Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

Tomcik, Kristyen; Ibarra, R; Sadhukhan, Sushabhan; Han, Yong; Tochtrop, Gregory P.; Zhang, Guo Fang

doi:10.1016/j.ab.2010.11.030

Cited by 54 publications

(43 citation statements)

References 31 publications

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“…The conditions for the GC were as follows: injector: 250°C (splitless injection of samples); oven: 60°C for 1.0 min; ramp, 15°C/min to 240°C; hold at 240°C for 10 min. In addition to better retention on the GC column and higher resolutions and sensitivity, the PFBBr derivatives do not disrupt the natural isotopomer distribution in NCI, because only the FA moiety is measured in NCI (47). The numerous advantages of PFPBr derivative under NCI conditions, including sensitivity and fragmentation pattern, enabled us to trace the isotopic incorporation of 13 C carbons from the [U- 13 C]glucose and to quantify a wide spectrum of FA ranging from C2 to C22 in both plasma and milk.…”

Section: Methodsmentioning

confidence: 99%

De novo synthesis of milk triglycerides in humans

Mohammad

Sunehag

Haymond

2014

American Journal of Physiology-Endocrinology and Metabolism

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Mohammad MA, Sunehag AL, Haymond MW. De novo synthesis of milk triglycerides in humans. Am J Physiol Endocrinol Metab 306: E838 -E847, 2014. First published February 11, 2014 doi:10.1152/ajpendo.00605.2013 de novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. Objective: To test the hypothesis that the incorporation of 13 C carbons from [U-13 C]glucose into fatty acids (FA) and glycerol in triglycerides (TG) will be greater: 1) in milk than plasma TG, 2) during a high-carbohydrate (H-CHO) diet than high-fat (H-FAT) diet, and 3) during feeding than fasting. Seven healthy, lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion, subjects received diets containing H-FAT or H-CHO diet for 1 wk. Incorporation of 13 C from infused [U-13 C]glucose into FA and glycerol was measured using GC-MS and gene expression in RNA isolated from milk fat globule using microarrays. Incorporation of 13 C2 into milk FA increased with increased FA chain length from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FAϾC16. During feeding, regardless of diets, enrichment of 13 C2 in milk FA and 13 C3 in milk glycerol were ϳ3-and ϳ7-fold higher compared with plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets, 25 and 6%, respectively, of medium-chain FA (MCFA, C6 -C12) in milk were derived from glucose but increased to 75 and 25% with feeding. Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. The human MG is capable of de novo lipogenesis of primarily MCFA and glycerol, which is influenced by the macronutrient composition of the maternal diet. fatty acid; glycerol; GC-MS; gene expression; lipogenesis; stable isotopes TRIGLYCERIDES (TG) make up 98% of the lipid content and, in humans, contributes 40 -50% of the total energy content (28). Total milk lipid content ranges, depending on the species, from 0 to 50% of the total milk volume (20). The fatty acid (FA) composition of the TG is dependents on the dietary FA composition, the dietary carbohydrate/lipid ratio and the species. Milk TG contains FA that are derived from three sources: de novo mammary gland (MG) synthesis, dietary lipids, and endogenous fat stores (adipose or hepatic lipids) (7). Data from a study comparing the milk fats from seven different species (4) revealed that the milk fats of humans, dogs, and guinea pigs are largely (92, 93, and 97%, respectively) made up of longchain FA (LCFA). The milk fats of horses contain large amounts (ϳ33%) of medium-chain FA (MCFA); however, the milk fats of cows, sheep, and goats are enriched (13-15%) in short-chain FA (SCFA) (4). The MG is one of three primary lipid-synthesizing organs in the body; the other two are liver and adipose tissue (48). In fact, many of the initial pathways of FA biosynthesis were defined using mammary tissue from lactating ruminants and rodents (1). The unique feature of FA synthesis in the MG is th...

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Section: Methodsmentioning

confidence: 99%

De novo synthesis of milk triglycerides in humans

Mohammad

Sunehag

Haymond

2014

American Journal of Physiology-Endocrinology and Metabolism

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“…The concentration and labeling of acetate and formate were assayed using negative chemical ionization of the pentafluorobenzyl derivatives (38). The concentrations and mass isotopomer distributions of acylCoA esters were assayed as in Ref.…”

Section: Methodsmentioning

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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“…The 13 C labeling of acetate (22) was a probe of the labeling of peroxisomal acetyl-CoA (18). The 13 C labeling of the C-1 ϩ 2 of ␤-hydroxybutyrate (BHB) was a probe of mitochondrial acetylCoA and was calculated from labeling of BHB and the C-3 ϩ 4 fragment according to our published method (14).…”

Section: Methodsmentioning

confidence: 99%

“…Correction of mass isotopomer distributions (MID) for natural enrichment was performed as in Ref. 22.…”

Section: Methodsmentioning

confidence: 99%

Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver, Investigated by Mass Isotopomer Analysis

Jin

Bian

Tomcik

et al. 2015

Journal of Biological Chemistry

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Background:We explored the compartmentation of n-dicarboxylate liver catabolism. Results: Dodecanedioate and azelate oxidation span peroxisomes and mitochondria; glutarate oxidation is mitochondrial; dodecanedioate is anaplerotic. Conclusion: Dodecanedioate is a potential substrate for anaplerotic therapy of reperfusion injury and some inborn disorders of metabolism. Significance: This metabolomic ϩ mass isotopomer strategy can be used to better characterize substrate catabolism in disease models.

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Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

Cited by 54 publications

References 31 publications

De novo synthesis of milk triglycerides in humans

De novo synthesis of milk triglycerides in humans

Metabolism of Levulinate in Perfused Rat Livers and Live Rats

Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver, Investigated by Mass Isotopomer Analysis

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