Metabolomics/Metabolites/Carbohydrates

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 * 1) Glucose
 * 2) Sucrose and Other Dissacharides
 * 3) Polysaccharides

=Carbohydrates=

KEGG MAPS
Examples of Carbohydrate Metabolites

Lactose: http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6134 (PubChem)

Glycogen: http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=439177 (PubChem)

Ribose http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=5779 (PubChem)

Glyceraldehyde http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=751 (PubChem)

Starch: http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=439341 (PubChem)

Websites

'''[http://www.whatislife.com/reader2/Metabolism/pathway/glycogen.html What is Life? Pentose Phosphate Pathway and Glycogen Metabolism] and [http://www.whatislife.com/reader2/Metabolism/pathway/glycolysis.html What is Life? Glycolysis and Gluconeogenesis]'''

 Summary:  This website gives a basic overview of carbohydrates and the role they play in the human body. It also covers metabolism of carbohydrates. Glucose oxidation can be seen in Glycolysis as well as the pentose-phosphate pathway while the regeneration of glucose is seen in gluconeogenesis. This website shows, step by step, the reactions of these pathways and the energy yield from each step. The second link for this website goes to a page covering glycogen metabolism, showing the net reactions for the synthesis and degradation of glycogen. Both links have sections on major control mechanisms for glucose and glycogen metabolism.

 Terms:  Glucosidases – enzymes involved in breaking down complex carbohydrates such as starch and glycogen

ascorbic acid – a sugar acid with antioxidant properties. Scurvy is the disease caused by a deficiency of vitamin C.

glucostat – removing glucose when offered in excess via glycogen synthesis and glycolysis and providing glucose when needed by gluconeogenesis

concomitant – accompanying in a passive way

hepatic lipase – lipolytic enzyme, synthesized by hepatocytes and found localized at the surface of liver sinusoid capillaries

phosphoglucomutase – an enzyme that transfers a phosphoryl group on a glucose monomer from the 1' to the 6' position in the forward direction or the 6' to the 1' position in the reverse

UTP – (Uridine triphosphate) - a pyrimidine nucleotide, consisting of the organic base uracil

Relation: This website clearly explains what is meant by carbohydrate metabolism. It shows the relationship between glycolysis and gluconeogenesis and the three enzymes that are different between the two pathways. It also shows major control methods for the metabolism of carbohydrates, specifically glucose and glycogen in this case. This site is a great resource to use for a more in depth coverage of glucose and glycogen metabolism.

The Human Metabolome Database

Summary: The Human Metabolome Database is a database that contains information about small metabolites. This information includes chemical composition, taxonomy, location in the body, normal levels of the metabolite, genomic sequence, pathways it is involved in, and other molecules it interacts with. This site has links to KEGG, PubChem, MetaCyc, ChEBI, PDB, Siss-Prot, and GenBank (see terms below). This website is particularly easy to use and has enormous amounts of information on each molecule. Just go to the main page and type in the name of the molecule you are looking for.

Terms: KEGG – Kyoto Encyclopedia of Genes and Genomes - bioinformatics resource for linking genomes to life and the environment

PubChem – Free database of chemical structures of small organic molecules and information on their biological activities

MetaCyc – Encyclopedia of Metabolic Pathways - contains over 900 pathways from more than 900 different organisms

ChEBI – Chemical Entities of Biological Interest (ChEBI) is a freely available dictionary of molecular entities focused on 'small' chemical compounds

PDB – Protein Data Bank - Archive of experimentally-determined, biological macromolecule 3-D structures from the Brookhaven National Laboratory

Swiss-Prot – manually curated biological database of protein sequences

GenBank – NIH genetic sequence database, an annotated collection of all publicly available DNA sequences

Relation: This website provides easy access to information on small molecules, such as carbohydrates, that would be useful in the study of metabolomics. You are able to determine what the normal levels of a metabolite is in the body and the pathways involved that could increase or decrease those levels. This would be helpful in screening for particular metabolic diseases.

University of Akron: Carbohydrate Metabolism

Summary: This is a very basic website outlying the principles of carbohydrate metabolism. It touches on the metabolite glucose including its synthesis and degradation pathways. It also touches upon other carbohydrates such as starch, glycogen, and maltose. This site describes what these metabolites are used for in the body and how they are a part of several different pathways. Biosynthesis and regulation of carbohydrate metabolites are also a part of this website.

Terms: Fructose – is a simple reducing sugar (monosaccharide) found in many foods

Aldolase – an enzyme that helps convert glucose into energy

Fermentation – the process of energy production in a cell under anaerobic conditions (without oxygen)

Propionate – is a naturally-occurring carboxylic acid

Biotin – vitamin H or B7, water-soluble B-complex vitamin, used in carboxylation reactions

NDP-Glucose – Nucleoside diphosphate-Glucose – activated intermediate

Glycosidic bond – certain type of functional group that joins a carbohydrate (sugar) molecule to another

Relation: This site shows the different ways that carbohydrate metabolites interact in our body and how they can by synthesized and broken down. This site is a great tool to visualize where certain metabolites are found and the pathways they are involved in. With many diagrams, this site is ideal for piecing together where carbohydrate metabolites fit in the grand scheme of things in the human body.

Peer-review Articles

A Model for Carbohydrate Metabolism in the Diatom Phaeodactylum tricornutum Deduced from Comparative Whole Genome Analysis

Summary: Diatoms are responsible for 20% of the earth’s carbon fixation. However, this mechanism is not well understood. This paper attempt to clarify this process by proposing a model for carbon fixation by diatoms. Little is also known about the synthesis and storage of Chrysolaminaran, the main carbohydrate in diatoms. This carbohydrate is secreted and is a major component of biofilms. In this paper, a model for the carbon concentrating mechanism, photorespiration, the oxidative and reductive pentose phosphate pathways and a model of the glycolytic reactions were generated. The model for the glycolytic reaction is shown below.

Link to glycolytic reaction proposed by this paper

Terms: Diatoms – major group of eukaryotic algae, and are one of the most common types of phytoplankton, most are unicellular

Rubisco – Ribulose-1,5-bisphosphate carboxylase/oxygenases - catalyzes the incorporation of CO2

Chrysolaminaran – the principle storage carbohydrate in diatoms

Chimeras – two or more different populations of genetically distinct cells

Endosymbiont – any organism that lives within the body or cells of another organism

Plastids – organelles that only exist in plant cells and photosynthetic protists, responsible for photosynthesis

in silico analysis – analysis performed using computers in conjunction with informatics capabilities

chlorenchyma cells – parenchyma cells that contain chlorophyll

periplasmic space – space seen between the plasma membrane and the outer membrane

Relation: Much is known about carbohydrate metabolism and the many different carbohydrate metabolites in the human body. However, it is surprising that little is known about how diatoms fix carbon dioxide since it is so important to our survival. This paper takes the first step in proposing a mechanism by which this takes place. Someday, we may find this very diagram in our textbooks.

Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress

Summary: In order to access this article, you must first complete a free registration with the journal of biology. This study is one of the first to develop a mathematical model that describes the observed experimental changes in metabolic flux. In response to reactive oxygen species, yeast will shift from glycolysis to the pentose-phosphate pathway as an immediate and protective response to ROS. This shift is made in order to maintain redox balance in the cell.

Terms: Deleterious – harmful, injurious

Triose-phosphate isomerase – TPI - catalyzes the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate

metabolic flux – the rate of flow of metabolites

Reactive oxygen species – ROS - oxygen ions, free radicals, and peroxides, both inorganic and organic

Perturbations – disturbance of motion, course, arrangement, or state of equilibrium

de novo – afresh, anew, beginning again

diamide – Any compound containing two amido groups united with one or more acid or negative radicals

Relation: It is well known that the human body will alter which pathway it uses (glycolysis or gluconeogenesis) depending on the carbohydrate status of the blood. This study has now shown that yeast will change from using glycolysis to the pentose phosphate pathway in response to reactive oxygen species. It is interesting to see how many different organisms have the ability to switch between pathways in order to counteract detrimental affects or to save energy.

Destabilization of ATP-sensitive potassium channel activity by novel KCNJ11 mutations identified in congenital hyperinsulinism

Summary: This paper demonstrates the importance of the ATP-sensitive potassium channel in controlling the amount of insulin secreted by pancreatic cells. It is shown that having a mutation in the genes encoding for this potassium channel can depolarize the membrane leading to an increase in the basal level of insulin secretion. This can lead to the disease, hyperinsulinism.

Terms: Hyperinsulinism – refers to an above normal level of insulin in the blood of a person or animal

Sulfonylurea – A class of oral hypoglycemic agents (pills and capsules taken to lower the level of blood glucose) by people with type 2 diabetes

Phosphoinositides – serve as direct local modulators or recruiters of the protein machineries that control membrane trafficking

Hypoglycemia – is the medical term for a pathologic state produced by a lower than normal level of glucose in the blood

Somatostatin – growth hormone inhibiting hormone

octreotide.- potent inhibitor of growth hormone, glucagon, and insulin

Chemiluminescence – the emission of light (luminescence) with limited emission of heat as the result of a chemical reaction

Exogenous – caused by factors from outside the organism or system

Relation: Throughout the study of metabolism, we learn that a pH and charge gradient is necessary to have across a membrane. In this case, the charge gradient is essential for proper signaling in the cell. With a lower depolarization, insulin is secreted at a higher basal level, leading to the disease of hyperinsulinism.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1694820.com

Summary: The main point of this peer review article is to discuss the glycolysis process, which results in the formation of ATP as the main fuel in the anaerobic eukaryote, Monocercomonoides. Both ATP and Glucose are metabolites that relate directly to glycolysis and would be included in the organism's metabalome. Since anaerobic eukaryotes lack a complete tricarboxylic acid cycle, they can only extract few molecule of ATP per molecule of glucose. This is opposite of human cells, which makes this article important as it discusses alternate versions of enzymes in glycolysis. In anaerobic eukaryotes, enzymes such as pyruvate orthophosphate dikinase and pyrophosphate-fructose 6-phosphate phosphotransferase were examined through horizontal gene transfers from the bacteria in question to other eukaryotes. Horizontal gene transfers are processes in which an organism transfers genetic material to another cell that is not its offspring. Analysis of the glycolytic pathway of Monocercomonoides resulted in identification of both single-gene trees and conserved HGT events that provide evidence for the monophyly of protists known as Ecavata. Shared horizontal gene transfer events between oxymonads and both Giardia and Trichomonas further supported this relationship. This relationship becomes an important resource as it focuses on using glucose metabolites of Monocercomonoides to determine its lineage.

Terms: Horizontal Gene Transfer- process in which an organism transfers genetic material to another cell that is not its offspring

Monophyly- a group of organisms that includes an ancestral species and all of its descendants.

Oxymonad- a group of flagellated protozoa found exclusively in the intestines of termites and other wood-eating insects.

Diplomonads- a group of flagellates, most of which are parasitic. Also classified as Protists.

Walking primers-is a sequencing method for sequencing large DNA fragments (between 1,3 and 7 kilobases) that works by dividing long sequences into several consecutive short ones.

Phylogenetic analysis- Analysis of the evolutionary connections between traits (for example, sequences)

Taxa- Any organism or group of organisms of the same taxonomic rank; for example, members of an order, family, genus, or species.

Relation: This resource is very closely related to our current coursework. As one would recall we had to memorize the complete glycolysis pathway, which is exactly what this article covered. It focused on the enzymes involved in the pathway, which are some that I have memorized. It also reviewed the process through an anaerobic prospective, which sheds light on how glycolysis can be altered but still produce ATP. This is very interesting as we mainly focused on just the one type of glycolysis.

Flux control and excess capacity in the enzymes of glycolysis and their relationship to flight metabolism in Drosophila melanogaster

Summary: The focus of this research article is to study the degree of flux control of several enzymes involved in the glycolytic pathway on flight performance of the fruit fly. The author’s were able to research this by “knocking out” the enzymes phosphoglucose isomerase, phosphoglucomutase, triosephosphate isomerase, hexokinase, pyruvate kinase, and glycogen phosphorylase and studying the effect each knockout had on wing beat frequency of Drosophila. Wing-beat frequency is an accurate measure of flux control because it is closely correlated with oxygen consumption and directly reflects the rate of the hydrolysis of ATP.

Terms: Dipteran - A member of the insect order Diptera (the two-winged, or "true," flies), characterized by the use of only one pair of wings for flight and the reduction of the second pair of wings to knobs used for balance. P-element - a transposon that is present in the fruit fly Drosophila melanogaster, and is used widely for mutagenesis and the creation of genetically modified flies used for genetic research. Hemolymph – insect “blood” Polymorphism - The occurrence of different forms, stages, or types in individual organisms or in organisms of the same species, independent of sexual variations. Dysgenic - Relating to or causing the deterioration of hereditary qualities in offspring. Introgression - Infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents.

Relevance: The research carried out in this article involves the glycolytic pathway, which we have covered in class. The authors researched the control of several of the enzymes involved in glycolysis on the regulation of flight in Drosophila. The authors tested the enzymes’ regulatory effectiveness by reducing each enzyme’s activity and calculating their flux controls on flight performance.

Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using (1,2-13C2) glucose

Summary: This paper focused on measuring the rate at which metabolites flow through the pathway (i.e. the flux) of the rat hepatic glucose metabolic network in both forward and reverse directions.

Terms: Ion exchange chromatography – A process that allows the separation of ions and polar molecules based on the charge properties of the molecules. Electron impact ionization – Method of mass spectrometry ionisation. The sample of interest is vaporized into the mass spectrometer ion source, where it is impacted by the a beam of electrons with sufficient energy to ionise the molecule. Isotopomer – (isotopic isomer) Isomer having the same number of each isotopic atom but differing in their positions. Outflux – Movement of metabolites out of the pathway. Influx – Movement of metabolites into the pathway. Anaplerosis - An intermediate enzyme reaction that replaces the concentration of vital reacting compound that has been depleted in an organism.

Relevance: The paper involves two major concepts that have been discussed in class. First, it uses a measurement of flux as a means to analyze the metabolic control of a pathway. Secondly, it mentions that, rather than existing in isolation, many pathways are in fact linked and integrated into each other through the sharing of substrates, activators, inhibitors etc, so that changes in one have an effect on the others.

Metabolic-flux profiling of yeasts S.cerevisiae and P.stipitis

Summary: This research explores the differences in metabolic regulation between 2 strains of yeast, S.cerevisiae and P.stipitis, when grown in media containing different concentrations of glucose. The central carbon metabolism of P.stipitis is largely unidentified, while that of S.cerevisiae is well documented. Metabolic-flux ratio analysis was used to identify active pathways in the central carbon metabolism of P.stipitis. Differences in flux ratios were indicative of differences in activity in pathways between the strains of yeast.

Terms: METAFoR (Metabolic-flux ratio) Analysis – a method to determine the rate at which a metabolite is produced NMR (nuclear magnetic resonance) spectroscopy – a technique used to identify molecules Chemostat Cultures – cultures grown in a static chemical environment anaplerosis – process of restoring depleted sources of metabolic intermediates respiro-fermentative metabolism – simultaneous execution of fermentation and respiration

Relevance: This research can be directly tied to the content of this course. It focuses on 3 metabolic pathways: glycolysis, pentose phosphate, and the citric acid cycle. The analysis of these pathways and subsequent comparison between 2 organisms is an investigation of metabolic regulation.

Articles and Web Pages for Review and Inclusion
Peer-Reviewed Article #1:

Transcriptomic and metabolomic profiling of Zymomonas Mobilis during aerobic and anaerobic fermentations
[http://www.biomedcentral.com/1471-2164/10/34 Yang S, Tschaplinski TJ, Engle NL, Carroll SL, Martin SL, Davison BH, Palumbo AV, Rodriguez M and Brown SD. BMC Genomics (2009) 10:34]

Reviewer: Shannon B

Main Focus: The main focus of the article was to understand how the aerobic and anerobic Z.mobilis fermentations mixture with ZM4s would affect their growth with and without oxygen.

Summary: Zhomana mobilis (ZM4) produces very closely to the theoretical yields of ethanol with high productivity that is specific. The recombinant strains are able to ferment both C-5 and C-6 sugars. ZM4 is achieved best under anaerobic conditions but is an aerotolerant organism. This particular DNA molecule is not well understood under the genetic and physiological responses to stresses. For Zhomana mobilis (ZM4), a type of bacteria, transcriptomic and metabolomic aerobic and anaerobic fermentations were demonstrated by microarray analysis. High-performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS) were also demonstrated to the experiment. When oxygen was not present, ZM4 absorbed glucose more rapidly, higher growth rate, and the major end product was ethanol. There were other end products that produced a great amount such as acetate, lactate, and acetoin. There was only 1.7% of ethanol shown aerobically as there was anaerobically. Certain gene expressions were not examined during the early exponential growth phase between aerobic and anaerobic conditions via microarray analysis. As fermentations continued there were greater differences in extracellular metabolite profiles. Under anaerobic conditions ZM4 contained lower levels of amino acids such as alanine, valine and lysine relating to aerobic conditions. In the stationary phase, the microarray analysis showed that 166 genes were differently expressed by more than a two-fold. ZM4 ferments glucose, fructose, and sucrose producing ethanol and carbon dioxide via the Enterner-Doudoroff (ED) pathway. A key enzyme was encoded by transcripts of the ED pathway genes. During the ZM4 fermentations the high oxygen concentration negatively influenced the fermentation performance and the maximum specific growth rates were not very different between the aerobic and anaerobic conditions. Although, oxygen did affect the physiology of the cells which lead to the buildup of metabolic byproducts which after awhile led to high differences in transcriptomic profiles in stationary phase.

Helpful links: http://www1.eere.energy.gov/vehiclesandfuels/pdfs/success/zmobilis_mar_2001.pdf

http://www.uniprot.org/taxonomy/542

New Terms
High-performance liquid chromatography (HPLC): is a form of column chromatography that separates, identifies, and quantifies compounds based on their idiosyncratic polarities and interactions with the column's stationary phase. (http://en.wikipedia.org/wiki/High_performance_liquid_chromatography)

Gas chromatography (GC): A chromatography that is used for separating and analyzing compounds that can be vaporized without decomposition. (http://en.wikipedia.org/wiki/Gas-liquid_chromatography)

Gas chromatography-mass spectrometry (GC-MS): identifies different substances within a test sample that combines gas-liquid chromatography and mass spectrometry. (http://en.wikipedia.org/wiki/GC-MS)

Microarray analysis: determines which genes in a cell are switched on at a certain point in time. Thousands of samples are measured in a single assay of many different types of assays that are used. (http://www.wisegeek.com/what-is-microarray-analysis.htm)

Lignocellulosic: "A compound of lignin and cellulose that occurs in the walls of xylem cells in woody tissue." (http://www.thefreedictionary.com/lignocellulose)

Entner-Doudoroff (ED) pathway: uses a set of enzyme that is different than what is used in glycolysis or the pentose phosphate pathway that catabolizes glucose to pyruvate. (http://en.wikipedia.org/wiki/Entner%E2%80%93Doudoroff_pathway)

Aerotolerant organism: An organism that is anaerobic and is capable of surviving or growing regardless of the presence of oxygen. (http://www.biology-online.org/dictionary/Aerotolerant)

Zymomonas mobilis: a bacterium that belongs to the genus Zymomonas.It's known for it's bioethanol-producing capabilities. "It was originally isolated from alcoholic beverages like the African palm wine, the Mexican pulque, and also as a contaminant of cider and beer in European countries." (http://en.wikipedia.org/wiki/Zymomonas_mobilis)

Relevance Statement: There are quite a lot of techniques used in this article which will be useful in the study of metabolism and different metabolites. For example, gas chromatography (GC), High-performance liquid chromatography, gas chromatography-mass spectrometry. As our course concentrates on different metabolic pathways these techniques are pretty helpful on understanding the metabolic measurements and results of these metabolic pathways. This article concentrates more on the aerobic and anaerobic fermentation. This gives us a better idea of where this metabolic pathway is applied in different organisms like the Zymomonas mobilis. Different bacteria have been studied such as Escherichia coli, Zymomonas mobilis, and Klebsiella oxytoca. Gram positive bacteria have also been studied such as Bacillus subtilis and corynebacterium glutamicum.