Quantifying rates of protein synthesis in humans by use of<sup>2</sup>H<sub>2</sub>O: application to patients with end-stage renal disease

Previs, Stephen F.; Fatica, Richard; Chandramouli, Visvanathan; Alexander, Robert R.; Brunengraber, Henri; Landau, Bernard R.

doi:10.1152/ajpendo.00271.2003

Cited by 85 publications

(134 citation statements)

References 31 publications

(39 reference statements)

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“…In addition, these techniques are not suited for human studies because they require the consumption of a fully 15 N-labeled diet. Recently we and others developed the 2 H 2 O metabolic labeling technique to measure protein turnover in free living animals (11)(12)(13)(14)(15). In rats that have been given intraperitoneal injection of 2 H 2 O, 2 H equilibrates with total body water and amino acids such as alanine within 10 and 20 min, respectively (16) (Fig.…”

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confidence: 99%

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Willard

Rachdaoui

et al. 2012

Molecular & Cellular Proteomics

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Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2 H 2 O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis.2 H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2 H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

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confidence: 99%

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Willard

Rachdaoui

et al. 2012

Molecular & Cellular Proteomics

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“…Plasma was collected from 2 ml blood samples in tubes containing EDTA. Corrections were made for dilution of water in the blood [31] by the water in which the EDTA was dissolved. Specific activities and 2 H enrichments in urinary glucuronide [25,32] The amount of glucuronide was determined in each 2-h collection [33,34].…”

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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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“…A major assumption of any tracer method is that the tracer and tracee are indiscriminately metabolized, therefore, after one stops administering a labeled precursor amino acid the labeling in the protein can only decrease if new protein is made in absence of labeled precursor amino acids. the protein is degraded; the labeling decreases because new proteins are being made from unlabeled precursors (Previs et al 2004;Waterlow 2006). We believe that it is possible to estimate protein breakdown using the following logic, changes in the abundance of a protein equal the rate of synthesis minus the rate of breakdown.…”

Section: Using Stable Isotope Tracers To Study Protein Synthesis and mentioning

confidence: 99%

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

Previs¹,

Zhou²,

Wang³

et al. 2012

Integrative Proteomics

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Quantifying rates of protein synthesis in humans by use of²H₂O: application to patients with end-stage renal disease

Cited by 85 publications

References 31 publications

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

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

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

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Quantifying rates of protein synthesis in humans by use of2H2O: application to patients with end-stage renal disease

Cited by 85 publications

References 31 publications

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling

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

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

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Quantifying rates of protein synthesis in humans by use of²H₂O: application to patients with end-stage renal disease