Assay of Low Deuterium Enrichment of Water by Isotopic Exchange with [U-13C3]Acetone and Gas Chromatography–Mass Spectrometry

Yang, Dongyan; Diraison, Frédérique; Beylot, M.; Brunengraber, Daniel Z.; Samols, Mark A.; Anderson, Vernon E.; Brunengraber, Henri

doi:10.1006/abio.1998.2632

Cited by 100 publications

(82 citation statements)

References 25 publications

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“…2 H Labeling of Body Water-The 2 H labeling of body water was determined by exchange with acetone (17). Briefly, samples were centrifuged for 1 min at ϳ12,000 ϫ g in a microcentrifuge, and 20 l of sample (or standard) was reacted with 2 l of 10 N NaOH and 4 l of a 5% (v/v) solution of acetone in acetonitrile for 24 h at room temperature.…”

Section: Analytical Proceduresmentioning

confidence: 99%

Triglyceride Synthesis in Epididymal Adipose Tissue

Bederman

Foy

Chandramouli

et al. 2009

Journal of Biological Chemistry

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The obesity epidemic has generated interest in determining the contribution of various pathways to triglyceride synthesis, including an elucidation of the origin of triglyceride fatty acids and triglyceride glycerol. We hypothesized that a dietary intervention would demonstrate the importance of using glucose versus non-glucose carbon sources to synthesize triglycerides in white adipose tissue. C57BL/6J mice were fed either a low fat, high carbohydrate (HC) diet or a high fat, carbohydrate-free (CF) diet and maintained on 2 H 2 O (to determine total triglyceride dynamics) or infused with [6,6-2 H]glucose (to quantify the contribution of glucose to triglyceride glycerol). The 2 H 2 O labeling data demonstrate that although de novo lipogenesis contributed ϳ80% versus ϳ5% to the pool of triglyceride palmitate in HC-versus CF-fed mice, the epididymal adipose tissue synthesized ϳ1.5-fold more triglyceride in CF-versus HC-fed mice, i.e. 37 ؎ 5 versus 25 ؎ 3 mol ؋ day ؊1 . The [6,6-2 H]glucose labeling data demonstrate that ϳ69 and ϳ28% of triglyceride glycerol is synthesized from glucose in HC-versus CF-fed mice, respectively. Although these data are consistent with the notion that non-glucose carbon sources (e.g. glyceroneogenesis) can make substantial contributions to the synthesis of triglyceride glycerol (i.e. the absolute synthesis of triglyceride glycerol from non-glucose substrates increased from ϳ8 to ϳ26 mol ؋ day ؊1 in HC-versus CF-fed mice), these observations suggest (i) the importance of nutritional status in affecting flux rates and (ii) the operation of a glycerol-glucose cycle.Epidemiological trends in the development of obesity (1-4) and its association with various diseases have generated interest in the study of lipid synthesis and mobilization. In particular, new methods have been developed for quantifying rates of triglyceride turnover in vivo (5-8). For example, using 2

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Section: Analytical Proceduresmentioning

confidence: 99%

Triglyceride Synthesis in Epididymal Adipose Tissue

Bederman

Foy

Chandramouli

et al. 2009

Journal of Biological Chemistry

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“…H enrichment in body water Enrichment in body water, measured as that in plasma water, was determined as described [30]. An aliquot of 50 μl plasma was spiked with 0.2 μl of acetone in 3.8 μl of acetonitrile and 2 μl of 10 N NaOH.…”

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confidence: 99%

Changes in hepatic glycogen cycling during a glucose load in healthy humans

et al. 2005

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Aims/hypothesis: Glycogen cycling, i.e. simultaneous glycogen synthesis and glycogenolysis, affects estimates of glucose fluxes using tracer techniques and may contribute to hyperglycaemia in diabetic conditions. This study presents a new method for quantifying hepatic glycogen cycling in the fed state. Glycogen is synthesised from glucose by the direct and indirect (gluconeogenic) pathways. Since glycogen is also synthesised from glycogen, i.e. glycogen→glucose 1-phosphate→glycogen, that synthesised through the direct and indirect pathways does not account for 100% of glycogen synthesis. The percentage contribution of glycogen cycling to glycogen synthesis then equals the difference between the sum of the percentage contributions of the direct and indirect pathways and 100. Materials and methods: The indirect and direct pathways were measured independently in nine healthy volunteers who had fasted overnight. They ingested 2 H 2 O (5 ml/kg body water) and were infused with [5-3 H] glucose and acetaminophen (paracetamol; 1 g) during hyperglycaemic clamps (7.8 mmol/l) lasting 8 h. The percentage contribution of the indirect pathway was calculated from the ratio of 2 H enrichments at carbon 5 to that at carbon 2, and the contribution of the direct pathway was determined from the 3 H-specific activity, relative to plasma glucose, of the urinary glucuronide excreted between 2 and 4, 4 and 6, and 6 and 8 h. Results: Glucose infusion rates increased (p<0.01) to ∼50 μmol kg −1 min −1 . Plasma insulin and the insulin : glucagon ratio rose ∼3.6-and ∼8.3-fold (p<0.001), respectively. From the difference between 100% and the sum of the direct (2-4 h, 54±6%; 4-6 h, 59±5%; 6-8 h, 63±4%) and indirect (32±3, 38±4, 36±3%) pathways, glycogen cycling was seen to be decreased (p<0.05) from 14±4% (2-4 h) to 4±3% (4-6 h) and 1±3% (6-8 h). Conclusions/interpretation: This method allows measurement of hepatic glycogen cycling in the fed state and demonstrates that glycogen cycling occurs most in the early hours after glucose loading subsequent to a fast.

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“…This has been demonstrated in test tube experiments where polymeric compounds were synthesized from monomers labeled with heavy atoms ( 13 (24), acetone labeled from deuterated water by keto-enol tautomerism (25), and trimethylphosphate labeled from 18 O-labeled water (26). In all these cases, precise values of precursor enrichment were calculated.…”

Section: Resultsmentioning

confidence: 98%

“…In all these cases, precise values of precursor enrichment were calculated. As a result, an important application of mass isotopomer analysis is the assay of the low isotopic enrichment of compounds that can be polymerized into a compound assayable by GC-MS (23,(25)(26)(27)(28).…”

Section: Resultsmentioning

confidence: 99%

In Vitro Modeling of Fatty Acid Synthesis under Conditions Simulating the Zonation of Lipogenic [13C]Acetyl-CoA Enrichment in the Liver

Bederman¹,

Kasumov²,

Reszko

et al. 2004

Journal of Biological Chemistry

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In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data.In the companion report (1) (2) of the activities of glycolytic (2-5) and lipogenic enzymes (7-9) (perivenous Ͼ periportal) versus the activity of cytosolic acetyl-CoA synthetase (periportal Ͼ perivenous) (10). Gradients of precursor enrichment were detected using isotopomer spectral analysis (ISA) (11). In addition, we found that fractional lipogenesis calculated by the two-isotopomer method (an algebraic method similar to that described by Chinkes et al. (12)) produces lower estimates of fractional synthesis than those produced by the best fit estimates of ISA. Although the "linear gradient" ISA model (see companion report (1) for model definitions) yielded a better fit than the "Single pool" ISA model, it was not possible to evaluate the effect of gradients on estimates of fractional synthesis. Thus, we could not quantitatively evaluate the performance of the ISA in comparison to the two-isotopomer method, because the true rates of fractional synthesis in the liver dog and rat liver perfusion study were unknown.The goal of the present study was to evaluate the differences in estimates of precursor enrichment and fractional synthesis calculated by the two-isotopomer method and ISA. We used an experimental model where both the gradient in precursor enrichment and the fractional synthesis are known. This was accomplished by in vitro preparations that simulated the zonation of acetyl-CoA enrichment. Lipogenesis from sub-populations of hepatocytes across the liver lobule was simulated, in parallel incubations, by synthesizing a fatty acid using purified fatty acid synthase (13,14) and [U-13 C 3 ]malonyl-CoA of varying enrichment. We used gradients of malonyl-CoA enrichment, because fatty acid synthesis involves the conversion to malonyl-CoA of all acetyl units added to the primer. We used [U-13 C 3 ]propionyl-CoA as a primer to avoid the possibility of contamination of our newly synthesized pentadecanoate with unlabeled pentadecanoate. In the presence of unlabeled malonyl-CoA, the process yields M3 2 [13,14, C 3 ]pentadecanoate. By monitoring the distribution of M3 to M15 isotopomers of pentadecanoate, we simulated in vitro the polymerization of six [13 C]acetyl units

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Assay of Low Deuterium Enrichment of Water by Isotopic Exchange with [U-13C3]Acetone and Gas Chromatography–Mass Spectrometry

Cited by 100 publications

References 25 publications

Triglyceride Synthesis in Epididymal Adipose Tissue

Triglyceride Synthesis in Epididymal Adipose Tissue

Changes in hepatic glycogen cycling during a glucose load in healthy humans

In Vitro Modeling of Fatty Acid Synthesis under Conditions Simulating the Zonation of Lipogenic [13C]Acetyl-CoA Enrichment in the Liver

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