Flooding and Continuous Method for Measuring Global Protein Synthesis
measurements of protein synthesis are applied widely in animal and human research and can be measured directly in vivo by the direct incorporation method using isotopically labeled amino acid tracers. Originally, this was done by administering the tracers either orally or intravenously in one bolus (9, 33), but subsequent work advanced the tracer method by applying a continuous intravenous tracer infusion that allowed for the calculation of the fractional rate of synthesis, termed k s (12, 34). This first version of the protein fractional synthesis rate (FSR) approach did account for the tracer abundance in the amino acid pool at the site of protein synthesis, i.e., in the tissue-specific precursor pool. However, the subsequent discussion (11, 20) showed that a significant error was apparent for the FSR calculation, as the tracer abundance in neither the circulation nor in the precursor pool had reached a steady state in the beginning of the constant infusion (12). By assuming that the tracee make up a single pool in the body, the enrichment of tracer obtained by a constant infusion will rise exponentially up to a steady level with a rate constant equal to the turnover rate of the tracee in the pool. For leucine, the steady-state level is not reached until after 10 h of continuous infusion (15). The rationale was that the tracer would have the time to equilibrate between watery body pools because of the slowly ascending enrichment obtained by the constant infusion, and thus, as long as the infusion period, hence the incorporation time, was markedly longer than the initial period with unequilibrated tracer abundance, the estimation of FSR was reasonable (11). Furthermore, it was concluded that the protein turnover measurement performed with the continuous tracer infusion was superior to that with the bolus administration (14). Therefore, the first continuous tracer infusion experiments in humans were exhaustively long (16). Matthews et al. (21) shortly after presented the primed, continuous infusion of tracer, meaning that a small tracer dose was injected just prior to the start of the continuous infusion. The priming dose resulted in an instantaneous elevation of the tracer abundance in the circulation, and when adjusted accordingly, tracer steady state was obtained immediately in the circulation, delivering the tracers to the free amino acid pools at the site of protein synthesis (21, 25). However, the instantaneous isotopic steady state obtained in the circulation did not reflect all pools of tracee. Time had to elapse to allow the amino acid tracer to equilibrate between circulation and the intracellular pools required to label the precursor pool for the protein synthesis.
The linear incorporation during the primed, continuous infusion protocol takes place only when isotopic steady state is present at the site of the real precursor pool; thus, at least two tissue/protein samples had to be obtained. The first could be obtained as soon as the isotopic steady state appeared in the real precursor pool, and the subsequent sample(s) had to be collected at a later time point as long as 1) adequate incorporation time between adjacent samplings was secured and 2) the isotopic steady state was maintained. The primed, continuous infusion protocol obtained general acceptance as it markedly shortened the trial duration and solved the inborn errors compared with previous protocols. However, the disadvantage was that a minimum of two biopsies has to be obtained.
The flooding approach was developed with the aim to meet the requirement for a single-biopsy approach and at the same time to have a good estimate of the precursor labeling (13, 22, 24, 29). Briefly, the principle is that tracer and tracee are mixed and injected in one bolus, resulting in supraphysiological concentrations. The tracer/tracee ratio (TTR) that is mixed in the bolus will then flood and equilibrate in all watery pools in the body, and therefore, a reliable estimate of the tracer enrichment in the intracellular pools can be measured by sampling from the circulation. Since it is only a single injection the turnover of the analyte will wash away the tracer, and the enrichment will drop first in the intracellular pools and then be somewhat delayed in the circulation. At some point after the flooding, the circulating enrichment will not reflect the enrichment any more in the intracellular pools. Therefore, the tracer incorporation time is very limited when the flooding approach is used, and hence, the absolute incorporation of tracer may become critical when proteins with slow turnover rates are studied. One solution would be to combine the continuous infusion method with a setting where the real precursor enrichment is known from the very beginning of the tracer administration period, as in the flooding approach. Burd and colleagues (3–5) recently reported data using the primed, continuous infusion approach, where they showed that the skeletal muscle FSR measured by a single-biopsy approach yielded comparable rates as when the two-biopsy approach was applied, provided that a longer (≥4 h) incorporation time was used. Although these authors claimed that they validated the protocol, they did so only by comparing the FSR values and not by assessing the precursor tracer enrichments, and concern about the validity of this approach has been presented (26).
Therefore, we wanted to compare the tracer enrichments in the muscle free pool after the conventional primed, continuous approach used by Burd and collegues (3–5), with a new technique using a flooding bolus as a prime, hypothesizing that the latter approach would be superior in labeling the muscle free pool early on. Furthermore, we wanted to evaluate whether or not the flooding bolus technique using phenylalanine as tracer in itself stimulates the muscle protein synthesis and evaluate to what overall extent this approach is feasible and reliable in practical usage for human physiological experiments.
METHODS
Subject Inclusion
Young, healthy, occasionally physically active (<2 times weekly) males were recruited for experimental trials through posters and advertisements at the official Danish web page for recruiting human volunteers (www.forsogsperson.dk). They were all recruited as being tracer naïve, i.e., never having received any stable isotopically labeled compounds, and all participants gave their written informed consent before being enrolled. The study was approved by the Copenhagen Ethics Committee (H-1-2010-007 and H-1-2012-102) and conformed to the code of the Helsinki Declaration. Prior to the experiments, all individuals had their anthropometric data determined and body mass index calculated and were scanned with dual-energy X-ray absorptiometry (DPX-IQ software version 4.6c; Lunar, Madison, WI) for determination of lean body mass. On the day prior to the trial, all subjects were instructed not to perform any strenuous exercise. On the experimental day, they arrived at the laboratory by car or public transportation after an overnight fast. Designs of experiments 1–3 are shown in Fig. 1.
Fig. 1.Design of experiments 1, 2, and 3.
Experiments
Experiment 1.
The first experiment aimed to investigate when the tracer, after being administered using a conventional primed, continuous infusion protocol, equilibrated between the plasma and the muscle free amino acids. Six males [age: 20.5 ± 0.4 yr (means ± SE); body mass index (BMI): 24.1 ± 1.1 kg/m−2; lean body mass (LBM): 68.4 ± 3.7 kg] were recruited. Before any tracer administration was started, background samples from various tissues were collected after prior preparation with lidocaine 1% and chlorhexidine-alcohol. First, a skin sample was obtained from the lateral part of the upper gluteal area using a 4-mm dermal biopsy punch (Miltex, York, PA). Next, a patellar tendon sample was obtained with a Bard magnum biopsy instrument (C. R. Bard, Covington, GA) from one leg using a disposable 14-g core tissue biopsy needle (Bard Peripheral Vascular, Tempe, AZ). Finally, a muscle biopsy was taken through a skin incision from the vastus lateralis (VL) muscle with a 5-mm Bergstrom needle using manual suction. All tissue samples were thoroughly washed in ice-cold saline, and all visible fat was removed before being frozen in liquid nitrogen and stored at −80°C. After these background samplings, an infusion protocol similar to the one used in the studies by Burd and colleagues (3–5) was started, i.e., a primed (2 μmol/kg LBM), continuous (3 μmol·kg LBM−1·h−1) infusion of ring-13C6-phenylalanine (see Fig. 1). After 10, 30, 60, 120, and 240 min, VL muscle biopsies were obtained, alternating between legs. Additionally, at 10, 20, 30, 60, 90, 120, 180, and 240 min, venous blood was drawn from an antecubital vein catheter placed in the arm contralateral to that receiving infusion. All blood was drawn into EDTA-coated vials and left on ice for 30 min before spinning (4°C, 3,060 g, 10 min). The extracted plasma was saved at −80°C for further analyses.
Experiment 2.
Next, we wanted to test whether a flooding bolus could be used to label the intramuscular pool instantaneously. Seven males (age: 22.4 ± 0.7 yr; BMI: 23.0 ± 1.0 kg/m−2; LBM: 61.7 ± 2.9 kg) were given a priming bolus over 1 min. The priming bolus contained a total of 1,665 mg of phenylalanine distributed with the relation corresponding to 12% TTR, i.e., 1,480 mg of unlabeled (8.97 mmol) and 185 mg of labeled ring-13C6-phenylalanine (1.08 mmol). The 12% TTR-enriched priming bolus was followed by a continuous infusion of ring-13C6-phenylalanine (8 μmol·kg LBM−1·h−1), which by experience we know will approximate an arterial 12% TTR enrichment. VL muscle biopsies were obtained after 10, 30, 60, 120, and 360 min, and blood samples were collected throughout the trial, following the procedures used in experiment 1 (See Fig. 1).
Experiment 3.
To test whether the flooding bolus with phenylalanine in itself affected the muscle protein synthesis rate, we conducted the third experiment. Five males were included (age: 20.5 ± 0.4 yr; BMI: 25.0 ± 2.1 kg/m−2; LBM: 60.5 ± 4.3 kg) and were given a primed (9 μmol/kg LBM), continuous (9 μmol·kg LBM−1·h−1) infusion of [1,2-13C2]leucine. After 120 and 300 min, VL muscle biopsies were obtained in the overnight-fasted condition to determine the FSR in the basal condition. Hereafter, a flooding bolus with 1,665 mg of phenylalanine, enriched ∼7% TTR with [15N]phenylalanine, was given followed by a continuous infusion (8 μmol·kg LBM−1·h−1) of unlabeled phenylalanine to exactly mimic the entire phenylalanine tracer infusion protocol used in experiment 2. The [15N]phenylalanine was added to the flooding bolus to follow its disappearance from the circulation over the course of the 3-h period. A third VL muscle biopsy was obtained at 480 min to determine the FSR over the 3-h period following the flooding bolus injection (see Fig. 1).
Investigating practicalities required for application of the single biopsy-approach.
To control for even natural abundances of the used stable isotopes in various proteins, we obtained tissue biopsies from the VL muscle, patellar tendon, and skin as well as a blood sample from tracer naïve subjects recruited for experiment 1 before the tracer infusion was started. The 13C natural abundances in phenylalanine and proline were determined in protein isolates from various tissues and blood.
We also tested whether washing and cleaning procedures of the raw muscle specimens affected the enrichment measured in the muscle free pool of the tracer amino acid. From the muscle biopsies obtained in either experiment 1 or experiment 2, we collected eight muscle specimens taken at isotopic steady state (>90-min infusion). After being obtained, muscle specimens were divided into two pieces; one was frozen directly in liquid nitrogen for further manual cleaning (cleaning sample) and another was washed carefully in sterile gaze with ice-cold saline (saline sample) before being quick-frozen in liquid nitrogen. Both muscle specimens were freeze-dried to visualize the cleanliness of the specimens. The cleaned sample was manually dissected free of blood under dry conditions after having been freeze-dried (32). Blood samples were collected from the same subjects at the same time point as the biopsies were taken. The muscle free amino acids and the plasma free amino acids were isolated from muscle specimens and plasma samples, respectively, following the procedures described below, and the phenylalanine tracer enrichments were determined.
Analyses
Plasma phenylalanine concentration and tracer enrichment were determined by using 200 μl of plasma, with a known amount of [U-13C9]phenylalanine added for use as an internal standard. Samples were acidified with 1 ml of 50% acidic acid before being poured over resin columns (AG 50W-X8 resin; Bio-Rad Laboratories, Hercules, CA) preconditioned with 1 ml of 50% acidic acid. After five washes with Milli-Q water, the purified amino acids were eluted with 2 × 1 ml of 4 M NH4OH. After being dried down under a stream of nitrogen, the purified amino acids were derivatized using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide + 1% tert-butyl-dimethylchlorosilane (Regis Technologies, Morton Grove, IL) and acetonitrile, with a mixing ratio of 1:1. The derivatives were separated on a CP-Sil 8 CB capillary column (30 m, 0.32 mm ID; coating, 0.25 μm) (ChromPack; Varian, Palo Alto, CA) using the programmed-temperature vaporization injection mode of a 1-μl sample. The tert-butyldimethylsilyl (t-BDMS) derivatives of the amino acids were analyzed on a triple-stage quadrupole-mass spectrometer (TSQ Quantum; Thermo Scientific, San Jose, CA) operated in electron ionization mode. The settings for the mass spectrometer were positive polarity, profile mode, scan range of 233.00–244.00 m/z, neutral loss mass of 56.00 m/z, scan time of 0.1 s, 10 V collision energy, and collision gas (Ar) pressure of 1.0 mTorr. Data processing, including peak areas and the TTR enrichment values, was carried out by MassRatio 2.72 (FBJ Engineering).
Plasma-free α-ketoisocaproic acid (KIC) purification was performed by following the previously described procedures (18), which were derivatized using pyridine and bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (Regis Technologies, Morton Grove, IL) and determining the [M + 2]/M ratio, corresponding to the ketoacid of the tracer [1,2-13C2]leucine of the fragment ion with m/z = 232 by analyzing the sample on gas chromatography-mass spectrometry (Thermo Quest Finnigan, Paris, France).
Muscle specimens of 10–20 mg wet wt were homogenized (Fastprep, 120A-230; Thermo Savant, Holbrook, NY) for 2 × 45 sec in 1.5 ml of ice-cold Milli-Q saline water. After a spin (4°C, 5,500 g, 10 min), the supernatant was transferred to new vials, acidified with 1.5 ml of 100% acidic acid, and poured over resin columns preconditioned with 1 ml of 50% acidic acid. The amino acids were then purified over resin columns and derivatized as their t-BDMS derivatives as described for the plasma free amino acids. The myofibrillar protein fraction was isolated from the remaining protein pellet by adding 1 ml of a homogenization buffer (0.02 M Tris, pH 7.4, 0.15 M NaCl, 2 mM EDTA, 0.5% Triton-X 100, and 0.25 M sucrose), with homogenizing at 2 × 45 s, and following the procedures reported previously (2). The analysis was performed on the GC-C-IRMS equipment (Finnigan Delta Plus, Bremen, Germany; see below).
Connective tissue protein and collagen, the most abundant protein in structural tissue, was isolated from tendon and skin samples obtained in tracer virgins by homogenizing them for 5 × 15 s in 1 ml of the homogenization buffer (Tris 0.02 M, pH 7.4, 0.15 M NaCl, 2 mM EDTA, and 0.05% Triton-X 100), leaving them for 2 h at 4°C, spinning (4°C, 1,600 g, 20 min), discarding the supernatant, and homogenizing the pellet for 1 × 15 s in a buffer (0.7 M KCl) that was then left overnight at 4°C. After a spin (4°C, 1,600 g, 20 min), the supernatant was discarded and the 1 ml of KCl buffer added again to the pellet, which was vortexed and left at 4°C for 2 h. After a spin (4°C, 1,600 g, 20 min), the pellet was washed once in 1 ml of 70% ethanol before being hydrolyzed in 6 M HCl at 110°C overnight. Hereafter, the liberated amino acids were purified over resin columns, NAP-derivatized, and analyzed on the GC-C-IRMS.
Mixed proteins were precipitated from 50 μl of plasma by adding 0.5 ml of ice-cold acetone. After incubation at 5°C for 2 h, the mixture was spun (4°C, 5,500 g, 10 min), the supernatant discarded, and the pellet washed once in 1 ml of 70% ethanol before being hydrolyzed, NAP-derivatized, and analyzed on the GC-C-IRMS.
Muscle specimens for freeze-drying weighed between 10 and 20 mg wet wt and were lyophilized under vacuum at −20°C for 3 days. Hereafter, the biopsies remained under vacuum and were kept at room temperature for 1 day before they were processed manually in a dry chamber under a microscope, allowing visualization of tissue constituents. From each specimen, visible blood, fat, and connective tissue infiltrations were dissected manually from the myofibrils. Thereafter, the manually cleaned muscle specimens were transferred to an Eppendorf tube and stored at −80°C until further preparation for measuring the enrichment of muscle free phenylalanine tracer (see description above).
The protein-bound amino acid 13C abundances were determined on the IRMS operated in the combustion mode. The NAP-derivatized constituent amino acids were isolated using gas chromatography on a CP-Sil 19 CB capillary column (60 m, 0.25 mm ID; coating 0.25 μm) (Agilent J & W) prior to combustion at 960°C. The combusted amino acid gases were analyzed for masses 44 and 45, corresponding to 12CO2 and 13CO2.
Calculations
The phenylalanine concentrations (experiment 3; Fig. 6) are reported as only the concentration of the unlabeled compound. Tracer enrichments were determined by subtracting the isotope ratio of a background sample from the isotope ratio of a labeled sample. The isotope ratios were converted to moles of tracer or trace molecule and are reported as the TTR. The abundance of 13C in phenylalanine from the IRMS analyses was converted to TTR from the δ-value by the following formula: 0.0112372 × (0.001 × δ + 1) × 100%. The FSR was calculated by using the standard equation ΔEprotein/Ēprecursor × Δtime, where ΔEprotein is the difference in tracer enrichment in the protein of interest between two different time points (interspersed by Δtime hours). Ēprecursor is the weighted average of the tracer enrichment at the site of the surrogate measurement of the real precursor pool.
The volume of distribution (VD) for the flood-primed amino acid was estimated by assuming that if the absolute amount of tracee and tracer in the flood prime was equilibrated between all watery pools in the body, the concentration measured in the circulation would be the same in all pools. Thus, VD was found by dividing the absolute amount of tracee and tracer in the flood prime by the peak concentration in plasma (15 min after the flood prime). For comparison, the body water content was calculated using the equation: BW [liters] = LBM [kg] × 0.73 (see, e.g., Ref. 35).
Statistical Calculations
When more than two sample points were reported, they were analyzed using a one-way ANOVA with repeated measurements, and when a significant overall effect was detected, post hoc Holm-Sidak tests were performed. When needed, t-tests were applied to compare only two data points. The statistical software Prism 6.0 (GraphPad) was used for all statistical tests. Data are presented either as individual data points or as means ± SE.
RESULTS
Experiment 1
By applying a conventional primed, continuous infusion protocol, the tracer enrichment in plasma increased instantaneously to 4.40 ± 0.27% at 10 min, dropped to 3.58 ± 0.17% at 30 min (P < 0.05), and rose again from 60 min up to a steady level not different from the enrichment at 240 min (1-way ANOVA with repeated measurements and post hoc test; Fig. 2A). During the same period of time, the muscle free tracer enrichment reached 1.29 ± 0.08% at 10 min (P < 0.05 compared with 240 min) and was still lower at 30 (P < 0.05) and 60 min (P < 0.10) compared with 240 min (Fig. 2A). The relative enrichments in the muscle free pool were not fully labeled during the initial hour: 65 ± 6% at 10 min (P < 0.05 vs. 240 min), 73 ± 3% at 30 min (P < 0.05 vs. 240 min), and 86 ± 5% at 60 min (P < 0.10 vs. 240 min) (see Fig. 2B). The abundance of carbon-labeled phenylalanine incorporated into myofibrillar proteins during the primed, continuous infusion is illustrated in Fig. 2C, showing that tracer was incorporated over time (P < 0.05, 1-way repeated-measures ANOVA) but that a significant increase in tracer abundance in myofibrillar proteins was detected only between 0 and 240, 10 and 240, and 30 and 240 min (P < 0.05; Fig. 2C) when the reported rate of tracer infusion was used. The myofibrillar FSRs based on various time intervals were calculated using the weighted intramuscular tracer enrichment. Poor agreement was found between 120-min interval FSRs (Fig. 2D), where better compliance was found when the incorporation time was extended (Fig. 2E).
Fig. 2. A: the conventional primed, continuous infusion protocol. Enrichments in plasma and muscle free pool. Ring-13C6-phenylalanine tracer-to-tracee ratio enrichment in venous plasma (●) and in the muscle free pool (■). A 1-way ANOVA with repeated measurements revealed that the enrichments in both plasma and the muscle free pool varied over time (P = 0.01 and P = 0.001, respectively), and the Holm-Sidak post hoc test comparing all values with the 240-min enrichment revealed significantly lower enrichments (P < 0.05) at 30 min in plasma and at 10 and 30 min in the muscle free pool (#) as well as borderline significant enrichments (†P = 0.057) at 60 min in the muscle free pool. Values are means ± SE. B: the conventional primed, continuous infusion protocol. Enrichments in the muscle free pool at 10, 30, 60, and 120 min relative to 240 min. One-way ANOVA with repeated measurements revealed a significant effect over time (P < 0.001), and the Holm-Sidak post hoc test comparing all prior values with the 240-min enrichment revealed significantly lower enrichment (P < 0.05) at 10 and 30 min (#) as well as borderline significant (†P = 0.05) at 60 min. Values are means ± SE. C: myofibrillar phenylalanine-derived carbon abundance during a primed, continuous infusion. A 1-way ANOVA revealed time effect (P < 0.01), and the subsequent Holm-Sidak post hoc tests revealed significantly improved abundances at 240 min compared with background (0 min), 10 min, and 30 min. Values are means ± SE. D and E: myofibrillar protein fractional synthesis rate (FSR) measured in vastus lateralis muscle from the primed, continuous infusion protocol using two 120-min time intervals (either a single-biopsy approach from 0 to 120 min or a 2-biopsy approach from 120 to 240 min; D) or either the single-biopsy (i.e., from 0 to 240 min) or the 2-biopsy approach over 180 min from 60 to 240 min (E). One paired measurement using the 2 protocols is represented by each ⧫. The diagonal lines are x = y.
Experiment 2
When a priming bolus is given, consisting of both tracer and tracee in the ratio that is expected to be present at steady state providing a continuous infusion, the tracer was introduced immediately into the muscle free space at a phenylalanine enrichment of 6.98 ± 0.70% at 10 min, which was similar to the mean of all measured time points (10, 30, 60, 120, and 360 min) of 6.52 ± 0.33% (P > 0.05; Fig. 3A). The venous plasma free phenylalanine enrichment was elevated by the bolus injection to 9.73 ± 0.27% at 10 min and thereafter, increasing steadily over the 6-h trial to peak at 300 min at 12.84 ± 0.25% (Fig. 3A).
Fig. 3. A: flood-primed, continuous infusion protocol. Enrichments in plasma and the muscle free pool. Ring-13C6-phenylalanine tracer-to-tracee ratio enrichment in venous plasma (●) and in the muscle free pool (■). One-way ANOVA with repeated measurements revealed that the enrichments in plasma varied over time (P < 0.0001), whereas in the muscle free pool the tracer enrichment remained unchanged (P = 0.63). The Holm-Sidak post hoc test for the plasma enrichments comparing all values with the 360-min enrichment revealed significantly lower enrichments (#P < 0.05) at all time points ≤240 min. Values are means ± SE. B: flood-primed, continuous infusion protocol. Enrichments in the muscle free pool at 10, 30, 60, and 120 min relative to 360 min. One-way ANOVA with repeated measurements revealed a steady relationship throughout the period (P = 0.57). Values are means ± SE. C: myofibrillar phenylalanine-derived carbon abundance during a flood-primed, continuous infusion. A 1-way ANOVA revealed time effect (P < 0.0001), and the subsequent Holm-Sidak post hoc tests revealed significantly improved abundances between time points with different letter symbols, which were ≥60 min. Values are means ± SE. D: plasma protein tracer enrichment during the flood-primed, continuous infusion protocol. ▲, Ring-13C6-phenylalanine tracer-to-tracee ratio. The slope of the line reflects the synthesis of the proteins, which is 6.6·10−6·min−1. When the precursor enrichment from the muscle free pool is used as seen in A, it corresponds to 0.62%/h. The intercept with the x-axis is 47 min. Values are means ± SE.
The muscle free pool of tracee was enriched instantaneously by the flood prime, and the relative enrichments compared with the enrichment at 360 min in the muscle free pool did not change throughout the 6-h flood-primed, continuous infusion protocol (P = 0.57; Fig. 3B).
The abundance of carbon-labeled phenylalanine incorporated into myofibrillar proteins during the flood-primed, continuous infusion is illustrated in Fig. 3C. The results show that tracer is incorporated continuously over time (P < 0.0001, 1-way repeated-measures ANOVA) and that the enrichment increases significantly over 60 min (0–60 min, P < 0.05, and 60–120 min, P < 0.05).
In an attempt to verify that the flood prime was superior in introducing the tracer into the intracellular pools enriching the real precursor pools, we measured the enrichments in mixed plasma proteins from blood samples obtained throughout the infusion protocol (Fig. 3D). These results demonstrated a linear incorporation of tracer into the plasma proteins, corresponding to an hourly rate of 0.62%. Furthermore, the estimated total time for complete synthesis of a protein and subsequent excretion and appearance in the circulation was 47 min (the time when the enrichment was still zero).
In experiment 2, we also determined the FSR in isolated myofibrillar proteins over either the initial 120-min period, the 120- to 360-min period, or the entire 360-min period using the flood-primed, continuous infusion approach. The results reveal that the myofibrillar FSR was of similar magnitude irrespective of what period was used for determination (P = 0.33; Fig. 4).
Fig. 4.Vastus lateralis myofibrillar protein FSR of each individual (⧫) measured over each of the 3 time intervals: either the initial 120-min or the whole 360-min period of the flood-primed, continuous infusion period (denoted 0–120 min and 0–360, respectively) or using only the 4-h time interval starting 120 min after the flood prime (120–360 min), allowing time-to-tracer equilibration between free pools, as when applying the conventional primed, continuous infusion protocol. Paired FSR measurements are connected by a black line. A 1-way ANOVA with repeated measures revealed that P = 0.33.
Experiment 3
Leucine tracer infusion resulted in a steady state of [13C]α-KIC enrichment of ∼5% in the venous site (Fig. 5A), and the fasting myofibrillar FSR was 0.079 ± 0.006%/h and was not altered significantly when performed after a phenylalanine flood-primed, continuous infusion (0.068 ± 0.009%/h, P = 0.23; Fig. 5B).
Fig. 5. A: experiment 3 venous plasma α-ketoisocaproic acid 13C enrichment. One-way ANOVA with repeated measurements revealed a highly significant effect; P < 0.0001. *Post hoc Holm-Sidak tests comparing all plasma enrichment values revealed significantly different enrichments (P < 0.05) at time points 300 to 480 compared with 120 min. Values are means ± SE. B: vastus lateralis myofibrillar protein FSR of each individual (⧫) measured in the basal state and over the initial 3 h after the flood-primed, continuous infusion of phenylalanine was given. A paired t-test revealed no difference in myofibrillar FSR (P = 0.23).
The basal, overnight-fasted-state plasma phenylalanine concentration was 53.1 ± 2.3 μM, which was increased to 254.7 ± 37.2 μM 15 min after the phenylalanine flood priming (Fig. 6). Hereafter, the concentration decreased over time but remained elevated partly because of the continuous infusion of phenylalanine. The plasma enrichment of [15N]phenylalanine, which was only added to the flood prime and thus revealed the fate of the amino acid's origination from this, was diluted slightly by the basal abundance of phenylalanine and was measured to 4.83 ± 0.30% 15 min after the flood prime (Fig. 6). The labeled phenylalanine disappeared over time from the circulation but was still present after 3 h (0.85 ± 0.07%; Fig. 6).
Fig. 6.Plasma phenylalanine concentration (Conc) and disappearance for 3 h after a flood prime. The addition of [15N]phenylalanine tracer to the flood prime bolus allowed the tracing of the disappearance of the phenylalanine in the flood prime bolus. Subsequent to the flood prime, a continuous infusion of unlabeled phenylalanine [8 μmol (1.32 mg)·kg lean body mass (LBM)−1·h−1] was started to mimic the approach described in experiment 2. Values are means ± SE. Enrich, enrichment.
The volume of distribution was estimated to be 42.5 ± 5.1 liters, which was similar to the calculated body water content, which was 41.5 ± 1.7 liters (P = 0.80; Table 1).
| VD and Body Water Content | Means ± SE |
|---|---|
| LBM, kg | 56.8 ± 2.3 |
| Water pool, liters | 39.8 ± 1.6 |
| Peak phenylalanine concentration, μmol/l | 255 ± 37 |
| VD, liters | 37.9 ± 4.5 |
Practicalities Required for Application of the Single-Biopsy Approach
In six tracer naïve individuals, background phenylalanine and proline 13C abundances were similar, with the only exception being a difference in [13C]phenylalanine between plasma proteins and skin-derived connective tissue proteins (Table 2).
| Phenylalanine δ-Value, Per Million (106) vs. PDB Limestone | Proline δ-Value, Per Million (106) vs. PDB Limestone | |||||||
|---|---|---|---|---|---|---|---|---|
| Subjects | Plasma protein | Skin | Patella tendon | Myofibrillar protein | Plasma protein | Skin | Patella tendon | Myofibrillar protein |
| Subject 1 | −35.750 | −35.474 | −35.167 | −35.961 | −35.262 | −35.278 | −36.834 | −35.820 |
| Subject 2 | −35.972 | −35.632 | −35.343 | −35.601 | −35.265 | −35.698 | −38.820 | −35.907 |
| Subject 3 | −35.787 | −35.487 | −36.325 | −35.881 | −35.896 | −35.723 | −35.586 | −36.069 |
| Subject 4 | −36.051 | −35.687 | −36.117 | −35.662 | −35.583 | −36.027 | −34.993 | −35.639 |
| Subject 5 | −35.682 | −35.220 | −37.091 | −35.701 | −35.382 | −35.863 | −35.864 | −35.176 |
| Subject 6 | −35.862 | −35.567 | −35.807 | −35.588 | −34.982 | −35.342 | −35.283 | −35.390 |
| Means ± SD | −35.851 ± 0.140 | −35.511 ± 0.165 | −35.975 ± 0.703 | −35.732 ± 0.154 | −35.395 ± 0.314 | −35.655 ± 0.293 | −36.230 ± 1.418 | −35.667 ± 0.335 |
| %CV | 0.39 | 0.46 | 1.95 | 0.43 | 0.89 | 0.82 | 3.91 | 0.94 |
Seven randomly selected raw muscle specimens were cleaned either by a wash in ice-cold saline wet gaze before freezing or by manual dissection after being freeze-dried prior to isolation of the free phenylalanine by standard procedures. The muscle free phenylalanine tracer enrichment was markedly lower than the plasma free enrichment at the same time point (1-way ANOVA, P < 0.0001). No difference was apparent in the muscle free phenylalanine tracer enrichment between cleaning procedures (post hoc tests, P > 0.05 and t-test, P = 0.23; Table 3).
| Enrichment TTR, % | Ratio to Plasma, % | |
|---|---|---|
| Plasma | 12.0 ± 0.5 | |
| Muscle specimen | ||
| Cleaned | 8.5 ± 0.7 | 70 ± 4 |
| Saline | 8.1 ± 1.0 | 67 ± 6 |
DISCUSSION
An important finding in the present study is that the lack of immediate labeling of the free amino acid pool in skeletal muscle when using the conventional primed, continuous protocol can be avoided by priming with a larger tracer/tracee bolus, i.e., the so called flood prime. Using the flood prime, the tracer is very quickly introduced into the tissue free pools and becomes available for the protein synthetic machinery. When prepared accordingly, steady-state enrichment can then be reached almost instantaneously and subsequently maintained with a continuous infusion, thereby securing one of the essential assumptions for applying the single-biopsy approach to measure the tissue protein fractional synthesis rate over a prolonged period of time with use of the continuous infusion approach. Furthermore, the required assessment of the tracer background abundance for either carbon-labeled phenylalanine or proline amino acids can, in tracer naïve individuals, be determined to be equally good in proteins from either muscle, tendon, skin, or blood for the measurement of protein synthesis in muscle and tendon tissues.
Immediate Introduction of Tracer Into the Intracellular Pools
Plasma protein tracer incorporation.
As an alternative way of determining whether the tracer was introduced in the intracellular pools immediately, we measured the increase in tracer abundance in a pool of fast-turning-over plasma proteins synthesized mainly in the liver (Fig. 3D). The line best describing the increase in enrichment in mixed plasma proteins was found to cross the x-axis at 47 min, which corresponds to the time for synthesizing the protein to secretion in to the circulation. Fu et al. (10) applied the primed, continuous infusion protocol and reported longer periods before they saw labeled protein appearing in the circulation (∼78 min for albumin, ∼50% of plasma proteins, and 112 min for fibrinogen, <10% of plasma proteins). In our mixed plasma proteins, the immunoglobulins (making up one-third of plasma proteins) are also present but are lacking in the report by Fu et al. (10). Having the slowest turnover rate of the three most abundant plasma proteins (10), it is unlikely that the immunoglobulins or other minor relative differences in plasma protein abundances can explain the twofold time difference, meaning that the time to excretion of labeled proteins is shortened by using the flood prime compared with the conventional prime protocol. This implies that when the flood prime is used, the tracer is more quickly introduced into the hepatic intracellular pools, becoming available for the synthetic apparatus.
Volume of distribution.
In addition, we assumed that the concentration measured in the circulation shortly after the flood prime would be present in all watery pools in the body. Knowing the absolute amount of amino acid in the flood prime, we calculated the VD that this amino acid amount had to be mixed in to reach the peak concentration measured at 15 min in the plasma (Fig. 6). In the present study, although the sample size was limited, we found that the VD was equal to the water content estimated from the lean body mass (Table 1). Thus, also by this comparison, we can conclude that the flooded amino acid is presumably distributed quickly and evenly in all watery pools in the body, which is in line with the conclusions in previous reports on flooding with amino acids (6, 22, 23).
Practicalities With the Use of the Flood-Primed, Continuous Infusion Approach Obtaining Only One Tissue Biopsy
Stimulation of myofibrillar FSR.
Although some amino acids are indeed themselves very potent stimulators of tissue protein synthesis and thus not wise choices for flooding tracers (e.g., leucine) (13, 27–29), other amino acids do not have this ability (29). Flooding with 1.5 g of phenylalanine in the present study did not stimulate myofibrillar protein FSR (Fig. 5B). That phenylalanine is not capable of stimulating muscle protein synthesis is in agreement with Caso et al. (7) but somewhat in contrast with findings by Smith et al. (29). However, both of these studies used much larger doses, 0.043 and 0.050 g phenylalanine/kg body wt, respectively, than we did (0.020 ± 0.002 g phenylalanine/kg body wt). Although contrasting results exist, it is obvious that the approximately threefold higher peak phenylalanine concentration reported after flooding with 0.05 g/kg (29) than in our protocol (Fig. 6) may have had an impact on the synthetic processes. The conflicting results in the literature, when using a larger dose, together with the absence of a stimulatory effect in the present study suggest that using a relatively small dose of phenylalanine does not in itself stimulate myofibrillar protein synthesis.
With the purpose to evaluate the reproducibility of the measured FSR values, we calculated the myofibrillar FSR in various time intervals throughout the 6-h flood-primed, continuous infusion protocol and found similar values whether these were based on measurements obtained in the initial (0–120 min) period, the prolonged period (0–360 min), or the late (120–360 min) period (Fig. 4). These results are clear, unequivocal, and as a protocol an improvement compared with the primed, continuous infusion protocol, albeit with lower infusion rates but in agreement with the literature (3, 5, 26), giving rather variable values of FSR in present study (Fig. 2, D and E).
Even background enrichments in tracer naïve individuals.
A convenient aspect of the applicability of the flood-primed, continuous infusion approach is that only one tissue biopsy is required, and this should be taken at the end of the tracer infusion to calculate the incorporation rate of tracer. Because of the natural abundances of all stable isotopes, the incorporation rate can only be validly calculated when the background abundance is known (26). Previously, it was shown that carbon 13 in leucine has the same natural abundance when originating from either plasma or muscle proteins (17). We extended this comparison to the amino acids phenylalanine and proline, both of which are expected to be more suitable as flood prime tracers, and more proteins/tissues (muscle, tendon, skin, and plasma protein). For proline, we found neither any significant difference between 13C abundances in different tissues nor any differences between individuals across tissues (Table 2). For phenylalanine, a small difference in 13C abundance was present when derived from plasma protein and skin connective tissue proteins (Table 2). The practical significance of this small difference is limited, as it will be relevant only in the case where skin protein synthesis rate is to be determined with the use of plasma proteins as a background measurement of [13C]phenylalanine abundance. The inclusion of skin and plasma proteins in the present comparison was done because both protein sources, being readily accessible, are used normally as background measurements for another tissue (e.g., either tendon or muscle). Thus, the use of an alternative background protein for skin protein synthesis measurements seems not to be relevant.
It should be emphasized that these samples were obtained in tracer naïve individuals (i.e., not exposed to this label before). If the used subjects have been exposed to a similar tracer before, it should be tested to see whether the background abundance is similar.
Sufficient Incorporation Time
To determine the least time necessary for detecting a significant increase in tracer abundance in myofibrillar proteins when applying the primed, continuous tracer infusion in the overnight-fasted and resting state, we applied various statistical evaluations. When using paired t-tests, borderline incorporations were found over 120 min (0–120 min, P = 0.068, and 120–240 min, P = 0.069), resulting in major variation in FSR measurements (Fig. 2D), whereas significant differences in tracer abundance appeared when incorporation was allowed for 180 min (from 60 to 240 min, P = 0.038), resulting in better compliance with FSR values (Fig. 2E). These data suggest that in the rested, fasted state, somewhere between 120 and 180 min is an absolute minimum time for incorporation between adjacent biopsies to detect differences in tracer abundance in myofibrillar proteins when the primed (2 μmol/kg LBM), continuous (3 μmol·kg LBM−1·h−1) tracer infusion rates are used. A theoretical estimation can also be made by making the following settings: FSR in the basal state = 0.05%/h; precursor enrichment = 2%; NAP-derivatized ring-13C6-phenylalanine; and an standard deviation on repeated analyses on the GC-C-IRMS equipment of 0.5 δ, meaning that differently labeled samples have to diverge with ≥1 δ to allow that a difference is measured in >95% of the analyses. We estimate that 160 min is the least time required for incorporation between adjacent biopsies. In the case of the flood-primed, continuous infusion using a continuous tracer infusion rate of 8 μmol·kg LBM−1·h−1 (2.7-fold higher rate than in experiment 1), our measurements revealed that >50 min was required between biopsies to obtain a detectable difference in tracer abundance. Thus, good agreement exists between the least necessary incorporation time periods based on the infusion rates by comparing experiments 1 and 2: >50 min × 2.7-fold higher infusion rate = ≥135 min (range 120–180 min). This emphasizes that 1) the infusion protocol settings are decisive for the necessary incorporation time periods and, 2) if any intervention that will increase the FSR over basal level is applied, the least necessary time will shorten. Hence, intervention type and infusion protocol settings should be taken into account when designing the biopsy time points.
Review of State of the Art
The priming dose was not expected to label all tracee pools instantaneously as the flooding dose, but it was expected to introduce the tracer into the circulation and from there equilibrate with other compartments over a subsequent period of time. Using the primed, continuous tracer infusion protocol, Wolfe and Chinkes (36) recommended, based on a long career of experiences and experiments, at least 60 min before the first (of 2 or more) biopsies were obtained. Volpi et al. (31) demonstrated that after 120 min a linear tracer incorporation was present using the primed, continuous infusion protocol. Hence, time must elapse to allow amino acids solely infused into only one pool in the body to equilibrate fully between pools, as the free amino acids do not make up a single pool in the living organism (1, 8, 19, 30). However, several studies placing the biopsy within the first hour or even before the tracer infusion start have been published (see Ref. 26). Recent work published by Burd and colleagues (3–5) even concludes that the FSR can be determined validly using a primed, continuous infusion protocol by taking only one biopsy as long as >210 min of total infusion time is allowed. However, no matter what the FSR values end up being, direct violations of the experimental requirements securing compliance with the underlying assumptions just increase variation and uncertainty of the measurements and should be avoided. Acknowledging that research questions asked nowadays are often more diverse, and searching for smaller differences than previously, which challenges trial protocols as well as analytical equipment, we encourage researchers to be cautious with too-short tracer infusion protocols and violations against underlying assumptions.
Applicability
In agreement with the literature investigating the ability of the flooding approach to label the various precursor pools and measure muscle protein synthesis (6, 13, 22), we report here that when mixing ∼1.5 g of phenylalanine enriched by ∼12%, we obtained an immediate enrichment in the muscle free pool of ∼7% and at the venous side of ∼10% (Fig. 3A). When the enrichment in the flood prime is adjusted to the infusion rate in a subsequent continuous infusion, the enrichments in the body water pool can be kept constant as long as the infusion runs. Thus, the flood-primed, continuous infusion protocol fulfills the requirements for a tracer infusion design by which the FSR of slow-turning-over proteins can be measured when only a single biopsy can be obtained. In addition, the flood-primed, continuous infusion protocol reduces the suffering from the patients/subjects, which by itself is an ethically wishful aim. The limitations are that, exactly because of the single-biopsy requirement, the protocol can presumably be applied only in tracer naïve persons, as any enriched background tracer abundance cannot be measured from the protein of interest. Furthermore, in patients with, e.g., sepsis or burns, where the turnover rate of amino acids is markedly different from that of healthy individuals, a certain infusion rate of tracer will result in very distinct steady-state enrichments. Thus, in such cases it is hard to mix the flood prime with the target enrichment, and thus the protocol may not be suitable for experiments in such patients.
Conclusion
We would recommend that when only one tissue biopsy can be obtained, either the flooding approach or the flood-primed, continuous infusion approach should be used instead of the primed, continuous infusion. When research questions demand a determination of a protein's synthesis rate over a short period of time, the flooding approach may be advantageous, as we with the present tracer infusion rate showed that comparable FSR values required at least a 1-h incorporation period after the priming. Beyond 90–120 min of trial time, we would prefer to use the flood-primed, continuous infusion protocol over the flooding protocol, as we claim that it copes better with the assumptions underlying the FSR approach. Also, if the protein of interest has a very slow turnover rate, the advantage of the continuous infusion protocol is that it can be extended for a long period, allowing more incorporation of tracer and thus going beyond the limit of detection for tracer incorporation.
GRANTS
This work was supported by the
DISCLOSURES
The authors declare no conflicts of interest, and there are no financial conflicts to disclose.
AUTHOR CONTRIBUTIONS
L.H. and M.K. conception and design of research; L.H., S.R., K.D., R.H.N., and J.B. performed experiments; L.H. and S.R. analyzed data; L.H., S.R., K.D., and M.K. interpreted results of experiments; L.H. prepared figures; L.H. drafted manuscript; L.H., S.R., K.D., R.H.N., J.B., and M.K. edited and revised manuscript; L.H., S.R., K.D., R.H.N., J.B., and M.K. approved final version of manuscript.
ACKNOWLEDGMENTS
A special thank you to Dr. Andreas Bornø, Clinical Metabolomics Core Facility, Rigshospitalet, Copenhagen, Denmark, for assistance with the mass spectrometry analyses and to Dr. Katja M. Heinemeier, Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark, for assistance with tendon specimen collection.
REFERENCES
- 1. . Precursor pools of protein synthesis: a stable isotope study in a swine model. Am J Physiol Endocrinol Metab 267: E203–E209, 1994.
Link | ISI | Google Scholar - 2. . The anabolic potential of dietary protein intake on skeletal muscle is prolonged by prior light-load exercise. Clin Nutr 32: 236–244, 2013.
Crossref | PubMed | ISI | Google Scholar - 3. . The reliability of using the single-biopsy approach to assess basal muscle protein synthesis rates in vivo in humans. Metabolism 61: 931–936, 2012.
Crossref | PubMed | ISI | Google Scholar - 4. . The single biopsy approach is reliable for the measurement of muscle protein synthesis rates in vivo in older men. J Appl Physiol 113: 896–902, 2012.
Link | ISI | Google Scholar - 5. . Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans. Nutr Metab (Lond) 8: 15, 2011.
Crossref | PubMed | Google Scholar - 6. . Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis. Am J Physiol Endocrinol Metab 282: E1029–E1038, 2002.
Link | ISI | Google Scholar - 7. . The increase in human muscle protein synthesis induced by food intake is similar when assessed with the constant infusion and flooding techniques. J Nutr 136: 1504–1510, 2006.
Crossref | PubMed | ISI | Google Scholar - 8. . Compartmental model of leucine kinetics in humans. Am J Physiol Endocrinol Metab 261: E539–E550, 1991.
Link | ISI | Google Scholar - 9. . The distribution pattern of sulfur-labeled methionine in the protein and the free amino acid fraction of tissues after intravenous administration. J Biol Chem 173: 355–361, 1948.
Crossref | PubMed | ISI | Google Scholar - 10. . Sequential purification of human apolipoprotein B-100, albumin, and fibrinogen by immunoaffinity chromatography for measurement of protein synthesis. Anal Biochem 247: 228–236, 1997.
Crossref | PubMed | ISI | Google Scholar - 11. . An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid. Biochem J 176: 402–405, 1978.
Crossref | PubMed | ISI | Google Scholar - 12. . The diurnal response of muscle and liver protein synthesis in vivo in meal-fed rats. Biochem J 136: 935–945, 1973.
Crossref | PubMed | ISI | Google Scholar - 13. . Measurement of the rate of protein synthesis in muscle of postabsorptive young men by injection of a 'flooding dose' of [1-13C]leucine. Clin Sci (Lond) 77: 329–336, 1989.
Crossref | PubMed | ISI | Google Scholar - 14. . The in vivo measurement of protein synthesis. Am J Clin Nutr 30: 1353–1354, 1977.
Crossref | PubMed | ISI | Google Scholar - 15. . Total protein synthesis in elderly people: a comparison of results with [15N]glycine and [14C]leucine. Clin Sci Mol Med 53: 277–288, 1977.
PubMed | Google Scholar - 16. . Measurement of muscle protein synthetic rate from serial muscle biopsies and total body protein turnover in man by continuous intravenous infusion of l-(alpha-15N)lysine. Clin Sci Mol Med 49: 581–590, 1975.
PubMed | Google Scholar - 17. . Baseline measurements for stable isotope studies: an alternative to biopsy. Biomed Environ Mass Spectrom 19: 176–178, 1990.
Crossref | PubMed | Google Scholar - 18. . Contraction intensity and feeding affect collagen and myofibrillar protein synthesis rates differently in human skeletal muscle. Am J Physiol Endocrinol Metab 298: E257–E269, 2010.
Link | ISI | Google Scholar - 19. . Functional heterogeneity of leucine pools in human skeletal muscle. Am J Physiol Endocrinol Metab 273: E564–E570, 1997.
Link | ISI | Google Scholar - 20. . Linear kinetic model to estimate protein synthesis rate after [14C]tyrosine infusion in dogs. FEBS Lett 79: 313–316, 1977.
Crossref | PubMed | ISI | Google Scholar - 21. . Measurement of leucine metabolism in man from a primed, continuous infusion of l-[1-13C]leucine. Am J Physiol Endocrinol Metab 238: E473–E479, 1980.
Link | ISI | Google Scholar - 22. . Measurement of protein synthesis in human skeletal muscle: further investigation of the flooding technique. Clin Sci (Lond) 81: 557–564, 1991.
Crossref | PubMed | ISI | Google Scholar - 23. . Response of protein synthesis in human skeletal muscle to insulin: an investigation with l-[2H5]phenylalanine. Am J Physiol Endocrinol Metab 267: E102–E108, 1994.
Link | ISI | Google Scholar - 24. . The effect of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem J 178: 373–379, 1979.
Crossref | PubMed | ISI | Google Scholar - 25. . Muscle protein synthesis measured by stable isotope techniques in man: the effects of feeding and fasting. Clin Sci (Lond) 63: 519–523, 1982.
Crossref | PubMed | ISI | Google Scholar - 26. . Timing of the initial muscle biopsy does not affect the measured muscle protein fractional synthesis rate during basal, postabsorptive conditions. J Appl Physiol 108: 363–368, 2010.
Link | ISI | Google Scholar - 27. . Flooding with l-[1-13C]leucine stimulates human muscle protein incorporation of continuously infused l-[1-13C]valine. Am J Physiol Endocrinol Metab 262: E372–E376, 1992.
Link | ISI | Google Scholar - 28. . Effect of a flooding dose of leucine in stimulating incorporation of constantly infused valine into albumin. Am J Physiol Endocrinol Metab 266: E640–E644, 1994.
Link | ISI | Google Scholar - 29. . Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein. Am J Physiol Endocrinol Metab 275: E73–E78, 1998.
Link | ISI | Google Scholar - 30. . Model to assess muscle protein turnover: domain of validity using amino acyl-tRNA vs. surrogate measures of precursor pool. Am J Physiol Endocrinol Metab 285: E1142–E1149, 2003.
Link | ISI | Google Scholar - 31. . Sequential muscle biopsies during a 6-h tracer infusion do not affect human mixed muscle protein synthesis and muscle phenylalanine kinetics. Am J Physiol Endocrinol Metab 295: E959–E963, 2008.
Link | ISI | Google Scholar - 32. . Tracers to investigate protein and amino acid metabolism in human subjects. Proc Nutr Soc 58: 987–1000, 1999.
Crossref | PubMed | ISI | Google Scholar - 33. . Adaptation of the rat to a low-protein diet: the effect of a reduced protein intake on the pattern of incorporation of l-[14C] lysine. Br J Nutr 20: 461–484, 1966.
Crossref | PubMed | ISI | Google Scholar - 34. . The effect of low protein diets on the turn-over rates of serums, liver and muscle proteins in the rat, measured by continuous infusion of l-[14C]lysine. Clin Sci 35: 287–305, 1968.
PubMed | ISI | Google Scholar - 35. . Total body water volumes for adult males and females estimated from simple anthropometric measurements. Am J Clin Nutr 33: 27–39, 1980.
Crossref | PubMed | ISI | Google Scholar - 36. . Isotope Tracers in Metabolic Research. Principles and Practice of Kinetic Analysis . Hoboken, NJ: Wiley-Liss, 2005.
Google Scholar
Source: https://journals.physiology.org/doi/full/10.1152/ajpendo.00084.2014
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