Metabolomics/Metabolites/Amino Acids

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Ribbon Structure of Glutamate Dehydrogenase

Interconverting between glutamate and 2-oxoglutarate is known to play a significant role in plant carbon (C) and nitrogen (N) metabolism, and can be catalyzed by glutamate dehydrogenase (GDH). However, GDH maintains trivial functionality, in plants. In an attempt to expound upon the role of GDH, researchers analyzed GDH expression in Arabidopsis thaliana tissues. Results indicated that Arabidopsis GDH gene expression regulation was dependent upon the type of organ or tissue analyzed and the cellular C concentration. In addition, gdh1-2 and gdh2-1 were identified as Arabidopsis mutants that were defective in GDH genes. These mutants were isolated, crossed to make a double knockout mutant, characterized as gdh1-2/gdh2-1, containing negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis of these mutants revealed that gdh1-2/gdh2-1 plants are more susceptible to C-deficient conditions, which indicated that the double-knockout mutant supports the catabolic role of GDH, and contributes to fuelling the TCA cycle during C starvation. When glutamate was provided as an alternative N source, there was a determined reduced rate of glutamate catabolism in gdh2-1 and gdh1-2/gdh2-1 plants that was indicated by the consequential growth retardation, of these mutants. There were significant differences among amino acid profiles obtained from WT and the gdh mutant plants, during prolonged dark conditions. These differences were evident when glutamate levels were discovered to increase in WT plants and decrease in gdh1-2/gdh2-1. In addition, identified contributing factors to GDH regulation were cellular carbohydrate, and ATP levels. Further evidence was provided by the abnormal accumulation of several amino acids that was observed in gdh1-2/gdh2-1 plants. These results conclude that GDH has a significant role in the breakdown of amino acids, during C-starvation.

GDH participation in amino acid breakdown is apparently significant to all organisms, during C deficiency. Although, plants are capable of sacrificing tissues and cells in order to support other tissues and cells that are more metabolically significant, for survival. This behavior was exemplified when the observed damage to rosette leaves was more severe than the damage that stems or shoot apical meristems incurred, during C deficient conditions. Mitochondrial localization of GDH was determined to directly affect GDH involvement in carbon skeleton funneling of amino acids into the TCA cycle, during C deficiency. In addition, the ability of GDH to bind to RNA, inhibiting in vitro RNA editing, was attenuated by increased levels of NADH, NADPH, and ATP. This provided further insight into the capability of GDH to bind to these molecules.


Review Article: Arginine deprivation and metabolomics: Important aspects of intermediary metabolism in relation to the differential sensitivity of normal and tumour cells Arginine deprivation is known to cause a wide range of tumor cell types to die due to the inability to convert urea cycle intermediates to arginine. In rapidly multiplying and growing organisms arginine is often the rate-limiting amino acid or "semi-essential" amino acid. Tumors have a high requirement for arginine and causes increase mitotic activity in animal tumors. Therefore, low arginine, slows proliferation and in malignant tumor cells low, but not completely deficient arginine concentrations cause cell death, however the pathway to death is not clear and apoptosis has been considered. Cell culture in vivo experiments were performed and it was demonstrated that homeostatic mechanisms existing in vivo to restore arginine levels were not present in vitro where there is no available citrulline. Arginine elimination was also assessed for cells where arginine was directly eliminated and for cells where elimination of arginine occurred via arginine degrading enzymes. In normal cell cultures made arginine deficient with arginine catabolizing enzymes, cell growth was recovered by citrulline, which was converted to arginine by the urea cycle, but none were resucued with the addition of argininge due to the catabolizing enzymes. In vivo the elimination of arginine without catabolic enzymes is not successful unless citrulline synthesis has been compromised such as in renal failure. Nevertheless, in vitro studies seem more promising. Under normal conditions the conversion rate of citrulline to arginine is too low to sustain tumor growth. The prospect of developing arginine anticancer drugs is very likely, although there is quite sometime before this can occur. Different enzymes will produce different products and these may impact cell metabolism differently. Metabolomics is relevant and could be useful in characterizing tumors and patients in the future. In addition metabolomics could be used to facilitate certain protocols in cancer therapy.

1. semi-essential amino acid- amino acids are ones that can sometimes be made internally if conditions are right. Arginine and histidine can be converted from other amino acids if needed. 2. citrulline- is an alpha amino acid and a key intermediate in the urea cycle. Citrulline is a substituted urea that is produced from ornithine and carbamoyl phosphate and also is produced from argininge as a byproduct catalyzed by nitric oxide synthase. When starved for arginine, organisms will convert ornithine to citrulline to yield arginine. 3. in vitro- is an experiment in a controlled environment outside living systems. 4. in vivo- experiments taking place inside an organism. 5. catabolism- metabolic process where molecules are broken down into smaller units and yield energy. 6. cell culture- the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. 7.. hepatocellular carcinoma- is a primary malignancy (cancer) of the liver. Most cases of HCC are secondary to either a viral hepatitides infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis). 8. stroma- is a nonfunctional connective framework found in cells, tissues, or organs. 9.. Argininosuccinate- Some cells produce argininosuccinic acid from citrulline and aspartic acid and use it as a precursor for arginine in the urea cycle. Argininosuccinic acid is a precursor to fumarate in the citric acid cycle via argininosuccinate lyase.

This article is related to our biochemistry metabolism class in that citrulline can be converted to arginine in the urea cycle. In the urea cycle, two amino groups enter the cycle as carbamoyl phosphate, which is formed in the mitochondrial matrix and as aspartate also formed in the mitochondrial matrix by transamination of oxolacetate and glutamate. Subsequently, the fist step is formation of citrulline from ornithine and carbomyl phosphpate. Citrulline is generated in the mitochondria then passes into the cytosol. Argininosuccinate is formed through citrullyl-AMP, an intermediate. Arginine is generated from argininosuccinate and fumarate is released, which can enter the citric acid cycle. From arginine, urea is produced and ornithine is regenerated.

Arginine- An essential Amino acid (pubchem) KEGG links:

Metacyc links: Peer Reviewed article: d-Serine-induced nephrotoxicity: a HPLC–TOF/MS-based metabonomics approach

The role in of D-serine as an endogenous modulator of the glycine site on the N-methyl-D- aspartate receptor in the brain of humans and experimental animals has recently been demonstrated. D-serine levels in the brain are analogous to glycine, however D-serine levels in some areas such as the stratium is 2.6 times higher than that of glycine. D- serine is known to cause selective damage to the renal proximal tubule cells and leads to proteinuria, glucosuria, and aminoaciduria. Aminoaciduria is the important product that cause onset of necrosis. The mechanism that D-serine produces renal injury in the rat is still unclear. D-serine is metabolized once it enters the pars recta region of the proximal tubule and alpha keto acid, ammonia and hydrogen peroxide are produced. Investigators of this paper used HPLC-MS metabonomics to study the characteristics of urinary metabolites related to D-serine induced nephrotoxicity. By HPLC-MS it was shown that D-serine caused a change in the urinary metabolic report. Metabolites such as proline, methionine, leucine, tyrosine and valine were observed in addition to an increase in acetyl carnitine. This information provided information of D-serine metabolism and its performed function in the kidney.

1. nephrotoxicity- is the poisonous effect on the kidney that may be caused by medications or toxins. 2. aminoaciduria- amino acids in the urine 3. proteinuria- protein in the urine 4. glucosuria- glucose in the urine 5. proximal tubule- is the portion of the duct system of the nephron leading from Bowman's capsule to the loop of Henle. 6. Perturbations- alteration of function induced by external or internal mechanisms. 7. halothane- is an anaesthetic that triggers malignant hyperthermia in susceptible animals or individuals. In this paper it was used to kill experimental rats. 8. exsanguinations- "bleeding to death"- fatal process of blood loss, after halothane treatment, exsnaguinations followed.

One way that this article is related to class is through the production of ammonia and hydrogen peroxide from D-serine as it enters the kidney. Catabolic production of ammonia is a threat to biochemical processes because of ammonia’s toxicity. The end stages of ammonia in humans includes a comatose state with cerebral edema and increased cranial pressure. To eliminate the cytosol of unwanted ammonia, reductive amination of alpha-ketogulturate to glutamate by glutamate dehydrogenase and conversion of glutamate to glutamine by glutamine synthetase is required. Both of these enzymes are available in the brain, however glutamine synthetase is more prominent for the pathway of ammonia removal.

D-serine- A nonessential Amino Acid (pubchem) KEGG links:

Metacyc links:

Peer Review Article: Combined Metabolomic and Genetic Approaches Reveal a Link between the Polyamine pathway and Albumin 2 in Developing Pea Seeds Pea (Pisum sativum) albumin 2 (PA2) is a protein from a seed that is not a classical storage protein, lacks a signal peptide, is not degraded on germination, and is poorly digested by farm animals and shows to be a potential allergen. Metabolomic approaches have been applied to investigate the biological function of the albumin protein PA2. The PA2 protein has four copies of a hemopexin repeat, which is related structurally to a group of mammalian regulatory proteins such as vitronectin. Due to this it has been thought that PA2 may function as a regulator of metabolic processes. In mammals, hemopexins act as scavengers and transporters of plasma heme that is toxic and may contribute in the homeostasis of nitric oxide. Investigators identified and characterized a mutant with a reduced PA2 in seeds that could provide ways of improving seed quality characteristics, including digestibility and reduced allergic reaction. Seeds lacking PA2 had more N content and higher levels of total seed protein and starch levels remained the same. Metabolites demonstrated a dramatic different in amino acid composition and polyamine content.. A lack of PA2 led to decreased amounts of the polyamine. Examining polyamine synthesis showed that the different spermidine amounts were due to changes in spermidine synthase and arginine decarboxylase. Researcher concluded that PA2 regulates polyamine metabolism. Polyamine- are organic compounds having two or more primary amino groups - such as putrescine, cadaverine, spermidine, and spermine - that are growth factors in both eucaryotic and procaryotic cells. Spermidine synthase- is an enzyme that catalyzes the transfer of the propylamine group from 5'-deoxy-5'-S-(3-methylthiopropylamine)sulfonium adenosine to putrescine in the biosynthesis of spermidine. Arginine decarboxylase- is an enzyme that catalyzes the chemical reaction of L-arginine and producing agmatine and carbon dioxide. Lyophilization- also called freeze-drying, is a method of drying that significantly reduces such damage. Dansylation- the process of using dansyl chloride- is a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green–fluorescent sulfonamide adducts. Dansyl chloride is widely used in protein sequencing and amino acid analysis This article is related to biosynthesis of nucleotides. Polyamines such as spermine and spermidine are involved in DNA packaging. They are derived from methionine and ornithine by a pathway starting out with the decarboxylation of ornithine, a precursor of arginine. Ornithine decarboxylase is a PLP requiring enzyme and that converts ornithine to putrescine. Propylaminotransferase I converts putrescine the spermidine and in a second propylaminotransferase recation[check spelling] spermidine is converted to spermine. S-adenosylmethioine provides propylamino groups for the propylamontransferase reactions.

Spermine- A biogenic polyamine formed from spermidine. It is found in a wide variety of organisms and tissues and is an essential growth factor in some bacteria. It is found as a polycation at all pH values. Spermine is associated with nucleic acids, particularly in viruses, and is thought to stabilize the helical structure. (pubchem)

Spermidine KEGG links:

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Peer Review Article: Transcriptomics and Metabolomics of Dietary Leucine Excess Previous studies has shown that leucine given to animals in diets with adequate protein levels had no problem with large doses of leucine. In this paper researchers analyzed rats that were fed for two weeks on a basal dies or with diets containing varying leucine concentractions including 1.5,5,10,15, and 30%. Also, the changes in gene expression in livers of the rats that were fed the basal diet or diets with 5% and 15% had leucine added and were analyzed by DNA microarrays. Data showed that there was little change in the expression of enzymes of leucine catabolic pathways but there were changes in enzymes associated with nitrogen metabolism and other downstream pathways of leucine catabolism. CAMC revealed that excess nitrogen clustered together with leucine and alpha-ketoisocaproate. In conclusion, excess leucine may be overloading nitrogen metabolism and urea or alpha-ketoisocaproate may be early markers for excessive leucine intake.

alpha-ketoisocaproate- Alpha-ketoisocaproate (KIC) is a keto acid of leucine (branched chain amino acid). Branched-chain keto acids (BCKAs) are very similar to branched-chain amino acids (BCAAs), the only difference being the presence of a keto group instead of an amino group. Thus, BCKAs can be viewed as ammonia-free sources of BCAAs. CAMC- Cluster analysis of multivariate correlations- is the classification of objects into different groups, or more precisely, the partitioning of a data set into subsets, so that the data in each subset share some common trait - often proximity according to some defined distance measure. SDH- succinate dehydrogenase is an enzyme complex bound to the inner mitochondrial membrane. It is the only enzyme that participates in both the citric acid cycle and the mitochondrial electron transport chain (Complex II). Ketoacids- are organic acids containing a ketone functional group and a carboxylic acid group DNA microarrays- is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection BUN- The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea This article is related to nitrogen metabolism that we learned in class. The biosynthetic pathways of amino acids and nucleotides share a requirement for nitrogen. The molecular nitrogen in the atmosphere is unavailable to most organisms until it is reduced. Fixation takes place in certain bacteria. The nitrogen cycle includes the formation of ammonia by the bacterial fixation previously noted of N2. The fixation of N2 as NH3 is done by the nitrogenase complex, which requires ATP. The reduced nitrogen is incorporated first into amino acids and then in other biomolecules including nucleotides. Glutamate and glutamine are the nitrogen donors. This article is also related to pathways of amino acid degradation. Leucine catabolism yields acetyl CoA and acetoacetyl Co-A.

Leucine- an essential amino acid (pubmed) KEGG links:

Metacyc links: Review Article: Biomarkers, Metabonomics, and Drug Development: Can Inborn Errors of Metabolism Help Understanding Drug Toxicity? Drug discovery and development has been rapidly expanding due to new technologies and advancements. This review article talks primarily about the role of metabonomics and its use in the drug development process and to facilitate the transition from preclinical to clinical applications. Investigators analyzed the knowledge gap between pharmaceutical companies and pediatric hospitals since there has been metabonomic applications already applied to screen and treat new born neonates who are born with inborn errors of metabolism . Inborn errors of metabolism disorders in humans have been linked to defects in enzymes catalyzing amino acids (other enzymes are also discussed catalyzing carbohydrates, fatty acids, nucleic acid, and urea cycle.) Amino acid metabolism disorders include phenylketonuria where phenylalanine hydroxylase is the defective enzyme, Tyrosinemia 1 where Fumarylacetoacetate hydrolase is the defective enzyme, Tyrosinemia 2 (Tyrosine aminotransferase), Urea cycle disorder (Arginosuccinate lysate), Maple syrup urine disease (Branched chain alpha- ketoacid dehydrogenase), homocystinuria (Cystathionine synthase), and alkaptonuria (homogentisic oxidase.) The available metabonomics biomarkers from the inborn errors of metabolism diseases should facilitate toxicologists characterize the mechanisms of drug induced toxicities and it is predicted in the review that clinical chemistry will be replaced by metabonomics. Hypoglycemia- lower than normal level of glucose (sugar) in the blood. Bilirubin- is a yellow breakdown product of normal heme catabolism. Its levels are elevated in certain diseases and it is responsible for the yellow colour of bruises and the brown colour of feces. Metabolic acidosis- is a process which if unchecked leads to acidemia (i.e. blood pH is low (less than 7.35) due to increased production of H+ by the body or the inability of the body to form bicarbonate (HCO3-) in the kidney. Sitosterolemia- is a rare autosomal recessively inherited lipid metabolic disorder. It is characterized by hyperabsorption and decreased biliary excretion of dietary sterols leading to hypercholesterolemia, tendon and tuberous xanthomas, premature development of atherosclerosis, and abnormal hematologic and liver function test results. Glaucoma- is a group of diseases of the optic nerve involving loss of retinal ganglion cells in a characteristic pattern of optic neuropathy. Staphyloma- A protrusion of any part of the globe of the eye; as, a staphyloma of the cornea. One way this article is related to our class discussions is through the of pathways of amino acid degradation. Many amino acids are neurotransmitters or precursors or antagonists of neurotransmitters. Genetic defects of amino acid metabolism can cause defective neural development and mental retardation and in most cases a specific intermediate accumulates. Both phenylketonuria and alkaptonuria were highlighted in our class discussions. Phenylketonuria is caused by the accumulation of phenylalanine hydroxylase, which is the first enzyme involved in the phenylalanine pathway. Phenylketonuria was one of the first inheritable metabolic defects discovered. Alkaptonuria is caused by the defective enzyme homogentistate dioxygenase. This is less severe than phenylketonuria but large amounts of homogentisate are excreted and black urine results upon oxidation. Arthritis is a concern with people who suffer from alkaptonuria.

homogentistate dioxygenase

Phenylalanine KEGG links:

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Peer Reviewed Article: Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic and computational systems analysis

The fruitfly is potential genetic model for studying hypoxia adaptation; however the metabolic source for intense hypoxia tolerance is not well understood in flies. Understanding cellular hypoxia adaptation is important in designing treatments for injuries caused by ischemia-repulsion, stroke, and myocardial infarction. Cell damage during hypoxia is generally caused by decreased pH, altered calcium concentrations, an increasing osmotic pressure, and damage to the mitochondrian which all influence decreased ATP. Metabolic characteristics were generated and it was shown that accumulation of lactate, alanine, and acetate may be major products of anaerobic fly metabolism. A model was built to show ATP producing pathways and then adaptation to hypoxia was analyzed in silico. Investigators concluded that the ability to convert pyruvate to acetate, alanine, and acetate might provide some hypoxia tolerance by improving the ATP/proton ratia and its glucose use.

Hypoxia- is a pathological condition in which the body as a whole (generalised hypoxia) or region of the body (tissue hypoxia) is deprived of adequate oxygen supply.

ischemia-reperfusion- Functional, metabolic, or structural changes, including necrosis, in ischemic tissues thought to result from reperfusion to ischemic areas of the tissue. Ischemia - A decrease in the blood supply to a bodily organ, tissue, or part caused by constriction or obstruction of the blood vessels.

in silico- is an expression used to mean "performed on computer or via computer simulation."

ANOVA- one way analysis of variance- is a collection of statistical models, and their associated procedures, in which the observed variance is partitioned into components due to different explanatory variables.

TCA cycle- this stands for the citric acid cycle.

Alanine is produced during hypoxia by transamination to pyruvate from another amino acid. In class we talked about alanine aminotransferase. In muscle and some other tissues that degrade amino acids for fuel, amino groups are collected in the form of glutamate by transamination. Glutamate is then converted to glutamine and transported to the liver or alternatively, it can be transferred to pyruvate by using alanin amino transferase. Alanine travels to the liver and in the cytosol of hepatocytes, alanine aminotransferase carries the amino group from alanine to alpha ketogluturate to form pyruvate and glutamate. This process is used to transport ammonia from the skeletal muscles to the liver.

Alanine- a non-essential amino acid

KEGG links:

Metacyc links: Retrieved from "" Views

AMINO ACIDS Metabolic engineering of a genetic selection system with tunable stringency. (

Summary: The main focus of this paper is the fine-tuning of the biosynthesis of small molecules by engineering metabolic flux within cells. In this case, it was achieved through provision of a regulable enzyme leading it down another parallel biopathway. Using this approach, the optimization of an in vivo selection system for the conversion of prephenate to phenylpyruvate (a step in the synthesis of phenylalanine) was achieved.

Terms: Metabolic Flux – the rate at which a certain metabolite is produced during a bioprocess.

Auxotrophy – the inability of an organism to synthesize a particular organic compound required for its growth.

Prototrophy - not requiring specific nutritional substances for normal metabolism and reproduction and not differing from the wild type in nutritional requirements.

Prephenate Dehydrogatase – catalyzes the conversion of prephenate to phenylpyruvate.

Site-directed Mutagenesis – The development of a mutation at a specific site that was targeted for the mutation.

Relevance: We have been studying how glycolysis and gluconeogenesis can be regulated in the cell, and this provides an alternate way to regulate a metabolic process and its flux by redirecting a specific metabolite down a parallel pathway.

Articles and Web Pages for Review and Inclusion


Peer-Reviewed Article #1:

Predicting metabolic biomarkers of human inborn errors of metabolism

Molecular Systems Biology 5 Article number: 263 Published online: 28 April 2009

Reviewer: Joseph B.

Main Focus


Using a computational model to predict biomarkers from gene knockouts.

New Terms

boundary metabolite
a metabolite that is known to be taken up or secreted between the intracellular and extracellular compartments. (source: http://)
exchange interval
a possible range of uptake and secretion flukes of a given boundary exchange interval (source: http://)
flux variable analysis
CBM method that computes the exchange interval. (source: http://)
boundary metabolite concentration in biofluid. (source: http://)
fraction of the predicted biomarkers that are correct when comparing the predicted accuracy with a random model. (source: http://)
fraction of the biomarkers that are correctly predicted when comparing the predicted accuracy with a random model. (source: http://)
an application of conservation of mass; accounts for material entering and leaving a system. (source: http://)
Constraint Based Modeling
the computational model used by the authors to predict the metabolic phenotypes of gene knockouts in microorganisms. (source: http://)
regulatory constraint
accounting for gene regulation (in the constraing based model). (source: http://)
Metabolic network
a database for metabolic pathways and diseases. (source: http://)



The authors describe the accuracy and potential usefulness of using a computational model to predict biomarkers of diseases caused by gene knockouts. In order to evaluate the model, they compared their predicted biomarkers with the known biomarkers published on the OMIM and HMDB databases. When the constraint based model was used for red blood cell metabolism, the authors found that the model predictions were accurate with a precision of .73 and a recall of .40. However, in some cases the CBM leads to false predictions. Such is true for argeninemia. The model fails to predict the elevated extracellular concentrations of arginine because of alternative arginine catabolic pathways. The alternative pathways cannot compensate for the main catabolic pathway due to low expression of certain enzymes involved in those reactions. Hence the CBM lacks a regulatory constraint. Some other reasons why this computational model gives false predictions is because of the simplifying assumptions in the model and the incompleteness of the metabolic network. Overall, the authors site that the probability of the model to predict correct biomarkers ranges from 6-15.8 times higher than a random model. These results are encouraging and the authors hope to use this model to predict biomarkers for gene knockouts that have not yet been documented to cause disease. Some needed improvements are integrating the model with larger human metabolic networks and integrating metabolic regulation with the model to improve the predictions.

Relevance to a Traditional Metabolism Course


This article discusses an analytic approach to predicting differing concentrations of metabolites resulting from gene mutations that lead to protein malfunction in pathways such as amino acid catabolism, glycolysis and the pentose phosphate pathway. A constraint based model was employed along with the OMIM and HMDB databases to measure the accuracy of predicted biomarkers. The future of the computational model described can possibly add a new dimension to the human genome project by predicting biomarkers of gene disruptions before the mutation has been identified.