Metabolomic analysis—Addressing NMR and LC-MS related problems in human feces sample preparation

Moosmang, Simon; Pitscheider, Maria; Sturm, Sonja; Seger, Christoph; Tilg, Herbert; Halabalaki, Maria; Stuppner, Hermann

doi:10.1016/j.cca.2017.10.029

Cited by 39 publications

(27 citation statements)

References 45 publications

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“…The MxP Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) was developed for human plasma samples and it is suitable for use with fecal samples; which could possibly explain why most of the metabolites were detected in plasma rather than in stool samples, and that plasma metabolites showed the strongest associations and diagnostic accuracy. Moreover, the total of stool metabolites identified in our study was 67% higher than that reported in the kit application note [59], probably due to the applied lyophilization process [60]. Therefore, this finding highlights the importance of prudent interpretation of fecal metabolomic data as the results obtained for stool samples in our study could differ if water content is not taken into account.…”

Section: Discussioncontrasting

confidence: 61%

Plasma and stool metabolomic biomarkers of non-alcoholic fatty liver disease in Argentina

Mazzini

Cook²,

Gorcsan³

et al. 2020

Preprint

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Background and Aims: Non-invasive biomarkers are urgently needed to identify patients with non-alcoholic fatty liver disease (NAFLD) especially those at risk of disease progression. This is particularly true in high prevalence areas such as Latin America. The gut microbiome and intestinal permeability may play a role in the risk of developing NAFLD and NASH, but the mechanism by which microbiota composition disruption (or dysbiosis) may affect NAFLD progression is still unknown. Targeted metabolomics is a powerful technology for discovering new associations between gut microbiome-derived metabolites and disease. Thus, we aimed to identify potential metabolomic biomarkers related to the NAFLD stage in Argentina, and to assess their relationship with clinical and host genetic factors. Materials and methods: Adult healthy volunteers (HV) and biopsy-proven simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) patients were recruited. Demographic, clinical and food frequency consumption data, as well as plasma and stool samples were collected. SNP rs738409 (PNPLA3 gene) was determined in all volunteers. HPLC and flow injection analysis with MS/MS in tandem was applied for metabolomic studies using the MxP Quant 500 Kit (Biocrates Life Sciences AG, Austria). Significantly different metabolites among groups were identified with MetaboAnalyst v4.0. Bivariate and multivariate analyses were used to identify variables that were independently related to NAFLD stage. Forward stepwise logistic regression models were constructed to design the best feature combination that could distinguish between study groups. Receiver Operating Characteristic (ROC) curves were used to evaluate models′ accuracy. Results: A total of 53 volunteers were recruited: 19 HV, 12 SS and 22 NASH. Diet was similar between groups. The concentration of 33 out of 424 detected metabolites (25 in plasma and 8 in stool) was significantly different among study groups. Levels of triglycerides (TG) were higher among NAFLD patients, whereas levels of phosphatidylcholines (PC) and lysoPC were depleted relative to HV. The PNPLA3 risk genotype for NAFLD and NASH (GG) was related to higher plasma levels of eicosenoic acid FA(20:1) (p<0.001). Plasma metabolites showed a higher accuracy for diagnosis of NAFLD and NASH when compared to stool metabolites. Body mass index (BMI) and plasma levels of PC aa C24:0, FA(20:1) and TG(16:1_34:1) showed high accuracy for diagnosis of NAFLD; whereas the best AUROC for discriminating NASH from SS was that of plasma levels of PC aa C24:0 and PC ae C40:1. Conclusion: A panel of plasma and stool biomarkers could distinguish between NAFLD and NASH in a cohort of patients from Argentina. Plasma biomarkers may be diagnostic in these patients and could be used to assess disease progression. Further validation studies including a larger number of patients are needed.

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Section: Discussioncontrasting

confidence: 61%

Plasma and stool metabolomic biomarkers of non-alcoholic fatty liver disease in Argentina

Mazzini

Cook²,

Gorcsan³

et al. 2020

Preprint

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“…Gut microbiota is associated with a variety of diseases and has become the subject of intensive research in recent years. Freeze drying of fecal samples has emerged as a useful tool for microbiota transplantation treatment and metabolome research (Moosmang et al 2019 ; Staley et al 2017 ), but comprehensive setups have not been published yet. The protocol provided here can be beneficial for laboratories utilizing high-performance liquid chromatography—mass spectrometry for metabolome analysis because removing liquid and volatile components from samples provides a highly pure, solvent free product without the degradation of components.…”

Section: Discussionmentioning

confidence: 99%

Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques

et al. 2021

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Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term stability of proteins and nucleic acids in lyophilized samples.Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to −80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.

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“…While working with dried samples is less laborious, more reproducible and prevents bacterial growth, it also results in a loss of detected metabolites, especially VOCs. Indeed, the effect of lyophilization has been compared with the use of fresh sample and results in a decrease in short chain fatty acids [76,77]. Therefore, Karu et al suggest that, unless volatile compounds are specifically targeted for quantification, fecal samples should be dried and weighed prior to storage or analysis.…”

Section: Sample Collectionmentioning

confidence: 99%

Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions

Smith

Villaret-Cazadamont

Claus³

et al. 2020

Metabolites

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Metabolomics has found numerous applications in the study of liver metabolism in health and disease. Metabolomics studies can be conducted in a variety of biological matrices ranging from easily accessible biofluids such as urine, blood or feces, to organs, tissues or even cells. Sample collection and storage are critical steps for which standard operating procedures must be followed. Inappropriate sample collection or storage can indeed result in high variability, interferences with instrumentation or degradation of metabolites. In this review, we will first highlight important general factors that should be considered when planning sample collection in the study design of metabolomic studies, such as nutritional status and circadian rhythm. Then, we will discuss in more detail the specific procedures that have been described for optimal pre-analytical handling of the most commonly used matrices (urine, blood, feces, tissues and cells).

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Metabolomic analysis—Addressing NMR and LC-MS related problems in human feces sample preparation

Cited by 39 publications

References 45 publications

Plasma and stool metabolomic biomarkers of non-alcoholic fatty liver disease in Argentina

Plasma and stool metabolomic biomarkers of non-alcoholic fatty liver disease in Argentina

Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques

Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions

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