We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of 2H2O, 2H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether 2H2O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of 2H2O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between 2H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that ∼50% of the plasma albumin that is synthesized over the course of 24 h is made within ∼5 h after eating (in rats trained to eat a complete 24-h ration of food in a single meal). Contrary to what we observed in vivo, feeding (the replenishment of cell culture medium) does influence the use of 2H2O for in vitro studies. In particular, since there can be slow equilibration of 2H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro.
- albumin turnover
- nutritional status
- stable isotopes
- gas chromatography-mass spectrometry
rates of protein synthesis can be determined using precursor-product labeling methods (34, 38). Namely, the rate of protein synthesis is determined by measuring the incorporation of a labeled amino acid into a protein. However, uncertainty regarding the identity and labeling of the true precursor often complicates the interpretation of data. Consequently, numerous approaches have been used to infer the true precursor labeling (the tRNA-bound amino acid; Ref. 32). For example, investigators have used different surrogates, e.g., infusing labeled leucine and measuring the labeling of plasma ketoisocaproate or infusing labeled glycine and measuring the labeling of urinary hippurate (34). An alternative method relies on the administration of a large bolus of a single amino acid (6), the principle being that, by flooding the system with a large quantity of an amino acid, the labeling of its plasma pool will reflect that of its intracellular pool. Although the various approaches have been applied, differing opinions have been expressed regarding the usefulness and validity of the methods (6, 27, 34, 38).
Perhaps the oldest, and least recognized, precursor-product labeling approach centers on the use of 2H2O (29). The principle is that, after the administration of 2H2O, 2H atoms distribute in body water and rapidly equilibrate with amino acids (e.g., alanine) before amino acids are incorporated into newly synthesized proteins (11, 12, 29). The 2H2O method offers several advantages over other precursor-product labeling techniques. First, provided that the labeling of a free amino acid equilibrates rapidly with body water before the amino acid is incorporated into newly synthesized protein, the precursor labeling (i.e., body water) is determined with confidence. Second, since 2H2O can be given orally, human studies are not limited to specialized research centers and surgical expertise is not necessary for studies in animals, which typically require catheterization. Third, 2H2O is relatively inexpensive and has a long half-life in body water, making it ideal for studying the synthesis of proteins with a slow rate of turnover.
We (24) recently demonstrated the usefulness of 2H2O for measuring rates of protein synthesis and degradation over a prolonged period in patients receiving hemodialysis. In our previous report (24), we measured the labeling of the α-hydrogen as well as the total hydrogens of alanine. In theory, precursor-product labeling ratios can be used to calculate protein synthesis by comparing the labeling of body water (the precursor) with that of alanine isolated from protein (the product, either the α-hydrogen or the total hydrogens, divided by the number of exchangeable atoms). The ability to use the total labeling of protein-bound alanine (as the product) offers a major advantage; i.e., since up to four hydrogens are replaced by deuterium before free alanine is incorporated into newly made protein, the sensitivity of the measurements is increased. This is particularly important in cases when protein synthesis is being studied over short intervals (e.g., after feeding) and/or when studying the turnover of proteins with long half-lives. Therefore, a primary objective of the current study was to carefully examine the equilibration of 2H in alanine in vivo and determine whether the total number of exchangeable hydrogens remains constant under changing conditions.
Although our goal was to study the regulation of protein synthesis by dietary factors in vivo, the simplicity of the 2H2O method made it attractive for studying protein synthesis in cell culture (11, 12). However, we hypothesized that the application of 2H2O in cell culture could be influenced by “feeding” (the replenishment of cell culture medium). For example, alanine found in cell culture medium, (i.e., the “food”) may slowly equilibrate with the 2H of the 2H2O that is added to medium. This is because cell culture studies often require that a relatively small number of cells be incubated in a large volume of medium, in contrast to the scenario observed in vivo where there is a small pool of alanine that undergoes a rapid turnover due to the large number of cells. Consequently, a lack of equilibration between the labeling of 2H in water and alanine could lead to an underestimation of protein synthesis. Therefore, a secondary objective of the current study was to examine the equilibration of 2H2O with alanine in cell culture. We report here on how feeding influences the use of 2H2O to measure rates of protein synthesis in vivo vs. in cells in culture.
MATERIALS AND METHODS
Chemicals and Supplies
Unless specified, all chemicals and reagents were purchased from Sigma-Aldrich. 2H2O (99.9 atom percent excess) and l-[2,3,3,3-2H]alanine (98 atom percent excess) were purchased from Cambridge Isotopes (Andover, MA). Ion exchange resins and HPLC columns were purchased from Bio-Rad (Hercules, CA). Gas chromatography-mass spectrometry supplies were purchased from Agilent Technologies (Wilmington, DE). Enzymes were purchased from Roche (Indianapolis, IN). Diets were purchased from Research Diets (New Brunswick, NJ). Cell culture medium [Dulbecco's modified Eagle's medium no. 11885–084 (DMEM)] was purchased from GIBCO (Carlsbad, CA). Anti-(4E-BP1) antibody was purchased from Bethyl Laboratories, and anti-phospho-S6 kinase[S6K1] (Thr389), anti-phospho-S6(Ser235/Ser236), and anti-phospho-S6(Ser240/Ser244) antibodies were purchased from Cell Signaling.
In Vivo Experiments
Equilibration of 2H2O between body water and free alanine.
Male Sprague-Dawley rats (360 ± 10 g, n = 20) were used to determine the equilibration of 2H2O in plasma alanine. Rats were fed normal chow (10% kcal from fat, 70% kcal from carbohydrate, and 20% kcal from protein) and fasted ∼18 h before the study. To enrich body water to ∼1.25% 2H, at time 0 rats (n = 18) were given an intraperitoneal bolus of 2H-labeled saline (10 μl/g body wt); the remaining rats (n = 2) were killed without 2H2O injection. Rats that received 2H2O were killed at 20, 40, 60, and 90 min postinjection (n = 2/time point); the remaining 10 rats were given an intraperitoneal bolus of alanine (2.0 g/kg, prepared by dissolving l-alanine in 1.25% 2H2O saline) and killed at various intervals. At the time of death rats were sedated using isoflurane, and blood was collected via cardiac puncture.
Influence of feeding on albumin synthesis.
Male Sprague-Dawley rats (376 ± 12 g, n = 40) were used to determine the influence of feeding on albumin synthesis. To ensure that a bolus of food was consumed, rats were trained for 10 days to eat their normal daily caloric requirement during a 2-h interval between 8 and 10 AM (7). At the end of the training period, rats consumed a normal ration of food (∼20 g) continuously over the 2-h feeding period. To maintain steady-state labeling of body water at ∼2.5% 2H, the night before the study all rats were given an intraperitoneal bolus of 2H-labeled saline (20 μl/g body wt) and then allowed free access to drinking water enriched to 5% 2H2O. Rats were killed at various time points after eating. To determine the influence of feeding on albumin synthesis, food was withheld from some rats on the experimental day. At the time of death, rats were sedated using isoflurane, blood was collected via cardiac puncture, and the liver was quick-frozen in liquid nitrogen.
Cell Culture Experiments
Equilibration of 2H2O between media water and free alanine.
Clone 9 cells (an epithelial cell line derived from livers of normal young male rats) were incubated in DMEM (an alanine-free culture medium). The production and equilibration of alanine labeling in the medium were measured over a 48-h period using two experimental designs. First, medium was changed at t = 0 and replaced with serum-free DMEM. Cells were then incubated for 24 h. After 24 h, 2H2O was added to achieve 10% 2H labeling, and the cells were incubated for another 24 h. Second, medium was changed at t = 0 to serum-free DMEM, 2H2O was immediately added, and cells were incubated for 48 h. Samples were collected at various time intervals, and the concentration and the labeling of free alanine in the medium was determined.
Influence of feeding on protein synthesis.
Once we determined how feeding influenced the equilibration of 2H with free alanine in the culture medium, we examined whether it was possible to accurately measure the rate of protein synthesis. Cells were incubated under four different conditions in DMEM (control, or plus 15% fetal calf serum, 50 μM anisomycin, or 5 mM azide). The interventions were chosen to stimulate (fetal calf serum) or inhibit (anisomycin or azide) protein synthesis (3, 31). At t = 0, medium was changed to serum-free DMEM, 2H2O was added to achieve 10% 2H labeling, and cell plates were randomized to one of the treatment groups described above. After a 24-h incubation, total protein was isolated and hydrolyzed in 6 N HCl at 100°C for 18 h. The 2H labeling of alanine incorporated into protein was measured.
2H labeling of water.
The 2H labeling of water was determined after exchange with acetone (35), with slight modifications. First, it was not necessary to decrease the ionization energy below 70 eV to obtain a correct basal (M + 1)/M signal ratio in acetone. Presumably the ionization effect(s) reported by Yang et al. (35) do not occur on our instrument. Second, assays were performed using 40 μl of sample or standard, 2 μl of 10 N NaOH, and 4 μl of a 5% (vol/vol) solution of acetone in acetonitrile.
Acetone was analyzed using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system. A DB17-MS capillary column (30 m × 0.25 mm × 0.25 μm) was used in all analyses. The temperature program is 60°C initial, increase by 20°C/min to 100°C, increase by 50°C/min to 220°C and hold for 1 min. The split ratio is 40:1 with a helium flow of 1 ml/min. Acetone elutes at ∼1.5 min. The mass spectrometer is operated in the electron impact mode. Selective ion monitoring of mass-to-charge ratio (m/z) 58 and 59 is performed using a dwell time of 10 ms/ion.
2H labeling of free alanine.
Plasma or cell culture medium (500 μl) was deproteinized by addition of 5 ml of methanol, and the supernatant was evaporated to dryness. The residue was dissolved in 1 ml of 0.6 N HCl and applied to an ion exchange column (hydrogen form). The column was washed with 20 ml of H2O, alanine was then eluted by washing with 20 ml of 4 N NH3OH, and the NH3OH fraction was then evaporated to dryness. (Note: in cases where the concentration of alanine was determined, a known amount of [2H4]alanine was added to the sample before processing.)
Free alanine was isolated from the liver by placing 1 g of tissue in 3 ml of 10% trichloroacetic acid (wt/vol). After homogenization, the supernatant was collected and evaporated to dryness. The residue was dissolved in 5 ml of water and applied to an ion exchange column (hydrogen form). The column was developed as described above.
Isolation of plasma albumin (18).
Plasma proteins were precipitated using 1,000 μl of 10% trichloroacetic acid (wt/vol)/200 μl plasma. The pellet was washed 3 times with 5% trichloroacetic acid (wt/vol). Albumin was extracted into 100% ethanol. The purity was verified in randomly selected samples using polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, ∼30 μg of a sample were dissolved in Laemmli buffer and then resolved on a 10% gel. The gel was first stained with Coomassie brilliant blue R-250 and then destained overnight.
Albumin was hydrolyzed for 18 h in 6 N HCl at 100°C. The hydrolysate was diluted to 0.6 N HCl, and alanine was isolated using ion-exchange chromatography (as described above). (Note: in cell culture studies, total protein was hydrolyzed for 18 h in 6 N HCl at 100°C. Alanine was then isolated as described above.)
GC-MS analyses of alanine.
To form its “methyl-8” derivative, alanine was reacted with N,N-dimethylformamide dimethyl acetal (Pierce, Rockford, IL; Ref. 28). Under electron impact ionization, this yields a molecular ion at m/z 158 (that contains all of the carbon-bound hydrogens) and a fragment ion at m/z 143 (that contains only the hydrogen bound to the α-carbon). The 2H labeling of alanine was determined using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system. A DB17-MS capillary column (30 m × 0.25 mm × 0.25 μm) was used in all analyses. The temperature program was 90°C initial, hold for 5 min, increase by 5°C/min to 130°C, increase by 40°C/min to 240°C, and hold for 5 min. The split ratio was 10:1 with a helium flow of 1 ml/min. Alanine elutes at ∼12 min. The mass spectrometer was operated in the electron impact mode. Selective ion monitoring of m/z 158, 159 and 160 (total 2H labeling of alanine) and 143 and 144 (2H labeling of the α-hydrogen of alanine) was performed using a dwell time of 10 ms/ion. Note: when [2H4]alanine is added to a sample (to determine the concentration of alanine), it is not possible to use the same sample to assay the concentration and the labeling since the [2H4]alanine yields a signal at m/z 144.
Molecular Signaling Proteins
To determine the phosphorylation state of eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1), ribosomal protein S6, and the 70-kDa S6 kinase S6K1 (3 important proteins involved in regulating the initiation of mRNA translation), 0.2 g of liver was homogenized in 7 volumes of homogenizing buffer (20 mM HEPES, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, and 50 mM β-glycerophosphate, pH 7.4) with a motor-driven homogenizer. Homogenate was centrifuged at 1,000 g for 3 min at 4°C. The resulting supernatant (200 μl) was mixed with an equal volume of SDS buffer [8 ml 0.5 M Tris base, 8 ml glycerol, 8 ml 10% SDS, 0.8 ml β-mercaptoethanol, 1.6 ml 4-bromophenacyl bromide, 5.6 ml water] and then subjected to protein immunoblot analysis, as described previously (1, 9, 20, 25, 33, 37). Diluted samples were then subjected to electrophoresis on a 12.5% polyacrylamide gel.
Phosphorylation of 4E-BP1 causes a decrease in the electrophoretic mobility of the protein on SDS-PAGE. Thus 4E-BP1 present in tissue extracts was separated into multiple electrophoretic forms during SDS-PAGE, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.
Phosphorylation of p70S6K1 at Thr389, a site whose phosphorylation is associated with maximal activation of the kinase, was measured.
Phosphorylation of ribosomal protein S6 at Ser235/Ser236/Ser240/Ser244 was assessed by protein immunoblot analysis using a mixture of two antibodies that specifically recognize the protein when it is phosphorylated on Ser235/Ser236 and Ser240/Ser244, respectively.
Protein immunoblots were developed using an Amersham ECL Western Blotting Kit. The chemiluminescence signal was visualized and quantified using a GeneGnome Bioimaging System running GeneTools software (Syngene).
To determine the effect of feeding, data from fasted rats were averaged and used to normalize the response observed in three fed rats killed at the same time. Data are expressed as the fold increase (means ± SE) in phosphorylated protein in fed vs. fasted rats. (Note: the meal was given between 0 and 2 h.)
The fractional rate of protein synthesis was not calculated for the in vivo studies. This was because of the nonlinear change in the labeling of plasma albumin (see results and discussion).
In cell culture studies, the fractional rate of protein synthesis was calculated using the equation where labelingtimex represents the total 2H labeling of alanine isolated from protein (34).
Statistical analysis was performed using a t-test.
Equilibration of 2H2O In Vivo
Figure 1 shows that the equilibration of alanine with body water is rapid; i.e., the label has largely equilibrated 20 min postinjection of 2H2O. The labeling of the α-hydrogen is approximately equal to that of water and the labeling of total hydrogen is approximately equal to 3.7 times that of water (as expected, since there are up to four exchangeable hydrogens in alanine). The results demonstrate that alanine labeling returns to near baseline values shortly (within ∼1 h) after an injection of a supraphysiological quantity of unlabeled alanine. The data also demonstrate that the labeling of the α-hydrogen and the total hydrogens equilibrate in a similar time course. [Note: the concentration of alanine increased from basal values of ∼1.2 mM between 0 and 90 min to ∼5.5 mM between 120 and 135 min and remained elevated for the duration of the experiment (∼2.5 mM at 240 min).]
Figure 2 demonstrates that albumin isolated from plasma migrates as a major band on acrylamide gel electrophoresis. There are two minor bands that represent a small amount of the total protein loaded onto the gel. These two bands are also found in commercially available albumin obtained from Sigma-Aldrich (not shown).
Figure 3, top, demonstrates that it is possible to maintain a constant labeling of body water and of free alanine over 24 h. The total labeling of plasma and intrahepatic alanine was ∼90% that of body water (×4, the theoretical maximum). The concentration of plasma alanine (not shown) and the labeling of both plasma and intrahepatic alanine remained constant over the 24-h period.
Because the precursor labeling is not substantially perturbed by feeding, steady-state equations can be used to calculate the rate of protein synthesis. For example, calculation of the rate of protein synthesis is typically done by measuring the change of labeling of a protein during the linear phase of isotope incorporation. However, in this study we do not calculate a rate of albumin synthesis because the protein labeling changes in a nonlinear manner over the experimental period (Fig. 3, bottom). Nevertheless, our data demonstrate that, when the physiological response is integrated over the complete experimental period, fed rats synthesize about two times more albumin compared with fasted rats (Fig. 3, bottom, the difference between the 0- and 24-h time points). Also, in fed rats, ∼50% of the plasma albumin that is synthesized and secreted in a 24-h period is made within the initial 5-h period after the food is ingested.
The data in Fig. 4 strongly support the isotope labeling data in Fig. 3, bottom. Namely, feeding increased the phosphorylation of 4E-BP1, p70S6K1 (Thr389), and ribosomal protein S6, three proteins that are involved in regulating the initiation of mRNA translation. This effect was strongest during the 5 h immediately following the meal and was diminished between ∼9.5 and 24 h. These changes suggest that the stimulation of protein synthesis by feeding is maximal immediately following the meal and that the rate of protein synthesis decreases thereafter, consistent with the changes in labeling of plasma albumin (Fig. 3, bottom).
Equilibration of 2H2O in Cell Culture
Table 1 shows the concentration and the labeling of free alanine in culture medium bathing Clone 9 cells for 48 h. Although alanine was not present in the culture medium that was added to the cells, the concentration of alanine increased over time, demonstrating that alanine is produced and secreted into the medium. If 2H2O is added 24 h after the medium is changed, there is a slow and incomplete equilibration between the 2H in water and that incorporated into free alanine. However, the addition of 2H2O simultaneously with the change in the medium results in complete equilibration between the labeling of water and free alanine.
Table 2 shows the influence of various conditions on the rate of protein synthesis in the cell culture model. The addition of fetal calf serum stimulated protein synthesis, whereas both anisomycin and azide inhibited protein synthesis. These observations are consistent with the respective conditions; i.e., addition of fresh serum is known to stimulate protein synthesis (31), and anisomycin inhibits protein synthesis (3). The effect of azide on inhibiting protein synthesis is not yet described but is consistent with the effect of azide as an agent that blocks oxidative phosphorylation and shifts cellular ATP production toward glycolytic flux (3). Presumably, in azide-treated cells, protein synthesis is limited because of decreased ATP production.
Four general approaches are available for studying protein dynamics in vivo (34). First, whole body nitrogen balance can be calculated by measuring nitrogen intake and loss (19). Second, the balance of amino acids across limbs and/or tissue beds can be determined by measuring the arteriovenous differences (2). Third, the turnover of specific proteins (e.g., plasma proteins) can be measured by intravenously injecting a labeled protein and measuring its biological decay (15). Last, precursor-product labeling techniques can be used to measure the synthesis of total and/or specific proteins (32, 38).
We used 2H2O to measure rates of protein synthesis, a precursor-product labeling technique (24). A primary goal of our research was to determine how dietary factors would influence protein synthesis. In the current study we directly examined the equilibration of alanine labeling during conditions that could perturb its labeling (e.g., during feeding) and subsequently examined whether this would impact studies aimed at understanding the influence of nutrient intake on measurements of protein synthesis in vivo. We discuss these observations in turn.
We previously observed that the labeling of plasma alanine reflects that of body water under near steady-state conditions (24). In the current study, we first determined whether a supraphysiological load of alanine would equilibrate with body water. We found that the labeling of alanine readily equilibrates with that of body water, even under conditions that result in a substantial perturbation in the plasma concentration of the amino acid (Fig. 1). Although we do not consider the exact mechanism or flux ratios that affect this equilibration, the observation presented here is consistent with our previous report and with the literature (22, 36). Specifically, the fractional turnover of plasma alanine is very rapid, and a large component of the turnover is due to transamination, which incorporates 2H into alanine (23). Therefore, one would expect equilibration of 2H between body water and free alanine under most conditions (Fig. 1). Because the labeling of the carbon-bound hydrogens of alanine (α and β) equilibrate in a similar manner, we sought to further explore and apply this observation under more physiological conditions. Namely, the ability to use the total labeling of alanine as that of the “product labeling” increases the sensitivity of measurements of protein synthesis; i.e., since about four hydrogens are exchanged, the labeling of newly made protein is about four times greater than that of the precursor.
We hypothesized that rapid exchange between 2H in body water and free alanine would allow us to measure the rate of protein synthesis in response to a meal. We tested our hypothesis in rats that were trained to eat their normal caloric requirement during a 2-h interval, which ensured that food was consumed as a reasonable bolus (7). We determined that the total 2H-labeling of free alanine (plasma and intrahepatic) remained virtually constant before and after a meal and that it was ∼90% equilibrated with that of body water multiplied by 4 (the theoretical maximum labeling) (Fig. 3, top).
Because a steady-state precursor labeling was achieved, steady-state equations could be used to calculate the rate of protein synthesis. However, application of those equations requires that one measure the change in labeling of a protein during the linear phase of isotope incorporation. As shown in Fig. 3, bottom, there was a nonlinear increase in the labeling of plasma albumin over 24 h in fed rats. To simplify the discussion, we considered a semiquantitative treatment of the data; the development of a mathematical formula to calculate protein synthesis under non-steady-state conditions is an area under investigation by our laboratory. We found that the labeling of plasma albumin was approximately twofold greater in fed vs. fasted rats 24 h following the meal (Fig. 3, bottom), and yet the precursor labeling was similar in both groups. Therefore, the integrative rate of albumin synthesis was approximately twofold greater in fed vs. fasted rats. Furthermore, the change in protein labeling (i.e., protein synthesis) over 24 h demonstrated that a large portion (∼50%) of plasma albumin was made within an ∼5-h period after the meal (Fig. 3, bottom; ∼50% of the total change in labeling of albumin occurred within the 5 h immediately following the meal). Our observation, that feeding stimulates albumin synthesis, is consistent with the reports of others (who demonstrated a doubling in the rate of albumin synthesis in fed vs. fasted subjects; Refs. 5 and 16) and is in agreement with the time course of changes in the molecular signaling mechanisms that control mRNA translation (Fig. 4).
Although the changes in the rate of albumin synthesis and the phosphorylation state of the initiation factors occur in a predictable manner, it is intriguing to note that the time course of change in the initiation factors is in strong agreement with the exponential change in the labeling of plasma albumin. For example, the “burst” in albumin synthesis that occurs immediately after the meal directly corresponds with a change in the phosphorylation state of the initiation factors. In addition, several hours after the meal, there is no difference between the rates of albumin synthesis in fed vs. fasted rats; likewise, there is no difference between the phosphorylation states of the initiation factors.
Although 2H2O can be used to study the influence of feeding on protein synthesis in vivo, we questioned whether 2H2O could also be used to measure rates of protein synthesis in vitro or whether feeding would have a negative impact on the reliability of the method. We hypothesized that the presence of unlabeled alanine in cell culture medium (i.e., the “food”) could limit the equilibration between the 2H labeling of the medium and free alanine. This is because cell culture studies often require that a relatively small number of cells be incubated in a large volume of medium. This is in contrast to what is observed in vivo, where there is a relatively small pool of alanine that undergoes a rapid turnover. The consequence of any lack of equilibration would be an underestimation of the true rate of protein synthesis.
We found that cells grown in alanine-free medium readily produce alanine (Table 1). Presumably, the alanine is formed during the metabolism of glucose and pyruvate. We demonstrated that the labeling of free alanine in the medium slowly approaches that of 2H2O in the medium if 2H2O is added 24 h after the medium is changed, i.e., when the concentration of free alanine reaches a substantial level (Table 1). Consequently, one cannot assume that the precursor labeling is that of the water in the medium and that the product labeling is that of total incorporated alanine divided by 3.7 (as we observed in vivo). However, we found that it is possible to obtain complete equilibration between the 2H labeling of water in the medium and free alanine, provided that 2H2O is added immediately after the medium is replaced with fresh alanine-free medium (Table 1). Under these conditions, the labeling of the α-hydrogen is equal to that of the water and the total labeling of alanine is ∼3.7 times that of the water (not shown). Presumably, this occurs because cells generate [2H]alanine during the metabolism of various substrates (e.g., the conversion of glucose and pyruvate to alanine). We found that the rate of protein synthesis could be measured when 2H2O was added immediately after the cells were washed and incubated in alanine-free medium (Table 2).
In summary, 2H2O can be used to measure rates of protein synthesis in vivo and in vitro. Under the conditions tested, there is rapid equilibration between the hydrogens of body water and those bound to the α- and β-positions of alanine in vivo. This demonstrates that one can reliably determine precursor-product labeling ratios (i.e., the precursor labeling is that of body water and the product labeling is that of protein-bound alanine divided by 3.7). As shown, the 2H2O method can be used to study how the rate of protein synthesis is affected by different factors in vivo (8, 14, 16, 17, 26). However, special considerations apply when 2H2O is used to study protein synthesis in vitro. In particular, feeding can lead to an underestimation of the rate of protein synthesis if proper adjustments are not made.
We anticipate that the relative simplicity of the 2H2O method will facilitate in vivo and in vitro studies aimed at understanding the regulation of protein synthesis. For example, the ability to easily and reliably measure the effect of dietary interventions on protein synthesis could facilitate patient management; e.g., in patients undergoing hemodialysis, better clinical outcomes may be achieved by stimulating protein synthesis and minimizing protein wasting (13, 30). The ability to use the total labeling of alanine in the protein product improves the sensitivity of the measurements of protein synthesis, i.e., since the product labeling (protein-bound alanine) is approximately four times that of the precursor (body water). Finally, measuring the incorporation of [2H]alanine into proteins offers advantages over the use of other amino acids that also become labeled during the exposure to 2H2O. For example, it is possible to measure the incorporation of [2H]glycine into proteins (10). However, since there are only two exchangeable hydrogens in glycine, the product labeling will only reach two times that of water (the precursor). Although one may consider glutamate a more attractive amino acid compared with alanine (since there are possibly 5 exchangeable hydrogens), the carbon-bound hydrogens of glutamate are labile and sensitive to nonspecific exchange during protein hydrolysis (S. F. Previs, unpublished observations).
This work was supported by the Mt. Sinai Foundation of Cleveland, OH, and National Institute of Diabetes and Digestive and Kidney Diseases grants DK-61994–01A1 (F. Ismail-Beigi), DK-13499 (S. R. Kimball), and DK-007319 (training fellowship to D. A. Dufner).
We thank Drs. V. Chandramouli and V. E. Anderson (Case Western Reserve University) for helpful discussions during the development of this work.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 by American Physiological Society