Measuring whole-body actin/myosin protein breakdown in mice using a primed constant stable isotope-infusion protocol

2003 ◽  
Vol 104 (6) ◽  
pp. 585-590 ◽  
Author(s):  
Yvonne L. J. VISSERS ◽  
Maarten F. VON MEYENFELDT ◽  
Valeria B. BRAULIO ◽  
Yvette C. LUIKING ◽  
Nicolaas E. P. DEUTZ
Keyword(s):  
Stable Isotope ◽  
Total Protein ◽  
Myofibrillar Protein ◽  
Whole Body ◽  
Constant Infusion ◽  
Protein Breakdown ◽  
Net Protein ◽  
Infusion Protocol

To measure actin/myosin protein breakdown, the 24 h excretion of Nτ-methylhistidine (3MH) is used. However, in mice, this method is invalid. Therefore we have developed a liquid chromatography-MS technique to measure the tracer/tracee ratio and concentration of 3MH in plasma, enabling an in vivo primed constant infusion protocol with a deuterated stable isotope of 3MH. We tested this model by giving a primed constant infusion of L-[3-methyl-2H3]histidine, L-[phenyl-2H5]phenylalanine and L-[phenyl-2H2]tyrosine to three anaesthetized experimental groups: mice receiving saline intraperitoneally (i.p.) (CON), mice receiving saline i.p. and starved for 9 h (STA), and mice receiving lipopolysaccharide i.p. and starved for 9 h (STA + LPS). The contribution of myofibrillar to total protein breakdown was significantly lower in the STA group than the CON group (30±4% and 54±14% respectively; P<0.05), and was significantly higher in the STA + LPS group than the STA group (52±7% and 30±4% respectively; P<0.05). Whole-body myofibrillar protein breakdown, total protein breakdown, protein synthesis and net protein breakdown were not different between the groups. We conclude that this in vivo primed constant stable isotope-infusion protocol can give valuable information about the role of actin/myosin protein breakdown in mice.

Compartmental model of leucine kinetics in humans

1991 ◽  
Vol 261 (4) ◽  
pp. E539-E550 ◽  
Author(s):  
C. Cobelli ◽  
M. P. Saccomani ◽  
P. Tessari ◽  
G. Biolo ◽  
L. Luzi ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Compartmental Model ◽  
Linear Time ◽  
A Priori ◽  
Normal Subjects ◽  
Whole Body ◽  
Constant Infusion ◽  
Individual Subject ◽  
Domain Of Validity

The complexity of amino acid and protein metabolism has limited the development of comprehensive, accurate whole body kinetic models. For leucine, simplified approaches are in use to measure in vivo leucine fluxes, but their domain of validity is uncertain. We propose here a comprehensive compartmental model of the kinetics of leucine and alpha-ketoisocaproate (KIC) in humans. Data from a multiple-tracer administration were generated with a two-stage (I and II) experiment. Six normal subjects were studied. In experiment I, labeled leucine and KIC were simultaneously injected into plasma. Four plasma leucine and KIC tracer concentration curves and label in the expired CO2 were measured. In experiment II, labeled bicarbonate was injected into plasma, and labeled CO2 in the expired air was measured. Radioactive (L-[1-14C]leucine, [4,5-3H]KIC, [14C]bicarbonate) and stable isotope (L-[1-13C]leucine, [5,5,5-2H3]KIC, [13C]bicarbonate) tracers were employed. The input format was a bolus (impulse) dose in the radioactive case and a constant infusion in the stable isotope case. A number of physiologically based, linear time-invariant compartmental models were proposed and tested against the data. The model finally chosen for leucine-KIC kinetics has 10 compartments: 4 for leucine, 3 for KIC, and 3 for bicarbonate. The model is a priori uniquely identifiable, and its parameters were estimated with precision from the five curves of experiment I. The separate assessment of bicarbonate kinetics (experiment II) was shown to be unnecessary. The model defines masses and fluxes of leucine in the organism, in particular its intracellular appearance from protein breakdown, its oxidation, and its incorporation into proteins. An important feature of the model is its ability to estimate leucine oxidation by resolving the bicarbonate model in each individual subject. Finally, the model allows the assessment of the domain of validity of the simpler commonly used models.

Muscle wasting in emphysema

1988 ◽  
Vol 75 (4) ◽  
pp. 415-420 ◽  
Author(s):  
W. L. Morrison ◽  
J. N. A. Gibson ◽  
C. Scrimgeour ◽  
M. J. Rennie
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Muscle Wasting ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Myofibrillar Protein ◽  
Whole Body ◽  
Protein Breakdown ◽  
Body Protein ◽  
Net Protein

1. We have investigated arteriovenous exchanges of tyrosine and 3-methylhistidine across leg tissue in the postabsorptive state as specific indicators of net protein balance and myofibrillar protein breakdown, respectively, in eight patients with emphysema and in 11 healthy controls. Whole-body protein turnover was measured using l-[1-13C]leucine. 2. Leg efflux of tyrosine was increased by 47% in emphysematous patients compared with normal control subjects, but 3-methylhistidine efflux was not significantly altered. 3. In emphysema, whole-body leucine flux was normal, whole-body leucine oxidation was increased, and whole-body protein synthesis was depressed. 4. These results indicate that the predominant mechanism of muscle wasting in emphysema is a fall in muscle protein synthesis, which is accompanied by an overall fall in whole-body protein turnover.

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

2008 ◽  
Vol 295 (6) ◽  
pp. E1315-E1322 ◽  
Author(s):  
Yvette C. Luiking ◽  
Marcella M. Hallemeesch ◽  
Marcel C. van de Poll ◽  
Cornelis H. C. Dejong ◽  
Wouter J. de Jonge ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
De Novo ◽  
Whole Body ◽  
No Production ◽  
Protein Breakdown ◽  
Otc Deficiency ◽  
Ornithine Transcarbamoylase ◽  
Net Protein ◽  
Lps Treatment

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spfash (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-15N2]arginine to l-[guanidine-15N]citrulline; de novo arginine production was measured as conversion of l-[ureido-13C-5,5-2H2]citrulline to l-[guanidino-13C-5,5-2H2]arginine. Protein metabolism was measured using l-[ ring-2H5]phenylalanine and l-[ ring-2H2]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30–50% ( P < 0.001), reduced de novo arginine production ( P < 0.05), reduced whole-body NO production to 50% ( P < 0.005), and increased net protein breakdown by a factor of 2-4 ( P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups ( P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.

scholarly journals Comprehensive assessment of post-prandial protein handling by the application of intrinsically labelled protein in vivo in human subjects

2021 ◽  
pp. 1-9
Author(s):  
Jorn Trommelen ◽  
Andrew M. Holwerda ◽  
Philippe J. M. Pinckaers ◽  
Luc J. C. van Loon
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Stable Isotope ◽  
Dietary Protein ◽  
Protein Metabolism ◽  
Muscle Protein ◽  
Human Subjects ◽  
Whole Body ◽  
Body Protein

All human tissues are in a constant state of remodelling, regulated by the balance between tissue protein synthesis and breakdown rates. It has been well-established that protein ingestion stimulates skeletal muscle and whole-body protein synthesis. Stable isotope-labelled amino acid methodologies are commonly applied to assess the various aspects of protein metabolism in vivo in human subjects. However, to achieve a more comprehensive assessment of post-prandial protein handling in vivo in human subjects, intravenous stable isotope-labelled amino acid infusions can be combined with the ingestion of intrinsically labelled protein and the collection of blood and muscle tissue samples. The combined application of ingesting intrinsically labelled protein with continuous intravenous stable isotope-labelled amino acid infusion allows the simultaneous assessment of protein digestion and amino acid absorption kinetics (e.g. release of dietary protein-derived amino acids into the circulation), whole-body protein metabolism (whole-body protein synthesis, breakdown and oxidation rates and net protein balance) and skeletal muscle metabolism (muscle protein fractional synthesis rates and dietary protein-derived amino acid incorporation into muscle protein). The purpose of this review is to provide an overview of the various aspects of post-prandial protein handling and metabolism with a focus on insights obtained from studies that have applied intrinsically labelled protein under a variety of conditions in different populations.

scholarly journals Comparison of different techniques for estimating rates of protein synthesis in vivo in healthy and bacteraemic rats

1985 ◽  
Vol 226 (1) ◽  
pp. 37-42 ◽  
Author(s):  
J J Pomposelli ◽  
J D Palombo ◽  
K J Hamawy ◽  
B R Bistrian ◽  
G L Blackburn ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Rectus Muscle ◽  
Stochastic Modelling ◽  
Whole Body ◽  
Constant Infusion ◽  
Second Phase ◽  
Sprague Dawley ◽  
Body Protein ◽  
To Receive

Previous studies have reported that use of a flooding dose of radiolabelled amino acid is a more precise technique than the constant infusion of tracer quantities for determining rates of protein synthesis in rapidly turning-over tissues in the rat. However, there has been little direct investigation comparing different methods under comparable conditions. Initially, 12 healthy male Sprague-Dawley rats, weighing approx. 100 g, were randomized to receive either a bolus intravenous injection of 100 mumol of L-leucine (containing 30 microCi of [1-14C]leucine)/100 g body wt., or a continuous 2 h tracer infusion of [14C]leucine. In the second phase of the experiment, 12 additional rats were intravenously injected with 1 × 10(8) colony-forming units of Pseudomonas aeruginosa and 16 h later randomized to receive one of two infusions described above. Total protein synthesis as well as fractional synthesis rates were determined in liver, rectus muscle and whole body. Synthesis rates measured in liver, muscle and whole body were significantly higher in bacteraemic rats than in healthy rats. The flooding-dose methodology gave significantly higher estimates of protein synthesis in the liver, skeletal muscle and whole body than did the continuous-infusion method using direct measurement of the acid-soluble fraction from the respective tissue. Indirect estimates of whole-body protein synthesis based on plasma enrichments and stochastic modelling gave the lowest values.

scholarly journals Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4097-4104 ◽  
Author(s):  
T Misaizu ◽  
S Matsuki ◽  
TW Strickland ◽  
M Takeuchi ◽  
A Kobata ◽  
...  
Keyword(s):  
Whole Body ◽  
Renal Handling ◽  
Sugar Chain ◽  
Erythroid Progenitor ◽  
Human Erythropoietin ◽  
Sugar Chains ◽  
Effective Transfer ◽  
Branched Structure

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

scholarly journals Dose-Dependent Increases in Whole-Body Net Protein Balance and Dietary Protein-Derived Amino Acid Incorporation into Myofibrillar Protein During Recovery from Resistance Exercise in Older Men

2019 ◽  
Vol 149 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Andrew M Holwerda ◽  
Kevin J M Paulussen ◽  
Maarten Overkamp ◽  
Joy P B Goessens ◽  
Irene Fleur Kramer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Resistance Exercise ◽  
Dietary Protein ◽  
Older Men ◽  
Myofibrillar Protein ◽  
Whole Body ◽  
Protein Balance ◽  
Dose Dependent ◽  
Net Protein ◽  
Myofibrillar Protein Synthesis

ABSTRACT Background Age-related decline in skeletal muscle mass is at least partly attributed to anabolic resistance to food intake. Resistance exercise sensitizes skeletal muscle tissue to the anabolic properties of amino acids. Objective The present study assessed protein digestion and amino acid absorption kinetics, whole-body protein balance, and the myofibrillar protein synthetic response to ingestion of different amounts of protein during recovery from resistance exercise in older men. Methods Forty-eight healthy older men [mean ± SEM age: 66 ± 1 y; body mass index (kg/m2): 25.4 ± 0.3] were randomly assigned to ingest 0, 15, 30, or 45 g milk protein concentrate after a single bout of resistance exercise consisting of 4 sets of 10 repetitions of leg press and leg extension and 2 sets of 10 repetitions of lateral pulldown and chest press performed at 75–80% 1-repetition maximum. Postprandial protein digestion and amino acid absorption kinetics, whole-body protein metabolism, and myofibrillar protein synthesis rates were assessed using primed, continuous infusions of l-[ring-2H5]-phenylalanine, l-[ring-2H2]-tyrosine, and l-[1-13C]-leucine combined with ingestion of intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled protein. Results Whole-body net protein balance showed a dose-dependent increase after ingestion of 0, 15, 30, or 45 g of protein (0.015 ± 0.002, 0.108 ± 0.004, 0.162 ± 0.008, and 0.215 ± 0.009 μmol Phe · kg−1 · min−1, respectively; P < 0.001). Myofibrillar protein synthesis rates were higher after ingesting 30 (0.0951% ± 0.0062%/h, P = 0.07) or 45 g of protein (0.0970% ± 0.0062%/h, P < 0.05) than after 0 g (0.0746% ± 0.0051%/h). Incorporation of dietary protein–derived amino acids (l-[1-13C]-phenylalanine) into de novo myofibrillar protein showed a dose-dependent increase after ingestion of 15, 30, or 45 g protein (0.0171 ± 0.0017, 0.0296 ± 0.0030, and 0.0397 ± 0.0026 mole percentage excess, respectively; P < 0.05). Conclusions Dietary protein ingested during recovery from resistance exercise is rapidly digested and absorbed. Whole-body net protein balance and dietary protein-derived amino acid incorporation into myofibrillar protein show dose-dependent increases. Ingestion of ≥30 g protein increases postexercise myofibrillar protein synthesis rates in older men. This trial was registered at Nederlands Trial Register as NTR4492.

Enhancement of tumor proteolysis by TNF-alpha: correlation of in vivo isotope estimates with growth

1991 ◽  
Vol 261 (1) ◽  
pp. R106-R116
Author(s):  
N. W. Istfan ◽  
P. R. Ling ◽  
G. L. Blackburn ◽  
B. R. Bistrian
Keyword(s):  
Protein Synthesis ◽  
Protein Metabolism ◽  
Whole Body ◽  
Independent Effect ◽  
Yoshida Sarcoma ◽  
Protein Breakdown ◽  
Body Protein ◽  
Breakdown Rates ◽  
Made In

To evaluate the accuracy of in vivo estimates of protein synthesis and breakdown, measurements of plasma and tissue leucine kinetics were made in rat tumor tissues at different conditions of growth by use of constant intravenous infusion of [14C]leucine. These measurements were made in Yoshida sarcoma tumors on days 10 and 13 after implantation, with and without tumor necrosis factor (TNF) infusion and on day 10 in Walker-256 carcinosarcoma. Expressed as micromoles of leucine per gram tissue, tumor protein breakdown increased (P less than 0.01) from 0.32 +/- 0.02 to 0.52 +/- 0.09 (SE) mumol/h, with progress of the Yoshida sarcoma tumor between days 10 and 13 after implantation. Similarly, TNF increased tumor proteolysis on day 10 (0.43 +/- 0.03 mumol.h-1.g-1, P less than 0.05 vs. day 10 control) but not on day 13 after implantation of the Yoshida tumor. Estimates of growth derived from the difference between protein synthesis and breakdown rates were not statistically different from those based on actual tumor volume changes in both tumor models. However, estimates of “whole body” protein metabolism (plasma leucine flux) were not affected either by tumor aging or by treatment with TNF. This study shows that in vivo estimates of tissue protein metabolism based on our [14C]leucine constant infusion model closely reflect the growth characteristic of that tissue. A cytotoxic perfusion-independent effect for intravenous TNF on growing tumor tissue is demonstrable as increased protein breakdown. Furthermore, the commonly used concept of whole body protein metabolism, derived solely from tracer dilution in plasma, is an oversimplification.

Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. Analysis of mechanisms responsible for the rejection of parasites in vivo

Parasitology ◽  
1981 ◽  
Vol 83 (3) ◽  
pp. 543-558
Author(s):  
Gina Moser ◽  
F. von Lichtenberg ◽  
A. Sher
Keyword(s):  
Schistosoma Mansoni ◽  
Passive Immunization ◽  
Early Response ◽  
Challenge Infection ◽  
Delayed Hypersensitivity ◽  
Whole Body ◽  
Hypersensitivity Response ◽  
Time Points

SUMMARYSchistosomula, surface labelled with trinitrophenyl (TNP) target antigens were tested for their susceptibility to killing by humoral- or cell-mediated anti-TNP effector mechanisms in vivo. It was found that mice passively immunized with anti-TNP serum effectively rejected an intravenous (i.v.) challenge infection with TNP-labelled schistosomula. In contrast, mice which demonstrated a strong TNP-specific, delayed hypersensitivity response to the haptenated larvae as evidenced by ear swelling, were unable to eliminate the same challenge infection. Significant passive immunization against TNP-labelled schistosomula was shown to require microlitre quantities of anti-TNP serum and could be conferred with an IgG fraction purified from the serum. The role of cells in the antibody-dependent rejection of TNP-labelled schistosomula was investigated using histopathological methods. In passively immunized mice, haptenated larvae elicited neutrophil-enriched focal reactions in the lungs and showed evidence of degeneration as early as 2 h after injection. These cellular reactions were not observed in recipients which had received prior whole-body irradiation. Nevertheless, by 24 h TNP-labelled larvae were found to have been killed in the lungs of the irradiated mice despite the absence of significant cellular attack. The above observations suggest that the antibody-dependent destruction of haptenated schistosomula results from two overlapping responses, an early response mediated by radio-sensitive cells and a second, radio-resistant response manifesting its effects at later time points. Since mice genetically deficient in the fifth component of complement fail to develop the later response, it probably reflects the effect of the lytic pathway of complement on the parasite.

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