Using Isotopic Tools to Dissect and Quantitate Parallel Metabolic Pathways

Sadhukhan, Sushabhan; Han, Yong; Zhang, Guofang; Brunengraber, Henri; Tochtrop, Gregory P.

doi:10.1021/ja100399m

Cited by 29 publications

(54 citation statements)

References 23 publications

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“…After identifying reactions which should have increased turnover based on changes to substrates, we investigated further evidence of pathway activation from hyperglycemia. Examination of these reactions generally showed increases in amine products (reactions 1,4,5,6,8). In certain cases (reactions 2, 3, and 7), the product was below our limit of detection (LOD) even though they were targeted for analysis.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Subsecond Absolute Quantitation of Amine Metabolites Using Isobaric Tags for Discovery of Pathway Activation in Mammalian Cells

Yuan

Anderson

et al. 2012

Anal. Chem.

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The absolute quantitation of amine metabolites from mammalian cell samples was achieved by combining amine standards, isobaric tags, and capillary liquid chromatography (LC) tandem mass spectrometry (MS/MS). Our approach allowed 32 specific amines to be analyzed within a single chromatographic run, with the generation of the calibration curve and absolute quantitation of each analyte taking less than 900 ms. Using this strategy, we determined the amine response of human aortic endothelial cells (HAECs) from a glucose challenge. The observed changes of the absolute concentration of these metabolites implied eight enzymatic reactions may change efficiency upon glucose treatment. Five of these reactions have been previously reported as being up-regulated in diabetic conditions. The remaining three reactions were analyzed by measuring the expression of these enzymes, with 66% showing increases. Our data indicate that rapid determination of absolute quantitation is useful in determining novel pathway activation. Furthermore, even though we determined the absolute quantity of 32 metabolites here, the number of analytes that can be measured by this method is limited mainly by commercial availability of amine standards.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Subsecond Absolute Quantitation of Amine Metabolites Using Isobaric Tags for Discovery of Pathway Activation in Mammalian Cells

Yuan

Anderson

et al. 2012

Anal. Chem.

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“…Our interest in levulinate arose from our recent study of the metabolism of 4-hydroxyacids, which are products of lipid peroxidation (C 9 , C 6 ) or drugs of abuse (C 4 , C 5 ) (5,6). We showed that 4-hydroxyacids with 5 or more carbons are metabolized by two new pathways.…”

mentioning

confidence: 99%

Metabolism of Levulinate in Perfused Rat Livers and Live Rats

Harris¹,

Zhang²,

Sadhukhan³

et al. 2011

Journal of Biological Chemistry

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Calcium levulinate (4-ketopentanoate) is used as an oral and parenteral source of calcium. We hypothesized that levulinate is converted in the liver to 4-hydroxypentanoate, a new drug of abuse, and that this conversion is accelerated by ethanol oxidation. We confirmed these hypotheses in live rats, perfused rat livers, and liver subcellular preparations. Levulinate is reduced to (R)-4-hydroxypentanoate by a cytosolic and a mitochondrial dehydrogenase, which are NADPH-and NADH-dependent, respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4-hydroxypentanoate. In livers perfused with [ 13 C 5 ]levulinate, there was substantial CoA trapping in levulinyl-CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA. This CoA trapping was increased by ethanol, with a 6-fold increase in the concentration of 4-phosphopentanoylCoA. Levulinate is catabolized by 3 parallel pathways to propionyl-CoA, acetyl-CoA, and lactate. Most intermediates of the 3 pathways were identified by mass isotopomer analysis and metabolomics. The production of 4-hydroxypentanoate from levulinate and its stimulation by ethanol is a potential public health concern.Levulinate (4-ketopentanoate) is a food additive allowed by the Food and Drug Administration. As a calcium salt, it has been used for many years as an oral and intravenous form of calcium administration (1, 2). It is listed in the pharmacopeias of a number of countries (USA, Europe, India, China, Japan, etc). It is also listed in quality control documents of the World Health Organization. The dry salt is sold on the internet as a non-prescription dietary supplement. To the best of our review of the literature, the catabolism of levulinate has not been investigated in mammalian cells. We found one report on the identification of the reduced form of levulinate, 4-hydroxypentanoate, in the urine of children with ␤-ketothiolase deficiency treated with intravenous calcium levulinate (3). We also found an unexplained report of induction of lactic acidosis in premature babies treated with oral calcium levulinate for hypocalcemia (4).Our interest in levulinate arose from our recent study of the metabolism of 4-hydroxyacids, which are products of lipid peroxidation (C 9 , C 6 ) or drugs of abuse (C 4 , C 5 ) (5, 6). We showed that 4-hydroxyacids with 5 or more carbons are metabolized by two new pathways. The first pathway involves the isomerization of 4-hydroxyacyl-CoAs to 3-hydroxyacylCoAs via 4-phosphoacyl-CoAs, a new class of acyl-CoA esters. After isomerization, the 3-hydroxyacyl-CoAs are catabolized via classical ␤-oxidation to acetyl-CoA and, in the case of odd-chain 4-hydroxyacids, to propionyl-CoA. The second pathway involves a sequence of ␤-, ␣-, and ␤-oxidation steps leading to acetyl-CoA, formate and, in the case of even chain 4-hydroxyacids, to propionyl-CoA.The five-carbon 4-hydroxypentanoate, as a racemic mixture, is a new drug of abuse used in lieu of 4-hydroxybutyrate, which is now a controlled substance in the United States (7-11). Based on our previous work...

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“…Tandem mass spectrometry provides information on the fragmentation, which is critical in the identification of new unknown metabolites. For example, the identity of 4-phosphopentanoyl-CoA, a metabolite of 4-hydroxypentanoic acid, is deduced by careful analyzing its fragmentation pattern in positive ionization mode of tandem mass spectrometry Sadhukhan et al, 2010). This example demonstrates the utility of product ion scan for identification of new metabolites.…”

Section: New Metabolite Discovery By Tandem Mass Spectrometrymentioning

confidence: 99%

“…HNE oxidation to 4-hydroxynonanoic acid is another major detoxification pathway. We identified two parallel catabolic pathways of 4-hydroxynonanoic acid in rat liver Sadhukhan et al, 2010). Two pathways are shown in Fig 8. The first round beta oxidation is the same for both pathways.…”

Section: Tandem Mass Spectrometry Combined With the Mass Isotopomer Amentioning

confidence: 99%