Measurement of muscle protein fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry

Yarasheski, Kevin E.; Smith, Kenneth; Rennie, Michael J.; Bier, Dennis M.

doi:10.1002/bms.1200211004

Cited by 83 publications

(75 citation statements)

References 21 publications

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“…However, this method still contains a number of laborious preparative steps. The reference method using modern GC-combustion-IRMS equipment, is therefore to measure such low enrichments directly in the hydrolysate after derivatization (Yarasheski et al 1992;Balagopal et al 1996). A disadvantage of the GC-combustion-IRMS method is the problem of dilution of 13 C label (C-1 of leucine) with natural C (of leucine and derivatization groups) generated during combustion.…”

Section: Amino Acid Incorporation Methods To Measure Protein Synthesimentioning

confidence: 99%

Tracers to investigate protein and amino acid metabolism in human subjects

Wagenmakers

1999

Proc. Nutr. Soc.

110

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Abbreviations: FSR, fractional synthesis rate; IRMS, isotope-ratio mass spectrometry; α-KIC, α-ketoisocaproic acid; MS, mass spectrometry; nb, net balance of tracer; NB, net balance of tracee; Ra, rate of appearance; Rd, rate of disappearance. The contribution of whole-body methods to the future gain of knowledge is expected to be limited due to the fact that most physiological disturbances have been investigated extensively, and due to the lack of information on the relative contribution of various tissues and proteins to whole-body changes.Tracer amino acid-incorporation methods are most suited to investigate these latter aspects of protein metabolism. These methods have shown that some tissues (liver and gut) have much higher turnover rates and deposit much more protein than others (muscle). Massive differences also exist between the fractional synthesis rates of individual proteins. The incorporation methods have been properly validated, although minor disagreements remain on the identity of the true precursor pool (the enrichment of which should be used in the calculations). Arterio-venous organ balance studies have shown that little protein is deposited in skeletal muscle following a proteincontaining meal, while much more protein is deposited in liver and gut. The amount deposited in the feeding period in each of these tissues is released again during overnight fasting. The addition of tracers to organ balance studies allows the simultaneous estimation of protein synthesis and protein breakdown, and provides information on whether changes in net protein balance are caused primarily by a change in protein synthesis or in protein breakdown. In the case of a small arterio-venous difference in a tissue with a high blood flow, estimates of protein synthesis and breakdown become very uncertain, limiting the value of using the tracer. An additional measurement of the intracellular free amino acid pool enrichment allows a correction for amino acid recycling and quantification of the inward and outward transmembrane transport. However, in order to obtain reliable estimates of the intramuscular amino acid enrichment and, therefore, of muscle protein synthesis and breakdown in this so-called three-pool model, the muscle should be freeze-dried and the resulting fibres should be freed from connective tissue and small blood clots under a dissection microscope. Even when optimal precautions are taken, the calculations in these tracer balance methods use multiple variables and, therefore, are bound to lead to more variability in estimates of protein synthesis than the tracer amino acid incorporation methods. In the future, most studies should focus on the measurement of protein synthesis and breakdown in specific proteins in order to understand the mechanisms behind tissue adaptation in response to various stimuli (feeding, fasting, exercise, trauma, sepsis, disuse and disease). The tracer laboratories, therefore, should improve the methodology to allow the measurement of low tracer amino acid enrichments in small amount...

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Section: Amino Acid Incorporation Methods To Measure Protein Synthesimentioning

confidence: 99%

Tracers to investigate protein and amino acid metabolism in human subjects

Wagenmakers

1999

Proc. Nutr. Soc.

110

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“…Labeled cultured media was serially diluted with unlabeled media to generate samples for a standard curve. A␤ was immunoprecipitated from the media, trypsin digested, and A␤ [17][18][19][20][21][22][23][24][25][26][27][28] fragments were analyzed on a LCQ-ESI-MS and the tandem mass spectra ions were quantitated using custom-written software. The predicted percentage labeled A␤ versus the measured value is shown with a linear regression line.…”

Section: In Vitro Labeling and Quantitationmentioning

confidence: 99%

“…and the presence of these naturally occurring isotopes, A␤ was enzymatically cleaved into fragments (A␤ 1-5 , A␤ 6 -16 , A␤ [17][18][19][20][21][22][23][24][25][26][27][28] , and A␤ 29 -40/42 ) using trypsin, before mass spectrometry analysis. The A␤ [17][18][19][20][21][22][23][24][25][26][27][28] fragment has a nominal mass of 1324.6 Daltons (D), contains one leucine residue, and was detected using MALDI-TOF-MS, as a singly charged species at m/z 1325.6 [MϩH] ϩ1 . The A␤ 6 -16 [17][18][19][20][21][22][23][24][25][26][27][28] peptide, and that this high level of 13 C 6 -leucine incorporation could be easily detected using MALDI-TOF-MS.…”

Section: In Vitro Labeling and Quantitationmentioning

confidence: 99%

“…The A␤ [17][18][19][20][21][22][23][24][25][26][27][28] fragment has a nominal mass of 1324.6 Daltons (D), contains one leucine residue, and was detected using MALDI-TOF-MS, as a singly charged species at m/z 1325.6 [MϩH] ϩ1 . The A␤ 6 -16 [17][18][19][20][21][22][23][24][25][26][27][28] peptide, and that this high level of 13 C 6 -leucine incorporation could be easily detected using MALDI-TOF-MS. This confirmed the specificity of the immunoprecipitation techniques in CSF and cell media and the ability to qualitatively identify labeled and unlabeled A␤ peptides.…”

Section: In Vitro Labeling and Quantitationmentioning

confidence: 99%

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Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

Bateman

Munsell

Chen

et al. 2007

J. Am. Soc. Mass Spectrom.

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In all biological systems, protein amount is a function of the rate of production and clearance. The speed of a response to a disturbance in protein homeostasis is determined by turnover rate. Quantifying alterations in protein synthesis and clearance rates is vital to understanding disease pathogenesis (e.g., aging, inflammation). No methods currently exist for quantifying production and clearance rates of low-abundance (femtomole) proteins in vivo. We describe a novel, mass spectrometry-based method for quantitating low-abundance protein synthesis and clearance rates in vitro and in vivo in animals and humans. The utility of this method is demonstrated with amyloid-␤ (A␤), an important low-abundance protein involved in Alzheimer's disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of A␤ from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human A␤ protein production and clearance rates. The method is sensitive and specific for stable isotope-labeled amino acid incorporation into CNS A␤ (Ϯ1% accuracy). This in vivo method can be used to identify pathophysiologic changes in protein metabolism and may serve as a biomarker for monitoring disease risk, progression, or response to novel therapeutic agents. The technique is adaptable to other macromolecules, such as carbohydrates or lipids. (J Am Soc Mass Spectrom 2007, 18, 997-1006

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“…8 They were converted to N-acetyl O-propyl (NAP) amino acid esters prior to analysis by GC=C=IRMS. 14,15 Determination of leucine enrichment by gas chromatograph=combustion=isotope ratio mass spectrometry (GC=C=IRMS). A Finnigan Mat Delta C isotope ratio mass spectrometer (Finnigan Mat, Bremen, Germany) coupled to an HP 5890 series II gas chromatograph (Hewlett Packard), was used to determine sample isotopic enrichment.…”

Section: Analytical Proceduresmentioning

confidence: 99%

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

et al. 2002

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AIMS=HYPOTHESIS:Mechanisms responsible for the decreased high-density lipoprotein (HDL) cholesterol level associated with insulin resistance in obese patients are not clearly understood. To determine the influence of insulin resistance at an early stage on HDL metabolism, we performed a stable isotope kinetic study of apolipoprotein (apo) A-I, in five obese insulin resistant women with normal fasting triglycerides and without impaired glucose tolerance, and in five age-matched control women. METHODS: Each subject received a 16 h constant infusion of L-[1-13 C]leucine at 0.7 mg=kg=h following a primed bolus of 0.7 mg=kg. RESULTS: ApoA-I fractional catabolic rate (FCR) was significantly increased in insulin-resistant women compared to controls (0.316 AE 0.056 vs 0.210 AE 0.040 per day, P < 0.01), indicating a significant 50% increase of apoA-I catabolism, leading to an important reduction of plasma apoA-I residence time (3.25 AE 0.59 vs 4.92 AE 1.11, P < 0.01). ApoA-I production rate tended to be higher in insulin resistant women than in controls (364 AE 77 vs 258 AE 60 mg=l=day, P ¼ 0.13), but the difference was not statistically significant. ApoA-I FCR was correlated with triglycerides during the fed state (r ¼ 0.69; P ¼ 0.026) and HDL triglycerides -esterified cholesterol ratio (r ¼ 0.73; P ¼ 0.016), suggesting that alteration of apoA-I metabolism in insulin resistance may be partly related to HDL enrichment in triglycerides. CONCLUSIONS: Our kinetic study shows that patients, at an early stage of insulin resistance (without impaired glucose tolerance nor fasting hypertriglyceridaemia), already have a significant alteration of apoA-I metabolism (increased apoA-I catabolism), which is consistent with the increased risk of atherosclerosis in this population.

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Measurement of muscle protein fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry

Cited by 83 publications

References 21 publications

Tracers to investigate protein and amino acid metabolism in human subjects

Tracers to investigate protein and amino acid metabolism in human subjects

Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

High-density lipoprotein apolipoprotein A-I kinetics in obese insulin resistant patients. An in vivo stable isotope study

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