Metabolomics/Analytical Methods/Mass Spectrometry/LC-MS

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LC-MS-BASED METABOLOMICS IN DRUG METABOLISM http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2140249

Summary: This paper describes techniques to identify xenobiotic metabolites and metabolic pathways. Currently, the primary techniques used to gather data are in vitro incubation assays in human and animal trials and in vivo radiotracing experiments. In vitro assays are performed using hepatocyte, merosome, recombinanat human P450s incubations and liver perfusion. These investigators, however, have shown that LC-MS based metabolomic techniques are helpful for xenobiotic metabolism. All living organisms perform some sort of metabolism to facilitate removal of foreign substances. Xenobiotic metabolites and metabolism has become a main componenet of drug metabolism and chemical toxicology.

Most widely used analytical instruments in metabolomic research are nuclear magnetic resonance (NMR), spectrometers, and mass spectroscopy (ms). Also used, but less frequently is electrochemistry or infrared spectroscopy. Prepared biological samples into a mass spectrometer can be used through direct injection, gas chromatography, liquid chromatography, and capillary electrophoresis. Liquid chromatography (LC-MS) is used the most because LC based samples result in lesser ion suppression and higher resolution.

This article explores applicable approaches using LC-MS based metabolomic techniques to facilitate problems in drug metabolism. Investigators compared the in vitro and in vivo methods in drug metabolism to LC-MS techniques and showed that LC-MS has an advantage because of its capacity to hold large datasets and is able to graphically represent metabolism samples as a way of classification. It is though that as LC-MS technology and MDA software increase in availability the use of LC-MS metabolomics will be widely used in xenobiotic metabolism.

Terms: -Xenobiotic metabolism- refers to the set of metabolic pathways that chemically modify xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as drugs and poisons.

-in silico- is an expression used to mean "performed on computer or via computer simulation." The phrase is coined from the Latin phrases in vivo and in vitro LC-MS- is liquid chromatography mass spectroscopy. It is a technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. LC-MS is frequently used in drug development at many different stages including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control

-Microsome- is a small vesicle that is derived from fragmented smooth endoplasmic reticulum (SER) produced when tissues such as liver are mechanically broken (homogenized). Microsomes contain the cell's cytochrome P450 (CYP) enzymes, involved in oxidative metabolism.

-software- Multivariate Data Analysis refers to any statistical technique used to analyze data that arises from more than one variable. This essentially models reality where each situation, product, or decision involves more than a single variable.

-XME polymorphisms- XMEs are xenobiotic metabolizing enzymes. Not much is know but they can be used in gene knock out models to help elucidate their role in xenobiotic metabolism and function.

-PCA analysis- Principal components analysis (PCA) is a technique used to reduce multidimensional data sets to lower dimensions for analysis. PCA is mostly used as a tool in exploratory data analysis and for making predictive models. PCA involves the calculation of the eigenvalue decomposition or Singular value decomposition of a data set, usually after mean centering the data for each attribute. The results of a PCA are usually discussed in terms of component scores and loadings.

-Cytochrome P450- abbreviated CYP, P450, and sometimes CYP450 is a very large and diverse superfamily of hemoproteins found in bacteria, archaea and eukaryotes. Cytochromes P450 use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer chains, called P450-containing systems.

-CYP1A2- a member of the cytochrome P450 mixed-function oxidase system, is involved in the metabolism of xenobiotics in the body. Expression of CYP1A2 appears to be induced by various dietary constituents.

Relevance: This article is related to some course material we have covered. Organisms are also exposed to compounds that they cannot use as foods/fuel and would be harmful if they accumulated in cells, as they have no metabolic function (xenobiotics). Xenobiotics are common in sewage treatment areas and can be attained through fish, drugs, and antibiotics.

The first phase of xenobiotic metabolism involves metabolic activation of xenobiotics (ie compounds that may be derived from drugs or environmental toxins). Several enzymes can take a part in this phase. We learned about redox reactions in chapter 13 and how electrons can be transferred as electrons, as hydride, as a hydrogen atom, or in combination with O2. Xenobiotics can be detoxified by a set of xenobiotic-metabolizing enzymes such as cytochrome P450 oxidase where an atom of oxygen is added to non-active hydrocarbons. In this case electrons are transferred in combination with O2. Another enzyme that could be used in metabolic activation is flavin monoxygenases, which catalyze reactions in where one of the two oxygen atoms of O2 is incorporated into the substrate. These flavin monoxygenases use reduced flavin nucleotides (FMNH2 or FADH¬2). We learned that flavin molecules are involved in carbon-carbon bonds and they can participate in 1 electron transfers. Furthermore, another enzyme that can contribute to metabolic activation is alcohol dehydrogenase. In chapter 14 we looked at the use of alcohol dehydrogenase in the production of ethanol in yeast by fermentation. Alcohol dehydrogenase converted acetaldehyde using NADH to ethanol and NAD+.

Articles and Web Pages for Review and Inclusion

Peer-Reviewed Article #1:

Dual Labeling of Metabolites for Metabolome Analysis (DLEMMA): A New Approach for the Identification and Relative Quantification of Metabolites by Means of Dual Isotope Labeling and Liquid Chromatography−Mass Spectrometry

Anal. Chem., 2009, 81 (22), pp 9257–9266'"

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Relevance to a Traditional Metabolism Course

Peer-Reviewed Article #2:

Ovarian cancer detection from metabolomic liquid chromatography/mass spectrometry data by support vector machines

BMC Bioinformatics 2009, 10:259'"

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Summary

Relevance to a Traditional Metabolism Course