications of the technique in understanding pathological processes of different anatomical origins, correlations with in vivo imaging, effectiveness of therapies, and progress in the HRMAS methodology.
Ethnopharmacological relevance:
Qizhiweitong particles (QZWT) which is derived from the Sinisan decoction in Shang Han Za Bing Lun, composed of Bupleurum chinenis, Paeonia obovata, Citrus aurantium L., Glycyrrhiza uralensis Fisch., Cyperus rotundus and Rhizoma Corydalis is a traditional Chinese medicine (TCM) treating gastrointestinal diseases. It have been used in clinical for years. It have been used in clinical for years. According to previous research, Bupleurum chinenis, Citrus aurantium, Cyperus rotundus in QZWT play the role of promoting gastric peristalsis, which consist of complex chemical constituents. The aim of this study is to probe the multiple effective components with gastrointestinal prokinetic efficacy in QZWT and investigate the multitarget integrated adjustment mechanism of QZWT curing atropine-induced gastrointestinal motility dysfunction mice.
Materials and methods:
One hundred and thirty two male mice were randomly divided into 11 groups, including control group, model group, Domperidone group, Mosapride group, QZWT group and six components groups. With gastric retention rate, rate of small intestine propulsion, serum content of GAS and MTL as indexes to evaluate the curing effect on gastrointestinal movement disorders caused by atropine in mice. A serum metabonomics method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) had been established to investigate the mechanism of QZWT and these components, and PCA and PLS-DA have been used to distinguish different groups and found potential biomarkers.
Results:
Four components from six present good prokinetic effects, including Bupleurum Polysaccharide, Citrus aurantium flavonoid, Citrus aurantium essential oil and Cyperus rotundus flavonoids. These components and QZWT regulate 5 potential biomarkers in the body, and primarily involved in 5 metabolic pathways. These potential biomarkers possess direct or indirect connections, each biomarker regulated by multiple components, each component adjusting multiple targets, and QZWT is nearly the sum of its components.
Conclusions:
This experiment deepened our understanding of insufficient gastrointestinal dynamics, confirmed that QZWT treating gastrointestinal disorders was through multicomponent, multitarget ways. These results fully reflect the multiple targets synergy characteristics of TCM.
Urinary tract infections impose substantial health burdens on women worldwide. Urinary tract infections often incur a high risk of recurrence and antibiotic resistance, and uropathogenic E. coli accounts for approximately 80% of clinically acquired cases. The diagnosis of, treatment of, and drug development for urinary tract infections remain substantial challenges due to the complex pathogenesis of this condition. The clinically isolated UPEC 83972 strain was found to produce four siderophores: yersiniabactin, aerobactin, salmochelin and enterobactin. The biosyntheses of some of these siderophores implies that the virulence of UPEC is mediated via the targeting of primary metabolism. However, the differential modulatory roles of siderophore biosyntheses on the differential metabolomes of UPEC and non-UPEC strains remain incompletely understood. In the present study, we sought to investigate how the differential metabolomes can be used to distinguish UPEC from non-UPEC strains and to determine the associated regulatory roles of siderophore biosynthesis. Our results are the first to demonstrate that the identified differential metabolomes strongly differentiated UPEC from non-UPEC strains. Furthermore, we performed metabolome assays of mutants with different patterns of siderophore deletions, and the data revealed that the mutations of all four siderophores exerted a stronger modulatory role on the differential metabolomes of the UPEC and non-UPEC strains relative to the mutation of any single siderophore and that this modulatory role primarily involved amino acid metabolism, oxidative phosphorylation in the carbon fixation pathway, and purine and pyrimidine metabolism. Surprisingly, the modulatory roles were strongly dependent on the type and number of mutated siderophores. Taken together, these results demonstrated that siderophore biosynthesis coordinately modulated the differential metabolomes and thus may indicate novel targets for virulence-based diagnosis, therapeutics and drug development related to urinary tract infections.
The interaction between biological systems and their environments is complex and often involves mechanisms beyond changes in the genome and/or the proteome at the metabolome level. Metabolomics is a field of science, which involves the comprehensive quantitative and qualitative profiling of multiple metabolites and their interactions with environmental variables of interest such as diet, disease, environment, or exposure to chemicals. Monitoring these metabolites represents the closest end-point to understand the cell dynamics at the functional level as compared to genomics or proteomics. Metabolomics is heavily applied in the identification of biomarkers for disease diagnosis and prognosis, drug toxicity and efficacy, genetic polymorphisms, drug metabolism, and fluxomics. These applications are discussed in detail in this chapter.
Breast cancer (BC) has the highest occurrence and mortality of all cancers that affect women with more than one million new cases each year across the globe. BC accounts for about one-quarter of all cancer-related deaths. Even though breast cancer is an aggressive and fatal disease, early detection and treatment can result in increased survival in more than three-quarters of diagnosed patients. In general, traditional diagnostic methods, such as ultrasonography and mammography, considerably increase t survival rates due to early disease detection. Although these traditional methods are useful, new strategies for early detection of breast cancer would likely reduce breast cancer mortality rates. Additional diagnostic imaging modalities, such as Computer Tomography (CT), Positron Emission Tomography (PET), and other types of scintigraphy techniques, have been used to identify the primary source of the cancer in metastatic cases, but none of these techniques is yet in routine clinical use. Among other imaging methodologies, Magnetic Resonance Imaging, Magnetic Resonance Spectroscopy (MRS) and Nuclear Magnetic Resonance (NMR) approaches are powerful tools for uncovering cancer biomarkers. In this review, we consider the current capabilities of magnetic resonance techniques in breast cancer research and highlight some milestones that are necessary to move early detection of breast cancer using such approaches into mainstream health care modalities.
Inflammation and metabolism are closely interlinked. Both undergo significant dysregulation following surgery for congenital heart disease, contributing to organ failure and morbidity. In this study, we combined cytokine and metabolic profiling to examine the effect of postoperative tight glycemic control compared with conventional blood glucose management on metabolic and inflammatory outcomes in children undergoing congenital heart surgery. The aim was to evaluate changes in key metabolites following congenital heart surgery and to examine the potential of metabolic profiling for stratifying patients in terms of expected clinical outcomes.
Laboratory and clinical study.
University Hospital and Laboratory.
Of 28 children undergoing surgery for congenital heart disease, 15 underwent tight glycemic control postoperatively and 13 were treated conventionally.
Metabolic profiling of blood plasma was undertaken using proton nuclear magnetic resonance spectroscopy. A panel of metabolites was measured using a curve-fitting algorithm. Inflammatory cytokines were measured by enzyme-linked immunosorbent assay. The data were assessed with respect to clinical markers of disease severity (Risk Adjusted Congenital heart surgery score-1, Pediatric Logistic Organ Dysfunction, inotrope score, duration of ventilation and pediatric ICU-free days).
Changes in metabolic and inflammatory profiles were seen over the time course from surgery to recovery, compared with the preoperative state. Tight glycemic control did not significantly alter the response profile. We identified eight metabolites (3-D-hydroxybutyrate, acetone, acetoacetate, citrate, lactate, creatine, creatinine, and alanine) associated with surgical and disease severity. The strength of proinflammatory response, particularly interleukin-8 and interleukin-6 concentrations, inversely correlated with PICU-free days at 28 days. The interleukin-6/interleukin-10 ratio directly correlated with plasma lactate.
This is the first report on the metabolic response to cardiac surgery in children. Using nuclear magnetic resonance to monitor the patient journey, we identified metabolites whose concentrations and trajectory appeared to be associated with clinical outcome. Metabolic profiling could be useful for patient stratification and directing investigations of clinical interventions.
AIM: To study liver cell apoptosis caused by the toxicity of selenium and observe the alteration of choline compounds using in vitro 9.4T high resolution magnetic resonance spectroscopy.
METHODS: Twenty male Wistar rats were randomly divided into two groups. The rats in the treatment group were intraperitoneally injected with sodium selenite and the control group with distilled water. All rats were sacrificed and the livers were dissected. 1H-MRS data were collected using in vitro 9.4T high resolution magnetic resonance spectrometer. Spectra were processed using XWINNMR and MestRe-c 4.3. HE and TUNEL staining was employed to detect and confirm the change of liver cells.
RESULTS: Good 1H-MR spectra of perchloric acid extract from liver tissue of rats were obtained. The conventional metabolites were detected and assigned. Concentrations of different ingredient choline compounds in treatment group vs control group were as follows: total choline compounds, 5.08 ± 0.97 mmol/L vs 3.81 ± 1.16 mmol/L (P = 0.05); and free choline, 1.07 ± 0.23 mmol/L vs 0.65 ± 0.20 mmol/L (P = 0.00). However, there was no statistical significance between the two groups. The hepatic sinus and cellular structure of hepatic cells in treatment group were abnormal. Apoptosis of hepatic cells was confirmed by TUNEL assay.
CONCLUSION: High dose selenium compounds can cause the rat liver lesion and induce cell apoptosis in vivo. High resolution 1H-MRS in vitro can detect diversified metabolism. The changing trend for different ingredient of choline compounds is not completely the same at early period of apoptosis.
To explore the potential of high-resolution magic angle spinning (HRMAS) H-1 nuclear magnetic resonance (NMR) spectroscopy for differentiation and metabolite characterization of hepatocellular carcinoma (HCC) and colorectal liver metastases (CRLM), we prospectively included 21 pathologically confirmed malignant hepatic tumors (8 HCC and 13 CRLM) and 26 non-tumorous hepatic parenchyma from 26 patients who underwent hepatic tumor resection. Using intact tissue samples obtained during surgery, HRMAS H-1 NMR spectroscopy was performed at 11.7 T. All observable metabolite signals were acquired using a water-presaturated standard one-dimensional Carr-Purcell-Meiboom-Gill sequence. Metabolomic profiles contributing to the differentiation of HCC and CRLM and of each tumor and non-tumorous hepatic parenchyma were represented by orthogonal partial least squares discriminant analysis (OPLS-DA) and loading plots. Metabolite intensity normalized by total spectral intensities in both tumors was compared using student's t tests. OPLS-DA and loading plots demonstrated good separation between tumors and non-tumorous hepatic parenchyma. The metabolomic characteristics of HCC showed separation from those of CRLMs according to OPLS-DA. Compared with CRLM, HCC showed significantly elevated levels of glucose (P < 0.01) and sn-Glycero-3-phosphocholine (P < 0.01), and decreased levels of hypoxanthine (P = 0.04). HCC and CRLM could be differentiated by the metabolic profile using HRMAS H-1 NMR spectroscopy.
Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have evolved as the most common techniques in metabolomics studies, and each brings its own advantages and limitations. Unlike MS spectrometry, NMR spectroscopy is quantitative and does not require extra steps for sample preparation, such as separation or derivatization. Although the sensitivity of NMR spectroscopy has increased enormously and improvements continue to emerge steadily, this remains a weak point for NMR compared with MS. MS- based metabolomics provides an excellent approach that can offer a combined sensitivity and selectivity platform for metabolomics research. Moreover, different MS approaches such as different ionization techniques and mass analyzer technology can be used in order to increase the number of metabolites that can be detected. In this chapter, the advantages, limitations, strengths, and weaknesses of NMR and MS as tools applicable to metabolomics research are highlighted.
Abstract Metabolomics in systems biology research unravels intracellular metabolic changes by high throughput methods, but such studies focusing on liver transplantation (LT) are limited. Microdialysate samples of liver grafts from donors after circulatory death (DCD; n=13) and brain death (DBD; n=27) during cold storage and post-reperfusion phase were analyzed through coulometric electrochemical array detection (CEAD) for identification of key metabolomics changes. Metabolite peak differences between the graft types at cold phase, post-reperfusion trends, and in failed allografts, were identified against reference chromatograms. In the cold phase, xanthine, uric acid, and kynurenine were overexpressed in DCD by 3-fold, and 3-nitrotyrosine (3-NT) and 4-hydroxy-3-methoxymandelic acid (HMMA) in DBD by 2-fold (p<0.05). In both grafts, homovanillic acid and methionine increased by 20%-30% with each 100 min increase in cold ischemia time (p<0.05). Uric acid expression was significantly different in DCD post-reperfusion. Failed allografts had overexpression of reduced glutathione and kynurenine (cold phase) and xanthine (post-reperfusion) (p<0.05). This differential expression of metabolites between graft types is a novel finding, meanwhile identification of overexpression of kynurenine in DCD grafts and in failed allografts is unique. Further studies should examine kynurenine as a potential biomarker predicting graft function, its causation, and actions on subsequent clinical outcomes.
Gliomas are the most common type of primary brain tumor. Radiation therapy (RT) is the primary adjuvant treatment to eliminate residual tumor tissue after surgery. However, the current RT guided by conventional imaging is unsatisfactory. A fundamental question is whether it is possible to further enhance the effectiveness and efficiency of RT based on individual radiosensitivity. In this research, to probe the correlation between radiosensitivity and the metabolite characteristics of glioma cells in vitro, a perchloric acid (PCA) extracting method was used to obtain water-soluble metabolites [such as N-acetylaspartate (NAA), choline (Cho), creatine (Cr) and succinate (Suc)]. Spectral patterns from these processed water-soluble metabolite samples were acquired by in vitro 14.7-T high-resolution (1) H MRS. Survival fraction analysis was performed to test the intrinsic radiosensitivity of glioma cell lines. Good (1) H MRS of PCA extracts from glioma cells was obtained. The radiosensitivity of glioma cells correlated positively with the Cho/Cr and Cho/NAA ratios, but negatively with the Suc/Cr ratio. Irradiation of the C6 cell line at different X-ray dosages led to changes in metabolite ratios and apoptotic rates. A plateau phase of metabolite ratio change and a decrease in apoptotic rate were found in the C6 cell line. We conclude that in vitro high-resolution (1) H MRS possesses the sensitivity required to detect subtle biochemical changes at the cellular level. (1) H MRS may aid in the assessment of the individual radiosensitivity of brain tumors, which is pivotal in the identification of the biological target volume. Copyright © 2014 John Wiley & Sons, Ltd.
Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer related mutations in HCC, integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. messenger RNA, mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry based metabolomics analysis of stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compared with non-tumor liver (p value < 0.0001). In addition, branched-chain amino acid metabolism (p value < 0.0001) and tricarboxylic acid metabolism (p value < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p value = 0.0013), succinate (p value = 0.0001), lactate (p value = 0.0114), glycerophosphoethanolamine (p value = 0.015), and inorganic phosphate (p value = 0.0001). However, (13) C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a (13) C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via (13) C labeling, to investigate targeted therapeutics against these enzymes, and to optimize the in vivo performance of (13) C MRI techniques. Copyright © 2014 John Wiley & Sons, Ltd.
31P high resolution nuclear magnetic resonance (NMR) spectroscopy was used to examine phospholipid metabolism and to analyze the phosphate-containing compounds in the bile in the transplanted liver recipients, the cholelithiasis patients’ and the living donors’ groups. Three signals of NMR spectrum of raw bile were determined: inorganic phosphate (Pi), lysophosphatidylcholine (LPtdC), and phosphatidylcholine (PtdC) in all investigated groups. Pi concentration was significantly higher in the recipients’ group than in the living donors’ group (Mann-Whitney test, p < 0.05). LPtdC and PtdC concentrations were significantly higher (Mann-Whitney test, p < 0.05) in the cholelithiasis patients’ group in comparison to the recipients’ group. Between the cholelithiasis patients’ group and the living donors’ group no significant differences in the three analysed compounds were found. The chemometric analysis for the 31P NMR spectral data set provided good classifications between the living donors’ and recipients’ groups and the poor one among all groups. Results of our study suggest that 31P NMR spectroscopy in vitro may be used for assessment of graft function, for the early signs of rejection and for the predisposition to gallstone formation.
In this work we have theoretically investigated photon absorption coefficient in ordered InAs/GaAs cubic quantum dot systems. We solved the Schrodinger equation associated with these structures, using a set of 13 × 13 × 13 plane waves at 12,167 equally spaced points of the Q space. We investigated the transitions between minibands arising from the conduction band, taking into account the different effective masses in each material in our calculations. The effects of the strain were included by taking a conduction band offset of 0.5 eV, corresponding to strained InAs in GaAs.
High-resolution magic angle spinning (HRMAS) is the combined application of high-resolution NMR experiments with dipolar- and susceptibility-reducing sample rotation to viscous or gel-like samples. It has generated significant insights into questions of structure in systems ranging from polymeric, inorganic and environmental samples, to foodstuffs, to whole cells and tissues, particularly in the burgeoning field of metabolite profiling. This review provides an updated look at the applications arising over the 8years that have passed, from 2003 to 2009, since our initial survey.
In vivo31 P magnetic resonance spectroscopy (MRS) was undertaken in 28 healthy adult individuals and 32 patients with hepatic malignancies of varying histology, using chemical shift imaging techniques. The mean peak area ratio (total range) of phosphomonoester (PME) to phosphodiester (PDE) in the health adult group was 0.23 (0.15–0.41). The mean (total range) PME/PDE ratio of the total patient group was 0.68 (0.15–2.38), which was significantly elevated (P < 0.001) compared to the mean of the healthy adult group. Liver biopsies, obtained at operation, were analysed using high-field in vitro MRS techniques in order to identify the contributions of aqueous-soluble metabolites to the multicomponent PME and PDE in vivo signals. Concentrations of phosphorylethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC) were measured. The in vitro spectrum of six samples of liver of normal histological appearance all showed a similar pattern of PE, PC, GPE and GPC. The in vitro spectrum of seven liver tumours of differing histology all showed an increase in PE and PC signals and a decrease in GPC and GPE signals. The in vitro results were compared with in vivo findings in five patients. The increase in PME/PDE observed in vivo represented, in part, an increase in PE and PC in the PME region and a decrease in GPE and GPC in the PDE region.
NMR spectroscopy of biofluids provides a wealth of information on the endogenous metabolic processes in an organism. Details of the various types of biofluid and the types of NMR experiment which are useful are given. The features of biofluid NMR spectra are described and practical details of spectral acquisition are also presented. However, the spectra are very complex and many resonances have not been assigned. Therefore, in order to focus on significant differences between a set of spectra from control organisms and from abnormals (e.g., humans with diseases or animals in toxic situations), recourse is made to pattern recognition or chemometric methods. This is exemplifed using NMR spectra of a number of different biofluids such as urine, blood plasma, and cerebrospinal fluid. This approach is encapsulated in the concept of metabonomics, a subject which can be regarded as complementary to studies of the genome (genomics) and the proteins in an organism (proteomics). Metabonomics is defined as “the quantitative measurement of the multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.” © 2000 John Wiley & Sons, Inc. Concepts Magn Reson 12: 289–320, 2000
Human livers with histologically proven cirrhosis were assessed using in vitro 31P NMR spectroscopy. Spectra were compared with those from histologically normal livers and showed significant elevations in phosphoethanolamine (PE) and phosphocholine (PC) and significant reductions in glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC). There were no significant differences in spectra from livers with compensated and decompensated cirrhosis. These results help to characterise the alterations in membrane metabolism in cirrhosis of the liver.
High-resolution 750 MHz 1H NMR spectra of control human blood plasma have been measured and assigned by the concerted use of a range of spin-echo, two-dimensional J-resolved, and homonuclear and heteronuclear (1H-13C) correlation methods. The increased spectral dispersion and sensitivity at 750 MHz enable the assignment of numerous 1H and 13C resonances from many molecular species that cannot be detected at lower frequencies. This work presents the most comprehensive assignment of the 1H NMR spectra of blood plasma yet achieved and includes the assignment of signals from 43 low M(r) metabolites, including many with complex or strongly coupled spin systems. New assignments are also provided from the 1H and 13C NMR signals from several important macromolecular species in whole blood plasma, i.e., very-low-density, low-density, and high-density lipoproteins, albumin, and alpha 1-acid glycoprotein. The temperature dependence of the one-dimensional and spin-echo 750 MHz 1H NMR spectra of plasma was investigated over the range 292-310 K. The 1H NMR signals from the fatty acyl side chains of the lipoproteins increased substantially with temperature (hence also molecular mobility), with a disproportionate increase from lipids in low-density lipoprotein. Two-dimensional 1H-13C heteronuclear multiple quantum coherence spectroscopy at 292 and 310 K allowed both the direct detection of cholesterol and choline species bound in high-density lipoprotein and the assignment of their signals and confirmed the assignment of most of the lipoprotein resonances.
High field 1H NMR spectroscopy was used for the rapid multicomponent analysis of low molecular wt compounds in urine in order to investigate the patterns of metabolic changes associated with early renal allograft dysfunction. Urine samples were collected daily for 14 days from 33 patients who underwent primary renal allograft transplantation, and analyzed by 500 and/or 600 MHz 1H NMR spectroscopy. All patients received 20 mg prednisolone and 5 mg/kg b.d. oral cyclosporin A (CsA) solution. In this study no patient showed clinical or histopathological evidence of CsA nephrotoxicity. For each patient the NMR-generated metabolite data were correlated with the clinical observations, graft biopsy pathology, and data from conventional laboratory techniques for assessing renal function. The NMR spectra of urine from patients with immediate functioning grafts were similar with respect to their patterns of amino acids, organic acids and organic amines, whereas the patients with delayed or non-functioning grafts showed significantly different metabolite excretion patterns. In longitudinal studies on individual patients there were increased urinary levels of trimethylamine-N-oxide (TMAO), dimethylamine (DMA), lactate, acetate, succinate, glycine and alanine during episodes of graft dysfunction. However, only the urinary concentration of TMAO was statistically significantly higher (P < 0.025) in the urine collected from patients during episodes of graft dysfunction (410 +/- 102 microM TMAO/mM creatinine) than in patients with good graft function (91 +/- 18 microM TMAO/mM creatinine) or healthy control subjects (100 +/- 50 microM TMAO/mM creatinine). These findings suggest that graft dysfunction is associated with damage to the renal medulla which causes the release of TMAO into the urine from the damaged renal medullary cells. This provides a possible novel urinary marker for post-transplant graft dysfunction. This study shows that NMR spectroscopy of biofluids, when used in combination with conventional laboratory techniques, is a valuable aid to renal transplant monitoring.
We have developed a quick, non-invasive method for measuring the ability of an isolated preserved liver to regenerate high energy phosphate nucleotides without the need for biopsy. Using 31P MRS we have monitored the hepatic energetics of intact cold preserved pig liver using standard clinical harvesting and storage techniques. Following cold storage for 2 h the livers were hypothermically reperfused with oxygenated modified University of Wisconsin preservation fluid. Prior to reperfusion MRS detectable adenosine diphosphate plus adenosine triphosphate was negligible; however, the spectrum showed intense resonances from phosphomonoesters and inorganic phosphate, as a consequence of adenosine triphosphate hydrolysis during cold preservation. Following a 10-min period of hypothermic reperfusion, regeneration of adenosine triphosphate occurred with a concurrent decline in inorganic phosphate and phosphomonoester, both of which are associated with adenosine triphosphate synthesis. The capacity of the liver to regenerate adenosine triphosphate following a 24-h period of cold storage was reduced by approximately 40% (p < 0.01) of the total amount achieved following the shorter cold storage time. Adenosine triphosphate regeneration rates were biphasic and were decreased upon prolonged storage, with the initial rate being reduced from 40.6 x 10(-2).min-1 (standard deviation (sd) 2.70 x 10(-2).min-1) to 14.8 x 10(-2).min-1 (sd; 2.4 x 10(-2).min-1) and the secondary rate from 1.77 x 10(-2).min-1 (sd; 0.18 x 10(-2).min-1) to 0.84 x 10(-2).min-1 (sd; 0.45 x 10(-2).min-1). MR images of the liver during the period of hypothermic reperfusion were also performed providing an assessment for the degree of hepatic vascular perfusion. This non-invasive, 31P MRS assessment of hepatic energetics in a clinically relevant animal model has great potential for the understanding of graft preservation injury.
In this review, metabonomics, a combination of data-rich analytical chemical measurements and chemometrics for profiling metabolism in complex systems, is described and its applications are reviewed. Metabonomics is typically carried out using biofluids or tissue samples. The relevance of the technique is reviewed in relation to other '-omics', and it is shown how the methods can be applied to physiological evaluation, drug safety assessment, characterization of genetically modified animal models of disease, diagnosis of human disease, and drug therapy monitoring. The different types of analytical data, mainly from nuclear magnetic resonance spectroscopy and mass spectrometry, are summarized. The outputs from a metabonomics study allow sample classification, for example according to phenotype, drug safety or disease diagnosis, and interpretation of the reasons for classification yields information on combination biomarkers of effect. Transcriptomic and metabonomic data is currently being further integrated into a holistic understanding of systems biology. An assessment of the possible future role and impact of metabonomics is presented.
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High-resolution magic angle spinning (MAS) 1H NMR spectra of small samples (ca. 8 mg) of intact rat liver are reported for the first time. One dimensional spectra reveal a number of large well-resolved NMR signals mainly from low to medium molecular weight compounds (generally <1000 Daltons) from a variety of chemical classes. A range of 2D MAS-NMR experiments were performed, including 1H J-resolved (JRES), 1H-1H total correlation spectroscopy (TOCSY) and 1H-13C heteronuclear multiple quantum coherence (HMQC) to enable detailed signal assignment. Resonances were assigned from α- and β-glucose, glycerol, alanine, glutamate, glycine, dimethylglycine, lysine, and threonine, together with phosphocholine, choline, lactate, trimethylamine-N-oxide (TMAO), and certain fatty acids. Well-resolved 1H NMR signals from glycogen (poly 1-4 α-glucose) were observed directly in intact liver using MAS-NMR spectroscopy. In addition, the resonances from the glycogen C1H proton in α(1→4) linked glucose units with either α(1→4) units adjacent or α(1→6) linked branches could be resolved in a high-resolution 1H NMR experiment giving direct in situ information on the ratio of α(1→4) to α(1→6) units. This indicates that despite the relatively high MW (>1,000,000 Daltons) there is considerable segmental motion in the glycogen molecules giving long 1H T2 relaxation times. Magn Reson Med 44:201–207, 2000. © 2000 Wiley-Liss, Inc.
NMR methods are being applied to study phospholipid metabolism of cancer cells by monitoring the resonances which appear in the 31P spectrum. This review, aside from considering the applicability of NMR to this specific pathway, raises the question of whether the phospholipid metabolite peaks observed by MR are indicators of cancer cell function or tumor response to treatment. After assessing the results from many investigations, it is concluded that there is no clear correlation and that a combination of techniques, including in vitro and extract studies, will be necessary for a more comprehensive evaluation of the in vivo data.
The metabolic consequences of xenobiotic-induced toxicity were investigated using high-resolution magic angle spinning (MAS) NMR spectroscopy of intact tissue. Renal papillary necrosis (RPN) was induced in Sprague-Dawley rats (n = 12) via a single i.p. dose of 250 mg/kg 2-bromoethanamine (BEA) hydrobromide. At 2, 4, 6, and 24 h after treatment with BEA, three animals were killed and tissue samples were obtained from liver, renal cortex, and renal medulla. Tissue samples were also removed at 2 and 24 h from matched controls (n = 6). 1H MAS NMR spectroscopic techniques were used to analyze samples of intact tissue (∼10 mg). Decreased levels of nonperturbing renal osmolytes (glycerophosphocholine, betaine, and myo-inositol) were observed in the renal papilla of BEA-treated animals at 6 and 24 h postdose (p.d.), concomitant with a relative increase in the tissue concentration of creatine. Increased levels of glutaric acid were found in all tissues studied in BEA-treated animals at 4 and 6 h p.d., indicating the inhibition of mitochondrial fatty acyl CoA dehydrogenases and mitochondrial dysfunction. Increased levels of trimethylamine-N-oxide occurred in the renal cortex at 6 h p.d. Changes in the metabolite profile of liver included an increase in the relative concentrations of triglycerides, lysine, and leucine. The novel application of 1H MAS NMR to the biochemical analysis of intact tissues following a toxic insult highlights the potential of this technique as a toxicological probe in providing a direct link between urinary biomarkers of toxicity and histopathological evaluation of toxicological lesions. Magn Reson Med 45:781–790, 2001. © 2001 Wiley-Liss, Inc.
A spin echo method adapted to the measurement of long nuclear relaxation times (T 2 ) in liquids is described. The pulse sequence is identical to the one proposed by Carr and Purcell, but the rf of the successive pulses is coherent, and a phase shift of 90° is introduced in the first pulse. Very long T 2 values can be measured without appreciable effect of diffusion.
Techniques for the correlation of 1H and 15N chemical shifts based on zero and double quantum NMR are discussed. The indirect determination of nitrogen shifts from the proton signals provides a dramatic gain in sensitivity over direct observation of 15N signals. Examples are given of applications of the methods to samples with natural abundance 15N concentration and to a sample of 0.7 mM 65% 15N enriched tRNAƒMet in H2O. Two-dimensional 15H15N shift correlation spectra of 0.1 M samples with natural abundance 15N concentration in a 5 mm sample tube can be obtained in measuring times on the order of 1 hr.
To assess the metabolic alterations induced by normothermic hepatic ischemia, 31P nuclear magnetic resonance analysis was performed on liver samples using perchloric acid extraction. In particular, phosphomonoesters and phosphodiesters, the intermediary metabolites of membrane phospholipid turnover, were characterized precisely and quantitated. Phosphocholine and phosphoethanolamine, the precursors of phospholipid anabolism, did not change, while the phosphodiesters decreased. In contrast, alpha-glycerophosphate, which is both a precursor of phospholipid synthesis and the intermediary product of phospholipid degradation, markedly increased following 30 min of normothermic ischemia. These findings suggest that cellular phospholipids are actively degraded during normothermic hepatic ischemia.
This study defined negative outcomes of solid organ transplantation, proposed a new classification of complications by severity, and applied the classification to evaluate the results of orthotopic liver transplantation (OLT).
The lack of uniform reporting of negative outcomes has made reports of transplantation procedures difficult to interpret and compare. In fact, only mortality is well reported; morbidity rates and severity of complications have been poorly described.
Based on previous definition and classification of complications for general surgery, a new classification for transplantation in four grades is proposed. Results including risk factors of the first 215 OLTs performed at the University of Toronto have been evaluated using the classification.
All but two patients (99%) had at least one complication of any kind, 92% of patients surviving more than 3 months had grade 1 (minor) complications, 74% had grade 2 (life-threatening) complications, and 30% had grade 3 (residual disability or cancer) complications. Twenty-nine per cent of patients had grade 4 complications (retransplantation or death). The most common grade 1 complications were steroid responsive rejection (69% of patients) and infection that did not require antibiotics or invasive procedures (23%). Grade 2 complications primarily were infection requiring antibiotics or invasive procedures (64%), postoperative bleeding requiring > 3 units of packed red cells (35%), primary dysfunction (26%), and biliary disease treated with antibiotics or requiring invasive procedures (18%). The most frequent grade 3 complication was renal failure, which is defined as a permanent rise in serum creatinine levels > or = twice the pretransplantation values (11%). Grade 4 complications (retransplantation or death) mainly were infection (14%) and primary dysfunction (11%). Comparison between the first and last 50 OLTs of the series indicates a significant decrease in the mean number of grade 1 and 2 complications. This was partially a result of better medical status of patients at the time of transplantation. Using univariate and multivariate analyses of risk factors, the best predictor of grade 1 complications was donor obesity; for grade 2 complications, the best predictor was a donor liver rewarming time of > 90 minutes, and for grade 3 and 4 complications, the best predictor was the APACHE II scoring system and donor cardiac arrest.
Standardized definitions and classifications of complications of transplantation will allow us to better evaluate and compare results of transplantation among centers and over time, and better compare effectiveness of new therapies. Orthotopic liver transplantation still is a procedure with high morbidity that requires careful analysis of risk factors to optimize selection of patients and organ sharing.
Spectral changes in human hepatic tumours and possible systemic effects of tumour on host liver were assessed by 31P and 1H in vitro NMR spectroscopy. The 1H and 31P spectra from liver tumour biopsies showed significant elevation in phosphoethanolamine, phosphocholine, taurine, citrate, alanine, lactate and glycine, and significant reduction in GPE (glycerophosphoethanolamine), GPC (glycerophosphocholine), creatine and threonine compared to histologically normal tissue. 31P-NMR spectra obtained from histologically normal tissue within tumour-bearing livers showed significant elevation in phosphoethanolamine and phosphocholine compared to data from liver biopsies from nontumour-bearing patients (pancreatitis). These results suggest that alterations in membrane metabolism in host liver can be detected by 31P-NMR.
The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.
High resolution 1H nuclear magnetic resonance (NMR) spectra using spinning at the magic angle (1H MAS NMR) have been obtained on intact normal and pathological kidney tissue samples from patients undergoing surgery for renal cell carcinoma (RCC). The spectra were measured on ca. 80 mg samples and provided high resolution 1H NMR spectra in which effects of dipolar couplings, chemical shift anisotropy and magnetic susceptibility differences are minimised thus yielding high spectral resolution. Conventional one-dimensional and spin-echo spectra and two-dimensional J-resolved, TOCSY and 1H-13C HMQC spectra were also measured on selected samples and these allowed the assignment of resonances of endogenous substances comprising both cytosolic and membrane components. The tumour tissues were characterised principally by an increased lipid content. These are the first reported results on human tumour tissues using this technique and the approach offers potential for the rapid classification of different types of tumour tissue.
In vivo hepatic phosphorus-31 magnetic resonance spectroscopy (MRS) provides non-invasive information about phospholipid metabolism.
To delineate MRS abnormalities in patients with chronic ductopenic rejection (CDR) and to characterise spectral changes by in vitro MRS and electron microscopy.
Sixteen liver transplant recipients (four with CDR; 12 with good graft function) and 29 controls (23 healthy volunteers; six patients with biliary duct strictures) were studied with in vivo 31P MRS. Peak area ratios of phosphomonoesters (PME) and phosphodiesters (PDE), relative to nucleotide triphosphates (NTP) were measured. In vitro MRS and electron microscopy were performed on biopsy specimens from five patients with CDR, freeze clamped at retransplantation. Phosphoethanolamine (PE), phosphocholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC) concentrations were measured.
The 12 patients with good graft function displayed no spectral abnormalities in vivo; the four patients with CDR showed significantly elevated PME:NTP (p < 0.01) and PDE:NTP ratios (p < 0.005). Patients with biliary strictures had significant differences in PME:NTP (p < 0.01) from patients with CDR, but not in mean PDE:NTP. In vitro spectra from CDR patients showed elevated PE and PC, mirroring the in vivo changes in PME, but reduced GPE and GPC concentrations were observed, at variance with the in vivo PDE findings. On electron microscopy, there was no proliferation in hepatocyte endoplasmic reticulum.
The increase in PME:NTP reflects altered phospholipid metabolism in patients with CDR, while the increase in PDE:NTP may represent a significant contribution from bile phospholipid.
High-resolution magic angle spinning (MAS) (1)H NMR spectroscopy has been used to investigate the biochemical composition of whole rat renal cortex and liver tissue samples. The effects of a number of sample preparation procedures and experimental variables have been investigated systematically in order to optimize spectral quality and maximize information recovery. These variables include the effects of changing the sample volume in the MAS rotor, snap-freezing the samples, and the effect of organ perfusion with deuterated saline solution prior to MAS NMR analysis. Also, the overall biochemical stability of liver and kidney tissue MAS NMR spectra was investigated under different temperature conditions. We demonstrate improved resolution and line shape of MAS NMR spectra obtained from small spherical tissue volume (12 microl) rotor inserts compared to 65 microl cylindrical samples directly inserted into the MAS rotors. D(2)O saline perfusion of the in situ afferent vascular tree of the tissue immediately postmortem also improves line shape in MAS NMR spectra. Snap-freezing resulted in increased signal intensities from alpha-amino acids (e.g., valine) in tissue together with decreases in renal osmolytes, such as myo-inositol. A decrease in triglyceride levels was observed in renal cortex following stasis on ice and in the MAS rotor (303 K for 4 h). This work indicates that different tissues have differential metabolic stabilities in (1)H MAS NMR experiments and that careful attention to sample preparation is required to minimize artifacts and maintain spectral quality.
The current shortage of donor organs in liver transplantation has led experienced transplant centers to use more "marginal" grafts. The development of a reliable technique of bile collection gives access to hepatic bile from donors and recipients for bile analysis to characterize the grafts. Proton nuclear magnetic resonance analysis has been applied to the study of bile for more than 30 years, showing encouraging results. This is the first study where proton nuclear magnetic resonance analysis has been applied to hepatic bile from selected liver grafts to evaluate its potential role in graft assessment. Hepatic bile was collected from eight liver donors (four with normal and four with steatotic grafts) during organ retrieval and four transplant recipients (two with good early graft function and two with primary dysfunction) immediately after graft reperfusion. A Varian Unity+ NMR spectrometer, operating at 11.7 Tesla (500 MHz for 1H), was used to obtain the proton nuclear magnetic resonance spectra. The results showed that the hepatic bile from steatotic grafts collected before transplantation had more intense phosphatidylcholine head group resonance than bile from normal grafts. It also showed slower clearance of University of Wisconsin solution in grafts with subsequent primary graft dysfunction, suggesting a slower recovery of bile secretion. These preliminary findings suggest that proton nuclear magnetic resonance analysis might help to differentiate the characteristics of bile acids and biliary lipids from normal and steatotic grafts. The monitoring of the resonance signal of University of Wisconsin solution washout, bile acid, and biliary lipid secretion may help to predict the development of primary graft dysfunction and avoid the need for retransplantation.
The metabolite profiles from livers of toxin-treated rats were investigated using high resolution 1H NMR spectroscopy of aqueous (acetonitrile/water), lipidic (chloroform/methanol) extracts and magic angle spinning (MAS)-NMR spectroscopy of intact tissue. Rats were treated with the model cholestatic hepatotoxin, alpha-naphthylisothiocyanate (ANIT, 150 mg/kg) and NMR spectra of liver were analysed using principal components analysis (PCA) to extract novel toxicity biomarker information. 1H NMR spectra of control aqueous extracts showed signals from a range of organic acids and bases, amino acids, sugars, and glycogen. Chloroform/methanol extracts showed signals from a range of saturated and unsaturated triglycerides, phospholipids and cholesterol. The MAS 1H NMR spectra of livers showed a composite of signals found in both aqueous and lipophilic extracts. Following ANIT treatment, 1H NMR-PCA of aqueous extracts indicated a progressive reduction in glucose and glycogen, together with increases in bile acid, choline, and phosphocholine signals. 1H NMR-PCA of chloroform/methanol extracts showed elevated triglyceride levels. The 1H MAS-NMR-PCA analysis allowed direct detection of all of the ANIT-induced tissue perturbations revealed by 1H NMR of extracts, enabling metabolic characterisation of the lesion, which included steatosis, bile duct obstruction and altered glucose/glycogen metabolism. MAS-NMR spectroscopy requires minimal sample preparation and, unlike 1H NMR spectroscopy of tissue extracts, does not discriminate metabolites based on their solubility in a particular solvent and so this is a particularly useful exploratory tool in biochemical toxicology.
High-resolution magic angle spinning (HR MAS) may develop into a new diagnostic tool for studying intact tissue samples, and several types of cancer have been investigated with promising results. In this study HR MAS spectra of breast cancer tissue from 10 patients have been compared to conventional high-resolution spectra of perchloric acid extracts of the same tissue type. The HR MAS spectra show resolution comparable to spectra of extracts, and two-dimensional techniques lead to identification of a majority of the constituents. More than 30 different metabolites have been detected and assigned. To our knowledge this is the most detailed assignment of biochemical components in intact human breast tissue. The spectra of intact breast cancer tissue differ from perchloric acid extracts by the presence of lipids and fewer signals in the low field region. HR MAS analysis of intact breast tissue specimens is a rapid method, providing spectra with resolution where relative quantification of the majority of the detected metabolites is possible.
The development of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for intact tissue analysis and the correlations between the measured tissue metabolites and disease pathologies have inspired investigations of slow-spinning methodologies to maximize the protection of tissue pathology structures from HR-MAS centrifuging damage. Spinning sidebands produced by slow-rate spinning must be suppressed to prevent their complicating the spectral region of metabolites. Twenty-two human prostatectomy samples were analyzed on a 14.1T spectrometer, with HR-MAS spinning rates of 600 Hz, 700 Hz, and 3.0 kHz, a repetition time of 5 sec, and employing various rotor-synchronized suppression methods, including DANTE, WATERGATE, TOSS, and PASS pulse sequences. Among them, DANTE, as the simplest scheme, has shown the most potential in suppression of tissue water signals and spinning sidebands, as well as in quantifying metabolic concentrations.
High-resolution 1H-NMR spectroscopy of serum and urine samples of an 11-year-old male living related orthotopic liver transplant recipient is reported. Serum glutamine increased to abnormal levels along with simultaneous abnormal excretion of urinary glutamine post-transplantation. High levels of glutamine in both blood and urine and concomitant reduced urea levels in urine were found to be evidence of impairment in urea cycle and compatible with persistently abnormal graft function. Thus glutamine levels in serum and urine, and urea in the urine as observed by 1H-NMR spectroscopy highlight their important roles in monitoring liver graft function; increased glutamine levels lead to brain damage, if untreated.
The exact nature of the interaction between small molecules and chromatographic solid phases has been the subject of much research, but detailed understanding of the molecular dynamics in such systems remains elusive. High-resolution (1)H magic-angle-spinning (MAS) NMR spectroscopy has been applied to the investigation of C18-bonded silica material as used in chromatographic separation techniques together with an adsorbed model analyte, p-xylene. Two distinct p-xylene and water environments were identified within the C18-bonded silica through the measurement of (1)H NMR chemical shifts, T(1) and T(2) relaxation times and diffusion coefficients, including their temperature dependence. The results have been analyzed in terms of two environments, p-xylene within the C18 chains, in slow exchange on the NMR time scale with p-xylene in a more mobile state adsorbed as a layer in close proximity to the C18 particles, but which is distinct from free liquid p-xylene. The techniques used here could have more general applications, including the study of drug molecules bound into phospholipid membranes in micelles or vesicles.
The previously observed improvement in spectral resolution of tissue proton NMR with high-resolution magic angle spinning (HRMAS) was speculated to be due largely to freeze-thawing artifacts resulting from tissue storage. In this study, 12 human prostate samples were analyzed on a 14.1T spectrometer at 3 degrees C, with HRMAS rates of 600 and 700 Hz. These samples were measured fresh and after they were frozen for 12-16 hr prior to thawing. The spectral linewidths measured from fresh and previously frozen samples were identical for all metabolites except citrate and acetate. The metabolite intensities of fresh and freeze-thawed samples depend on the quantification procedures used; however, in this experiment the differences of means were <30%. As expected, it was found that tissue storage impacts tissue quality for pathological analysis, and HRMAS conditions alone are not sufficiently destructive to impair pathological evaluation. Furthermore, although storage conditions affect absolute metabolite concentrations in NMR analysis, relative metabolite concentrations are less affected.
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