Comparison of bolus injection and constant infusion methods for measuring muscle protein fractional synthesis rate in humans

Eighteen pure-bred steers (live weight 350 kg) from each of two breeds, Aberdeen Angus (AA) and Charolais (CH), were split into three equal groups (six animals each) and offered three planes of nutrition during a 20-week period. The same ration formulation was offered to all animals with amounts adjusted at 3-week intervals to give predicted average weight gains of either 1·0 kg/d (M/M group) or 1·4 kg/d (H/H group). The remaining group (M/H) were offered the same amount of ration as the M/M group until 10 weeks before slaughter when the ration was increased to H. Data on animal performance, carcass characteristics and fibre-type composition in skeletal muscle are presented elsewhere (; ). On three occasions (17, 10 and 2 weeks before slaughter) the animals were transferred to metabolism stalls for 1 week, during which total urine collection for quantification of Nτ-methylhistidine (Nτ-MeH) elimination was performed for 4 d. On the last day, animals were infused for 11 h with [2H5] phenylalanine with frequent blood sampling (to allow determination of whole-body phenylalanine flux) followed by biopsies from m. longissimus lumborum and m. vastus lateralis to determine the fractional synthesis rate of mixed muscle protein. For both breeds, the absolute amount of Nτ-MeH eliminated increased with animal age or weight (P < 0·001) and was significantly greater for CH steers, at all intake comparisons, than for AA (P < 0·001). Estimates of fractional muscle breakdown rate (FBR; calculated from Nτ-MeH elimination and based on skeletal muscle as a fixed fraction of live weight) showed an age (or weight) decline for M/M and H/H groups of both breeds (P < 0·001). FBR was greater for the H/H group (P = 0·044). The M/H group also showed a lower FBR for the first two measurement periods (both at M intake) but increased when intake was raised to H. When allowance was made for differences in lean content (calculated from fat scores and eye muscle area in carcasses at the end of period 3), there were significant differences in muscle FBR with intake (P = 0·012) but not between breed. Whole-body protein flux (WBPF; g/d) based on plasma phenylalanine kinetics increased with age or weight (P < 0·001) and was similar between breeds. The WBPF was lower for M/M compared with H/H (P < 0·001) based on either total or per kg live weight0·75. Muscle protein fractional synthesis rate (FSR) declined with age for both breeds and tended to be higher at H/H compared with M intakes (intake × period effects, P < 0·05). Changing intake from M to H caused a significant increase (P < 0·001) in FSR. The FSR values for AA were significantly greater than for CH at comparable ages (P = 0·044). Although FSR and FBR responded to nutrition, these changes in protein metabolism were not reflected in differences in meat eating quality (Sinclair et al. 2000).

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Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00185.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. E666-E671 ◽

Cited By ~ 22

Author(s):

Gordon I. Smith ◽

Dennis T. Villareal ◽

Bettina Mittendorfer

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Muscle Tissue ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Fat Free Mass ◽

Synthesis Rate ◽

Tissue Fluid ◽

Fractional Synthesis Rate ◽

Fractional Synthesis

The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein·kg fat-free mass−1·h−1 × 2.5 h) by simultaneous intravenous infusions of [5,5,5-2H3]leucine and either [ring-13C6]phenylalanine or [ring-2H5]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were ∼20% greater ( P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 ± 0.005%/h; fed: 0.080 ± 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 ± 0.004 and 0.066 ± 0.005%/h, respectively). The feeding-induced increase in the FSR (∼20%; P = 0.011) was not different with leucine and phenylalanine tracers ( P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used ( P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.

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Assessment of the mathematical issues involved in measuring the fractional synthesis rate of protein using the flooding dose technique

Clinical Science ◽

10.1042/cs0840177 ◽

1993 ◽

Vol 84 (2) ◽

pp. 177-183 ◽

Cited By ~ 17

Author(s):

David L. Chinkes ◽

Judah Rosenblatt ◽

Robert R. Wolfe

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Intracellular Concentration ◽

Upper And Lower Bounds ◽

Constant Infusion ◽

Synthesis Rate ◽

Protein Synthesis Rate ◽

Underlying Assumption ◽

Fractional Synthesis Rate ◽

Fractional Synthesis

1. The fractional synthesis rate of protein is commonly measured by either the constant infusion method or the flooding dose method. The two methods often give different results. 2. An underlying assumption of the traditional flooding dose formula is that the protein synthesis rate is not stimulated by the flooding dose. A new formula for calculation of the fractional synthesis rate is derived with the alternative assumption that the protein synthesis rate is stimulated by an amount proportional to the change in the intracellular concentration of the infused amino acid. The alternative formula is: where EB and EF are the enrichments of bound and free amino acid, respectively (atom per cent excess), and C=1-(EF/EI), where EI is the enrichment of the infusate. This approach defines the lowest possible value for the fractional synthesis rate. The traditional equation gives a maximal value for the fractional synthesis rate. 3. When data from the literature are considered, the fractional synthesis rate of muscle protein as calculated by the constant infusion technique falls between the values of fractional synthesis rate calculated by the two flooding dose formulae when leucine is the tracer, suggesting that a flooding dose of leucine exerts a stimulatory effect on the rate of protein synthesis, but that the increase is not as great as the increase in the intracellular concentration of leucine. 4. The precision of the formula for the calculation of fractional synthesis rate is limited by the accuracy of the underlying assumptions regarding the effect of the flooding dose on the fractional synthesis rate. At present, the best approach would appear to be the use of both equations to calculate the upper and lower bounds of the true fractional synthesis rate.

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Comparison of Arterial-Venous Balance and Tracer Incorporation Methods for Measuring Muscle Fractional Synthesis and Fractional Breakdown Rates

Journal of Burn Care & Research ◽

10.1093/jbcr/irab059 ◽

2021 ◽

Author(s):

Joshua L Hudson ◽

Matthew Cotter ◽

David N Herndon ◽

Robert R Wolfe ◽

Elisabet Børsheim

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Stable Isotope ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Constant Infusion ◽

Synthesis Rate ◽

Skeletal Muscle Protein ◽

Breakdown Rates ◽

Fractional Synthesis

Abstract Loss of muscle mass in response to injury or immobilization impairs functional capacity and metabolic health, thus hindering rehabilitation. Stable isotope techniques are powerful in determining skeletal muscle protein fluxes. Traditional tracer incorporation methods to measure muscle protein synthesis and breakdown are cumbersome and invasive to perform in vulnerable populations such as children. To circumvent these issues, a two-bolus stable isotope amino acid method has been developed; although, measured rates of protein synthesis and breakdown have not been validated simultaneously against an accepted technique such as the arterial-venous balance method. The purpose of the current analysis was to provide preliminary data from the simultaneous determination of the arteriovenous balance and two-bolus tracer incorporation methods on muscle fractional synthesis and breakdown rates in children with burns. Five were administered a primed-constant infusion of L-[ 15N]Threonine for 180 minutes (Prime: 8 µmol/kg; constant: 0.1 µmol·kg -1·min -1). At 120 and 150 minutes, bolus injections of L-[ring- 13C6]Phenylalanine and L-[ 15N]Phenylalanine (50 µmol/kg each) were administered, respectively. Blood and muscle tissue samples were collected to assess mixed muscle protein synthesis and breakdown rates. The preliminary results from this study indicate there is no difference in either fractional synthesis rate (mean ± SD; arteriovenous balance: 0.19 ± 0.17 %/h; tracer incorporation: 0.14 ± 0.08 %/h; P = 0.42) or fractional breakdown rate (arteriovenous balance: 0.29 ± 0.22 %/h; tracer incorporation: 0.23 ± 0.14 %/h; P = 0.84) between methods. These data support the validity of both methods in quantifying muscle amino acid kinetics; however, the results are limited and adequately powered research is still required.

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The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats

Biochemical Journal ◽

10.1042/bj2350329 ◽

1986 ◽

Vol 235 (2) ◽

pp. 329-336 ◽

Cited By ~ 136

Author(s):

M M Jepson ◽

J M Pell ◽

P C Bates ◽

D J Millward

Keyword(s):

Protein Synthesis ◽

Protein Metabolism ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Synthesis Rate ◽

Liver Protein ◽

Fractional Synthesis Rate ◽

Protein Mass ◽

Fractional Synthesis ◽

Fasted Rats

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.

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Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia–hyperaminoacidaemia in healthy young and middle-aged men and women

Clinical Science ◽

10.1042/cs20100597 ◽

2011 ◽

Vol 121 (6) ◽

pp. 267-278 ◽

Cited By ~ 168

Author(s):

Gordon I. Smith ◽

Philip Atherton ◽

Dominic N. Reeds ◽

B. Selma Mohammed ◽

Debbie Rankin ◽

...

Keyword(s):

Amino Acid ◽

Cell Size ◽

Muscle Protein ◽

Synthesis Rate ◽

Middle Aged ◽

Anabolic Response ◽

Acid Infusion ◽

Fractional Synthesis Rate ◽

Amino Acid Infusion ◽

Fractional Synthesis

Increased dietary LCn−3PUFA (long-chain n−3 polyunsaturated fatty acid) intake stimulates muscle protein anabolism in individuals who experience muscle loss due to aging or cancer cachexia. However, it is not known whether LCn−3PUFAs elicit similar anabolic effects in healthy individuals. To answer this question, we evaluated the effect of 8 weeks of LCn−3PUFA supplementation (4 g of Lovaza®/day) in nine 25–45-year-old healthy subjects on the rate of muscle protein synthesis (by using stable isotope-labelled tracer techniques) and the activation (phosphorylation) of elements of the mTOR (mammalian target of rapamycin)/p70S6K (p70 S6 kinase) signalling pathway during basal post-absorptive conditions and during a hyperinsulinaemic–hyperaminoacidaemic clamp. We also measured the concentrations of protein, RNA and DNA in muscle to obtain indices of the protein synthetic capacity, translational efficiency and cell size. Neither the basal muscle protein fractional synthesis rate nor basal signalling element phosphorylation changed in response to LCn−3PUFA supplementation, but the anabolic response to insulin and amino acid infusion was greater after LCn−3PUFA [i.e. the muscle protein fractional synthesis rate during insulin and amino acid infusion increased from 0.062±0.004 to 0.083±0.007%/h and the phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) levels increased by ∼50%; all P<0.05]. In addition, the muscle protein concentration and the protein/DNA ratio (i.e. muscle cell size) were both greater (P<0.05) after LCn−3PUFA supplementation. We conclude that LCn−3PUFAs have anabolic properties in healthy young and middle-aged adults.

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Timing of the initial muscle biopsy does not affect the measured muscle protein fractional synthesis rate during basal, postabsorptive conditions

Journal of Applied Physiology ◽

10.1152/japplphysiol.00957.2009 ◽

2010 ◽

Vol 108 (2) ◽

pp. 363-368 ◽

Cited By ~ 18

Author(s):

Gordon I. Smith ◽

Dennis T. Villareal ◽

Charles P. Lambert ◽

Dominic N. Reeds ◽

B. Selma Mohammed ◽

...

Keyword(s):

Muscle Protein ◽

Protein Labeling ◽

Synthesis Rate ◽

Leucine Incorporation ◽

Fractional Synthesis Rate ◽

Population Average ◽

Constant Rate ◽

Initial Biopsy ◽

Fractional Synthesis ◽

The Difference

The muscle protein fractional synthesis rate (FSR) is determined by monitoring the incorporation of an amino acid tracer into muscle protein during a constant-rate intravenous tracer infusion. Commonly two sequential muscle biopsies are obtained some time after starting the tracer infusion. However, other protocols, including those with an initial biopsy before starting the tracer infusion to measure the background enrichment and those with only a single biopsy after several hours of tracer infusion have been used. To assess the validity of these approaches, we compared the muscle protein FSR obtained by calculating the difference in [ ring-2H5]phenylalanine and [5,5,5-2H3]leucine incorporation into muscle protein at ∼3.5 h after starting the tracer infusion and 1) at 60 min; 2) before starting the tracer infusion (background enrichment); 3) a population average muscle protein background enrichment; and 4) by measuring the tracer incorporation into muscle protein at ∼3.5 h assuming essentially no background enrichment. Irrespective of the tracer used, the muscle protein FSR calculated from the difference in the muscle protein labeling several hours after starting the tracer infusion and either the labeling at 60 min or the background enrichment were not different (e.g., 0.049 ± 0.007%/h vs. 0.049 ± 0.007%/h, respectively, with [2H5]phenylalanine; P = 0.99). However, omitting the initial biopsy and assuming no background enrichment yielded average FSR values that were ∼15% (with [2H5]phenylalanine) to 80% (with [2H3]leucine) greater ( P ≤ 0.059); using a population average background enrichment reduced the difference to ∼3% ( P = 0.76) and 22% ( P = 0.52) with [2H5]phenylalanine and [2H3]leucine, respectively. We conclude that during basal, postabsorptive conditions, valid muscle protein FSR values can be obtained irrespective of the timing of the initial biopsy so long as the protein labeling in two sequential biopsies is measured whereas the single biopsy approach should be avoided.

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Determination of human muscle protein fractional synthesis rate: an evaluation of different mass spectrometry techniques and considerations for tracer choice

Journal of Mass Spectrometry ◽

10.1002/jms.3387 ◽

2014 ◽

Vol 49 (8) ◽

pp. 674-680 ◽

Cited By ~ 13

Author(s):

Andreas Bornø ◽

Carl J. Hulston ◽

Gerrit van Hall

Keyword(s):

Mass Spectrometry ◽

Muscle Protein ◽

Human Muscle ◽

Synthesis Rate ◽

Fractional Synthesis Rate ◽

Fractional Synthesis

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Evidence for increased synthesis of lipoprotein(a) in the nephrotic syndrome.

Journal of the American Society of Nephrology ◽

10.1681/asn.v981474 ◽

1998 ◽

Vol 9 (8) ◽

pp. 1474-1481

Author(s):

M G De Sain-Van Der Velden ◽

D J Reijngoud ◽

G A Kaysen ◽

M M Gadellaa ◽

H Voorbij ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Synthesis Rate ◽

Fractional Synthesis Rate ◽

Lipoprotein A ◽

Control Subjects ◽

Apolipoprotein A ◽

Catabolic Rate ◽

Fractional Synthesis ◽

The Mean

In patients with the nephrotic syndrome, markedly increased levels of lipoprotein(a) (Lp(a)) concentration have been frequently reported, and it has been suggested that this may contribute to the increased cardiovascular risk in these patients. The mechanism, however, is not clear. In the present study, in vivo fractional synthesis rate of Lp(a) was measured using incorporation of the stable isotope 13C valine. Under steady-state conditions, fractional synthesis rate equals fractional catabolic rate (FCR). FCR of Lp(a) was estimated in five patients with the nephrotic syndrome and compared with five control subjects. The mean plasma Lp(a) concentration in the patients (1749+/-612 mg/L) was higher than in control subjects (553+/-96 mg/L). Two patients were heterozygous for apolipoprotein(a) (range, 19 to 30 kringle IV domains), whereas all control subjects were each homozygous with regard to apolipoprotein(a) phenotype (range, 18 to 28 kringle IV domains). The FCR of Lp(a) was comparable between control subjects (0.072+/-0.032 pools/d) and patients (0.064+/-0.029 pools/d) despite the wide variance in plasma concentration. This suggests that differences in Lp(a) levels are caused by differences in synthesis rate. Indeed, the absolute synthetic rate of Lp(a) correlated directly with plasma Lp(a) concentration (P < 0.0001) in all subjects. The present results demonstrate that increased synthesis, rather than decreased catabolism, causes elevated plasma Lp(a) concentrations in the nephrotic syndrome.

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