The application of 2H2O to measure skeletal muscle protein synthesis
© Gasier et al; licensee BioMed Central Ltd. 2010
Received: 26 January 2010
Accepted: 21 April 2010
Published: 21 April 2010
Skeletal muscle protein synthesis has generally been determined by the precursor:product labeling approach using labeled amino acids (e.g., [13C]leucine or [13C]-, [15N]-, or [2H]phenylalanine) as the tracers. Although reliable for determining rates of protein synthesis, this methodological approach requires experiments to be conducted in a controlled environment, and as a result, has limited our understanding of muscle protein renewal under free-living conditions over extended periods of time (i.e., integrative/cumulative assessments). An alternative tracer, 2H2O, has been successfully used to measure rates of muscle protein synthesis in mice, rats, fish and humans. Moreover, perturbations such as feeding and exercise have been included in these measurements without exclusion of common environmental and biological factors. In this review, we discuss the principle behind using 2H2O to measure muscle protein synthesis and highlight recent investigations that have examined the effects of feeding and exercise. The framework provided in this review should assist muscle biologists in designing experiments that advance our understanding of conditions in which anabolism is altered (e.g., exercise, feeding, growth, debilitating and metabolic pathologies).
Skeletal muscle comprises ~40-45% of the total mass and ~60% of the total body protein in humans ; thus, it is no surprise that skeletal muscle accounts for ~30-45% (dependent upon feeding and/or activity) of whole body protein metabolism . As a result of the high metabolic activity of skeletal muscle, research has centered on determining its role in the pathogenesis of many debilitating and chronic diseases (e.g., AIDS, cancer, sarcopenia, starvation, trauma and sepsis and several metabolic disorders). This is accomplished by examining changes that occur in gene expression, transcription factors and signal transduction pathways; however, the structure and function of skeletal muscle may largely be dependent on the renewal of protein. Thus, direct measurement of muscle protein synthesis can provide relevant information regarding the metabolic state of the tissue, and therefore, should be included as an outcome variable when examining anabolic/catabolic conditions.
Direct incorporation methods using tracers such as [13C]leucine or [13C]-, [15N]-, or [2H]phenylalnine have traditionally been used to measure tissue specific protein synthesis and are generally accepted for providing reliable measurements. The principal is that by providing a known amount of labeled (tracer) and unlabeled amino acid (tracee) the tracer and tracee will mix with the endogenous pool and become incorporated into protein over time. The tracer and tracee are generally administered as a primed-constant infusion (a priming bolus of tracer and tracee is administered and is continuously provided at a lower concentration to maintain enrichment), or as a flooding dose (a supraphysiological bolus of tracer and tracee is provided over seconds to minutes). Protein synthesis can be determined by measuring the enrichment (tracer/tracee) of the protein against the enrichment of the precursor (i.e., precursor:product labeling ratios). However, two central concerns that have existed with the precursor-product method have been identification and measurement of the true precursor (aminoacyl-tRNA), and the inability to conduct the tracer experiments in a free-living environment over an extended period of time, an integrative/cumulative assessment.
To circumvent the first concern in regards to isolating and measuring the aminoacyl-tRNA (a small pool with a rapid turnover), investigators have used surrogates (i.e., plasma and/or protein-free compartments), or implemented the flooding dose approach. Researchers that have directly compared the labeling ratios of the surrogates to the tRNA-bound amino acid (e.g., leucyl or phenylalanyl) have reported inconsistent findings as to which surrogate most accurately reflects the aminoacyl-tRNA [3–6]. In addition, although the flooding dose  can minimize labeling gradients by saturating the uptake of the amino acid by a tissue(s), questions such as whether muscle protein synthesis is stimulated when flooding with specific amino acid tracers [8, 9] exists when conducting the experiment in the fasted state. Therefore, researchers should consider the potential consequences when using various tracers with different approaches (primed-constant infusion vs. flooding dose) and interpret the data accordingly (i.e., qualitative comparisons).
The second concern, a controlled setting and short duration tracer infusion protocols, have not allowed for tracer experiments to be conducted under free-living conditions over extended periods of time. More specifically, the experiments require a controlled laboratory environment for the placement of cannulas to infuse the tracers and extract blood, and to insure that steady state enrichments are maintained over the course of the study. In addition, tracer studies are normally conducted following an over-night fast, and if nutrition is provided during the study additional tracer is needed to minimize dilution of the precursor. Although controlled environments are necessary to determine if a specific independent variable(s) influences muscle protein synthesis, one must consider the likelihood that the data will reflect a subject's unnatural behavior.
An alternative, but less familiar approach that can bypass these potential limitations is the use of deuterium oxide (2H2O), which was first described by Hans Ussing in 1941 . Ussing discovered that by providing 2H2O to mice and rats, one could determine a rate of protein renewal by the incorporation of 2H atoms (i.e., as 2H-labeled amino acids) into protein; including liver, kidney, skeletal muscle, and specific proteins such as myosin. He concluded that if 2H2O was provided to an animal and the concentration was maintained then the 2H would accumulate into protein over time allowing for the determination of protein synthesis rates. Ussing thought that this occurred by the transfer of 2H with the H of amino acids through transamination reactions and similar processes, which would then be incorporated into protein . Additionally, he suggested that the measurement of this process would be easiest if the exchange between 2H with amino acids occurred very rapidly so that their 2H content would not change throughout the incorporation process as long as a consistent 2H2O concentration was maintained . Interestingly, follow-up studies were not published, perhaps due to the difficulty in obtaining deuterium oxide during this time period.
In this review, we will discuss the application of 2H2O to measure skeletal muscle protein synthesis with known perturbations of interest to the field of muscle biology, feeding and exercise. Emphasis will be placed on the methods to aid researcher's in future experimental design. Although the focus of this review is skeletal muscle protein, the information contained within can be extended to study specific proteins within muscle (e.g., cytosolic, membrane, mitochondria and myofibrillar) or other tissues of interest. Furthermore, the application of 2H2O can be extended to investigate lipid  and glucose kinetics , cell proliferation , and total energy expenditure (when combined with 18O) . Finally, it is not our intention to undermine the numerous investigations that have, and currently employ protocols using labeled amino acids as tracers, as it is our opinion that the experimental question should determine what tracer method to use.
Mechanism of action
The use of 2H2O is similar to the use of other precursor:product labeling schemes, a labeled tracer is provided and its incorporation in the product is determined. In the case of 2H2O, the administered precursor (water) labeling is M+2 yet one measures M+1 in the product. Namely, the di-deutero labeled water that is given rapidly dissociates to yield mono-isotopically labeled water (2HHO), thus the equilibration of 2H-label with various amino acids (e.g., alanine) leads to the generation of singly-labeled species.
2H2O has been administered to small animals (mice and rats) by an intraperitoneal injection (~10-20 μl·g body mass-1 with ~99.9% 2H2O) [15–17], orally to humans (~5 ml·kg body mass-1 with 70% 2H2O) , and to fish simply by addition to the aquarium water (~2.0 to 4.0% 2H2O) . The goal is to rapidly obtain and then maintain a certain level of 2H-labeling in body water (e.g., ~2.0 mole percent excess, MPE, or greater in rodents and fish, and ~0.5% in humans), which allows a steady-state incorporation into end-products. Higher enrichments can be achieved in humans (e.g., ~2.0%), however, a slower increase in 2H-labeled body water (no more than 0.5% increase per day) should be considered to minimize the chances of experiencing nausea and vertigo [20, 21]. If the purpose of the experiment is to measure synthesis rates over days to weeks, then providing maintenance 2H2O is recommended and can easily be accomplished by adding the isotope to drinking water . Although 2H-labeling can be measured by a variety of methods (e.g., isotope ratio mass spectrometry  and nuclear magnetic resonance ), we have mainly focused on gas chromatography-mass spectrometry (GC-MS) methods herein since our lab is specialized in those techniques (discussed in "Measurement").
Another important point that needs to be addressed is the lack of influence that feeding has on 2H-labeling gradients. For instance, Dufner et al.  provided a supraphysiological bolus of unlabeled alanine (2 g·kg body mass-1) to rats following 90 min of 2H2O administration and measured the 2H-labeling of body water, and the α-hydrogen and total hydrogen's of alanine. Although the bolus of unlabeled material affected the plasma concentration of alanine (i.e., it increased from basal values of ~1.2 mM to ~5.5 mM between 120 and 135 min and remained elevated for the duration of the experiment (~2.5 mM at 240 min) the labeling of the α - and the total hydrogens equilibrated in a similar time course. In further support of this, Belleto et al.  reported that feeding did not modify the kinetics of plasma protein-free, or plasma protein-bound 2H-labeled alanine in both the portal and peripheral circulation. These data suggest that feeding does not result in dilution of the precursor and that the 2H-labeling of body water provides an accurate representation of 2H-labeled protein-free alanine and is more easily determined. Additionally, there is no reason to believe that a stressor such as exercise would influence 2H-labeling gradients and, in fact, we reported similar 2H-labeling of body water between exercised and sedentary cage control animals . Even in a disease state, such as chronic kidney failure where fluid changes occur, we did not observe any differences between the 2H-labeling of the α-hydrogen of alanine and that of body water . Taken together, experiments can be designed to address questions aimed at determining the influence of perturbations (e.g., feeding and exercise) without concerns of precursor fluctuations.
There may be scenarios where the use of 2H2O may not be the most appropriate tracer. One specifically is when the hypothesis is aimed at determining the influence of an independent variable(s) on the rate of protein synthesis over a very short period of time (e.g., minutes). In this case, the flooding dose is likely more suitable. More specifically, although 2H2O rapidly equilibrates with body water and labels alanine, within ~20 minutes , time is needed for 2H-alalnine to incorporate into protein, which is dependent upon the turnover rate.
An important question that investigators may consider a concern, "What affects the limit of 2H2O that can/should be administered?" requires a consideration of physiological and analytical factors. First, although we have typically maintained less than 3% 2H in body water in rodents, it appears that one can maintain ~5% 2H-labeling in body water for prolonged periods in rodents without any negative influences/side effects. In humans, nausea and vertigo have been reported in subjects who ingested a dose of 2H2O sufficient to acutely enrich body water to ~0.5%. These transient side effects can be avoided by instructing subject to ingest a few smaller doses over a short time period; for example, if the goal is to have a subject drink 200 ml of 2H2O, rather than give this is a single glass of water, subjects may drink 50 ml every 30 min. In regards to concerns for analytical windows, providing more tracer will obviously yield more 2H-labeling in products over a given time. At the dose we have been using (e.g., ~0.5% in humans and ~2% in rodents) there is reasonable incorporation into mixed muscle proteins. To improve the quantification of low levels of 2H-labeling in products (e.g., ~0.15% excess labeling), one can selectively integrate ion chromatograms ; an alternative is to use GC-pyrolysis-IRMS .
Another concern regarding the use of 2H2O centers on the half-life of the tracer and the ability to repeat studies. For example, the half-life of 2H in body water in healthy humans is ~7 to 10 days. Therefore, one might consider ~2 months before repeating an investigation in the same subject(s) if the goal is to allow ~5 half-lives to elapse and eliminate most of the tracer. Although we are not aware of any cross-over studies regarding protein synthesis in which subjects were studied more than at one time, we have previously demonstrated that one could measure gluconeogenesis by performing 'back-to-back' experiments in rats given 2H2O . Namely, although the half-life of water in a rat is ~3-4 days, we were able to administer a low dose of 2H2O on day 1 (e.g., to enrich the pool to ~0.05%) and then a higher dose of 2H2O on day 2 (e.g., to enrich the pool to ~0.5%). We expect that this logic would be practical to use in studies of protein synthesis, especially if one had access to a GC-pyrolysis-IRMS instrument .
Effects of feeding & exercise
Using 2H2O to study the effects of feeding and exercise on muscle protein synthesis
Determine if obesity impairs protein synthesis following nutrient ingestion
Diet-induced obesity impairs nutrient stimulated MPS
Determine the effects of acute fasting & chronic food restriction on protein synthesis
Male Wistar rats (2-3 mo old)
Acute fasting (20 h) and chronic food restriction (7 d) impaired MPS
Compare flooding dose vs. 2H2O to measure protein synthesis with acute resistance exercise
Male Sprague- Dawley rats (5-6 mo old)
4 & 24 hours
Methodologies compare qualitatively, and more muscle protein is synthesized over 24 h vs. 4 h, however, synthesis rates were higher when measured over 4 hours
Determine if chronic resistance exercise attenuates acute exercise stimulated protein synthesis
Male Sprague- Dawley rats (5-6 mo old)
Resistance exercise training alters the anabolic (MPS) response to acute exercise
Determine the effects of high-intensity resistance exercise when energy & macronutrient content controlled
Male Humans (20-24 y old)
High-intensity resistance exercise stimulated myofibrillar, but not mixed MPS
To date, there have only been two investigations that have measured the acute effects of feeding on muscle protein synthesis using 2H2O [33, 34]. In the first experiment , the purpose was to determine whether diet- induced obesity impairs tissue-specific FSR (a somewhat controversial topic) in C57BL/6 mice following 9-weeks of consuming either a high-carbohydrate or high-fat diet. In the second experiment , the rationale was to examine the effects of acute fasting (20 h) vs. chronic food restriction (7 days) on FSR in rats. In both designs, the FSR measurements were made over 5-6 h with feeding, since there is minimal concern for labeling gradients when using 2H2O, data were obtained with a certain degree of confidence. Both studies provided new insight into two feeding extremes: 1) diet induced obesity results in an attenuation in the feeding induced stimulation of muscle protein synthesis in mice, and 2) both acute fasting and chronic food restriction significantly reduce muscle protein synthesis in rats.
In regards to examining the effects of exercise (resistance based) with 2H2O, our group has conducted several experiments in rats [17, 27] and, most recent, in humans (unpublished observations). The questions that our group wanted to address using 2H2O that we could not with other isotopes alone were: 1) is a single bout of high-intensity resistance exercise anabolic in rats, 2) does chronic resistance exercise result in an anabolic "plateau", and 3) does a single session of high-intensity resistance exercise in humans stimulate muscle protein synthesis when energy and macronutrient ingestion is controlled and the experiment is conducted in the fed state. In the first experiment , we measured FSR over 25 min with L-[2,3,4,5,6-3H] phenylalanine (flooding dose) and with 2H2O over 4 and 24 h with an exhaustive session of resistance exercise. Though the conclusions drawn were not different with any of the measurements made (i.e., there was no exercise effect), we determined that measurements made over various time points reveal different values. More specifically, the FSR from the 24 h measurements were less than the 4 h measurements when expressed as %·h-1; however, a greater percentage of the pool was obviously synthesized over 24 h. As a result of these data, results from different tracer protocols should be compared "qualitatively," which was suggested by Smith et al.  following their observations of a 20% increase in FSR with [5,5,5-2H3]leucine vs. [2H5]- and [13C6]phenylalanine. Additionally, the expression of FSR over the course of a tracer experiment (e.g., % renewed over 24 h vs. %·h-1 when measured over 24 h) may better explain the metabolic state of the tissue. In our second experiment , we measured muscle protein synthesis at a time that has been determined to be anabolic in rats following resistance exercise (16 h following the previous bout ) using L-[2,3,4,5,6-3H] phenylalanine and observed no differences from the control groups; however, we observed significant elevations in FSR with 2H2O measured over 36 h. Thus, had we not employed an integrative assessment using 2H2O we would have falsely concluded that chronic resistance exercise training diminishes the anabolic effect. In our third study (unpublished observations) we assessed the effect of a high-intensity bout of resistance exercise in the fed state with energy and macronutrients controlled over 24 h in humans. Although we did not find a stimulatory effect of exercise on FSR in the mixed vastus lateralis, there was a significant effect on the myofibrillar FSR with exercise. Thus, the findings from this study emphasize that skeletal muscle protein is a heterogeneous pool, and although mixed muscle should be reflective of the myofibrillar fraction, this may not always be the case; we expect that future studies should focus on specific fractions or proteins within skeletal muscle.
The use of 2H2O is perhaps the oldest  and least invasive method for determining muscle protein synthesis, yet has not been regularly employed. There is considerable interest as to the rate at which muscle protein(s) are renewed over extended periods of time with inclusion of normal biological processes and environmental influences, and these experiments can be pursued with 2H2O. Our group's experiments have provided a starting point for determining the effects of feeding and exercise on "integrative" muscle protein synthesis, and hope that our description of the fundamental principles behind using 2H2O will assist others with its application to address questions of interest to the field.
We would like to extend our gratitude to Stephanie R. Anderson, Ilya R. Bederman, Emmanuelle Belleto, Michel Beylot, Danielle A. Dufner and Celvie L. Yuan for their work in this area that contributed the completion of this review.
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