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Am J Physiol Endocrinol Metab 292: E1340-E1347, 2007. First published January 16, 2007; doi:10.1152/ajpendo.00488.2006
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Determination of protein replacement rates by deuterated water: validation of underlying assumptions

Emmanuelle Belloto,1 Frédérique Diraison,1 Alexandra Basset,2 Gwenola Allain,1 Pauline Abdallah,1 and Michel Beylot1,2

1Institut National de la Santé et de la Recherche Médicale U499, Faculté Réne Theodore Hyacinthe, Laennec, and 2ANIPHY, Faculté Rockefeller, University Claude Bernard Lyon1, Lyon, France

Submitted 11 September 2006 ; accepted in final form 5 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
H2O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. 2H2O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during 2H2O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after 2H2O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of 2H2O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by 2H2O administration. All of these results support 2H2O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.

protein synthesis; stable isotope; mass spectrometry; alanine

BODY PROTEINS ARE CONTINUOUSLY SYNTHESIZED and degraded. These synthesis and degradation rates are highly variable among individual proteins and are modified by physiological factors and pathological situations. Since maintainance of adequate amounts of whole body protein store, but also of a given protein in plasma or tissue, is essential for health, much effort has been devoted to the measurement of protein synthesis rate, particularly in humans, and several approaches have been proposed (26, 28). Most measure protein synthesis using the precursor-product method, i.e., by following the incorporation into proteins of a labeled amino acid after acute (bolus) or prolonged (infusion) administration of this labeled molecule. A central problem in this method is to correctly measure the enrichment in the true precursor pool, the tRNA-bound amino acid for protein synthesis. Enrichment of the precursor by the tracer administered is usually measured in the plasma, the only pool that can be easily sampled, particularly in humans, as ethical considerations limit access to tissues. Assuming that enrichment of amino acid in the plasma pool reflects enrichment in the true precursor pool is questionable. It has been proposed that measuring the enrichment of plasma {alpha}-ketoisocaproate (KIC) during the infusion of labeled leucine (21), or of urinary hippurate during the infusion of labeled glycine (8), would give a correct estimate of the enrichment in the true precursor pool. Alternatively, one can give a large bolus of a labeled amino acid, since by flooding the system with a large amount of amino acid the labeling in plasma and in the intracellular pools should be comparable (13). However, these methods have been questioned (22). Last, one possibility is to administer the labeled amino acid continuously for several half-lives of the protein investigated; the label in the protein will eventually reach a plateau equal to the enrichment of the precursor pool. However, since the labeled amino acid is usually given intravenously to obtain a stable enrichment level in the precursor pool, this approach to measuring the enrichment in the precursor pool can be reasonably used only for proteins with a short half-life.

Deuterated water is now used for measuring several aspects of glucose and lipid metabolism (2, 5, 7, 10, 12, 14, 16, 18, 23, 24). Its use for estimating protein synthesis, initially proposed by Ussing (25), has been recently revived and developed (2, 6, 11, 19). The basis is that, after administration of deuterated water, 2H atoms distribute rapidly in whole body water and label free amino acids through various enzymatic reactions. Transamination in particular will label all free amino acids, including essential ones, at their {alpha}-carbon (17). Labeled amino acids are then incorporated into protein during their synthesis. 2H2O is an attractive tracer that offers important advantages. First, it can be given orally, making studies in humans and animals much easier than with tracers needing intravenous infusion. Second, it is a safe, relatively inexpensive tracer that can be administered for long periods (up to several weeks), with stable enrichment levels in plasma water (12), making it appropriate for studying molecules with slow turnover rates. In principle, the incorporation of any amino acid into proteins could be used for measuring protein synthesis. In practice, alanine is attractive and convenient, as it has four possible sites of incorporation of deuterium (17); this results in a much higher enrichment level (≤4 times) in free alanine than in body water, increasing the sensitivity and the accuracy of the measurements of enrichments in newly synthesized protein in the presence of safe enrichment levels in water. The usefulness of this method for measuring protein synthesis rate in vivo in animals and in humans has been recently demonstrated (2, 6, 11, 19). This method needs, like other precursor-product methods, determination of the enrichment in the precursor pool. This can be achieved by measuring enrichment in plasma water, provided that n, the number of effective incorporation sites of deuterium in the chosen free amino acid, is known. Multiplying enrichment in plasma water by n gives the enrichment in the precursor pool. The n can be determined by the mass isotopomer distribution analysis of the labeled amino acid incorporated into the protein studied (6). However, this needs relatively high enrichment levels in body water to have reliable measurements of enrichment for the m + 2 isotopomer. One can use for the calculation previously published n values, assuming that this value is constant, whatever the protein studied, the tissue synthesizing it, and the situation investigated. This assumption is supported for alanine by the results of Bush et al. (6) but will need further examination. A more direct possibility is to measure the enrichment of the free amino acid in plasma. Labeling by deuterium occurs during intracellular enzymatic reactions, and the only source of label in plasma is intracellularly labeled amino acid. Therefore, enrichment in plasma should reflect enrichment in tissues. However, this will be true if 1) intratissular enrichment is homogenous, i.e., if reactions resulting in deuterium labeling proceed in all tissues at a rate high enough compared with endogenous supply of unlabeled alanine, through, for example, proteolysis, to prevent any dilution of the label in a tissue compared with another one; 2) exchange rates between the tissular and plasma pools of alanine are sufficiently high to allow rapid equilibration even in situations of entry of unlabeled alanine (e.g., after ingestion of a protein meal). Therefore, a first aim of the present study was to compare, during 2H2O administration to rats, deuterium labeling in plasma and in various tissues in the presence or absence of food ingestion. We also determined whether plasma alanine enrichment in humans is modified or not by feeding or glucose ingestion. Another point is whether or not after 2H2O administration there is rapid equilibration of water and alanine enrichment between the portal and peripheral circulation. Previous studies show that stable levels of both plasma water and alanine (11) enrichments are rapidly obtained in peripheral circulation during 2H2O administration in rats. However, deuterated water used in metabolic studies is usually orally ingested or intaperitoneally injected. This could result, particularly after the initial loading dose of 2H2O, in higher labeling of plasma water, and possibly of plasma free alanine, in portal than in peripheral circulation. In this situation, peripheral plasma alanine enrichment would not correctly reflect liver free alanine enrichment. To our knowledge, no comparison between portal and peripheral plasma enrichments has been performed. Therefore, a second aim was to compare, in rats, enrichments of water and alanine in portal and peripheral circulation after 2H2O administration. Such a direct comparison cannot be performed in humans; however, we compared in humans kinetics of enrichments of apoB100-VLDL by trideuterated leucine and deuterated alanine in subjects infused with [2H3]leucine and receiving 2H2O. We reasoned that if there is in humans a significant difference between peripheral and liver alanine enrichment during 2H2O administration, this would reflect in the kinetics of apoB100-VLDL enrichment by deuterated alanine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and Supplies

2H2O was from Cortec (Paris, France) and [5,5,5-2H3]leucine from Eurisotop (St. Aubin, France). Cation exchange resin (AGWX4, hydrogen form) was from Bio-Rad (Hercules, CA). Unless specified, all chemical and reagents were from Sigma (St. Louis, MO) or Interchim (Montluçon, France). Rats were supplied by Charles River (L'Arbresle, France).

Rat Experiments

All experiments were performed in accordance with the Guiding Principles in the Care and Use of Animals.

Comparison of deuterium enrichments in portal and peripheral plasma water and alanine after bolus oral administration of 2H2O. In these experiments, male Wistar rats were given, at 0900, in the fed state, a loading dose of 2H2O (10 ml/kg) by gavage. Blood samples (1.5 ml) were collected from portal vein and inferior cava vena in anesthetized rats (pentobarbital sodium 6 mg/100 g body wt ip) 10, 20, 30, and 45 min after the bolus of 2H2O (n = 3 at each experimental point). All blood samples were immediately centrifuged, and plasma was stored at –30°C.

Kinetics of enrichments in plasma water, plasma and tissue free alanine, and plasma and tissue protein-bound alanine during administration of 2H2O. In a first series of experiments, 18 male Wistar rats received, between 0800 and 0900, a loading dose of 2H2O (10 ml/kg ip, isotonic with NaCl). They then had free access to drinking water enriched (3%) with 2H2O. All of these rats had their food removed at 0600 the previous day. Nine rats were kept fasting during the experiment (fasted group), and the other nine rats had free access to food after the bolus injection of 2H2O (fed group; these rats ate 4–8 g of diet depending on the length of the experiment). At 2, 4, and 8 h after the intraperitoneal bolus of 2H2O, three rats from each group (fasted and fed) were anesthetized (pentobarbital sodium ip). The abdomen was rapidly opened and blood quickly sampled from the portal vein and the inferior cava vena (1.5 ml), and thereafter samples of skeletal muscle (flexor digitorium superficialis), liver, and the left ventricle of the heart were successively and quickly (within 2–3 min) collected, snap-frozen in liquid nitrogen, and stored at –80°C until analysis.

In a second series of experiments, 10 male Wistar rats received at 0800 the same loading dose of 2H2O. They then had free access to drinking water enriched with 2H2O (3%) and to food. Blood (inferior vena cava) and tissue (liver, skeletal muscle, and left ventricle) samples were collected in anesthetized rats 24 h (5 rats) and 80 h (5 rats) after the loading dose of 2H2O.

Studies in Humans

All subjects gave their written, informed consent after a full explanation of the purpose, nature, and risks of the studies. Protocols were approved by a local ethics committee.

In the first study, 10 healthy control subjects (5 men, 5 women, aged 22 to 50 yr, BMI 20 to 24 kg/m2) received in the postabsorptive state a loading dose of 2H2O (3 g/l body water). They then ingested water enriched (6 g/l) with 2H2O (150 ml/h for 12 h). Peripheral venous blood was sampled every hour until 2000. Five subjects remained in the fasting state, whereas the others ingested glucose every hour (1 g/kg at 1 h, followed by ingestion of 20 g of glucose every hour, diluted in water enriched with deuterated water, until the end of the test).

In a second study, 5 control subjects (3 men, 2 women) ingested, after an initial blood sampling, in the evening, a loading dose of 2H2O (3 g/l body water, one-half at 2000. after dinner, one-half at 2200). They then drank during the following 60 h only water enriched with 2H2O (6 g/l) and consumed their usual diet. Blood was collected 12, 20, 36, 44, and 60 h after the loading dose (at 0800 in the postabsorptive state and at 1600 in the fed state).

In a third study, 5 control subjects (3 men, 2 women) received at 0800, in the postabsorptive state, an intravenous bolus injection of [5,5,5-2H3]leucine (10 µmol/kg) followed by a continuous 10-h intravenous infusion (0.13 µmol·kg–1·min–1). They also ingested a loading dose of 2H2O (same dose as in previous protocols: one-half at 0800, one-half 30 min later) followed by the free ingestion during the following 10 h of water enriched (6 g/l) with 2H2O. They remained in the fasting state until the end of the study. Peripheral blood samples were collected before the bolus and then every hour for 10 h for the various determinations.

Analysis

Metabolite and hormone determinations. Plasma glucose and insulin concentrations were measured in humans by enzymatic method and radioimmunoassay, respectively (4).

2H labeling. 2H enrichment [isotopic enrichment (IE)] in plasma water was determined as previously described (27). For the enrichment of free alanine in plasma, plasma was deproteinized by perchloric acid (6%), and after centrifugation the supernatant was neutralized by K2CO3 (3 M). After acidification (pH = 2) by 1 N HCl, this supernatant was applied to a cation exchange column (AGWX4, hydrogen form). The column was washed with water, and amino acids were then eluted with 4 N NH3OH. The eluate was dried before preparation of the t-butyldimethylsilyl derivative of alanine (20). One microliter was injected into a gas chromatograph (HP5890; Hewlett-Packard, Palo Alto, CA) equipped with a 25-m fused silica capillary column (OV1701; Chrompack, Bridgewater, NJ) and interfaced with an HP5971A mass spectrometer (Hewlett-Packard) working in electron impact mode. The carrier gas was helium. Operating conditions were as follows: injector 240°C, split ratio 1/10, oven 80°C for 4 min, then rising at 20°C/min to 250°C with a final plateau of 5 min. Alanine elutes at ~10 min. Ions of m/z 260 and 261 were collected, and enrichment was calculated from standards run along with the samples. For measuring free alanine enrichment in tissues, 100–200 mg of tissue were homogenized in 1 ml of perchloric acid (6%), and the supernatant was used for purification and preparation of the derivative of free alanine as described for plasma. For the alanine of plasma or tissue proteins, proteins were precipitated by perchloric acid, and after centrifugation the precipitate was washed three times with water before hydrolysis of proteins by 6 N HCl (15 h at 100°C). The hydrolysate was diluted to 0.6 N HCl before purification of alanine as described above. In some experiments in humans, deuterium labeling was also measured in plasma glucose and lactate. These molecules were purified from neutralized perchloric acid extracts of plasma by ion exchange chromatography (3, 15) before preparation of the bisbutylboronate acetyl derivative of glucose (4) and t-butyldimethylsilyl derivative of lactate (20). For measurement of enrichment of apoB100-VLDL by trideuterated leucine and by deuterated alanine, plasma VLDL were first purified by ultracentrifugation, and apoB100 was separated from other proteins by polyacrylamide gel electrophoresis (9). Purified apoB100 was then hydrolyzed as described above. Purification of alanine and leucine and preparation of their t-butyldimethylsilyl derivatives were performed as described above. For leucine, ions of m/z 200 (unlabeled leucine), 201 (monodeuterated leucine, incorporation of leucine labeled by deuterated water), and 203 (trideuterated leucine, labeling by the labeled leucine infused) were selectively monitored. In experiments with infusion of trideuterated leucine, enrichment of plasma {alpha}-KIC was measured as previously described (1). All IE are expressed as mole percent excess (MPE).

Calculations

Results are shown as means ± SE. Comparisons of enrichments between portal and peripheral venous plasma or between plasma and tissues were performed by two-tailed Student's t-test for paired values. When appropriate, kinetics of labeling of plasma water, plasma, and tissue free alanine by deuterium, of tissue or plasma proteins by deuterated alanine, and of apoB100-VLDL by deuterated alanine and leucine were estimated by nonlinear curve fitting of the experimental points assuming a single exponential rise to maximal value: IEt = IEmax(1 – e–kt), with k being the fractional replacement rate. For muscle and left ventricle, protein synthesis rates were estimated by linear fitting using plateau values of free alanine enrichments as the final enrichment value in proteins. Statistics and curve fitting were performed using Prism 4.0 (GraphPad, San Diego, CA) and Sigma Plot 5.0 (SPSS, Chicago, IL) software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiments in Rats

Comparison of plasma water and alanine IE in portal and peripheral circulation after bolus oral administration of 2H2O. As in previous studies (11), stable IE in both plasma water and alanine were obtained at 20 min in peripheral plasma with a ratio of total alanine over water enrichment of 3.7:4.0. These IE were comparable in portal and peripheral circulation at 20, 30, and 45 min. However, at 10 min, plasma water and alanine IE were moderately higher (P < 0.05) in portal than in peripheral plasma. These values in peripheral plasma were also slightly lower from those observed at plateau (20–45 min) (Fig. 1).


Figure 1
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  		Fig. 1. Deuterium enrichment in plasma water (circles) and free alanine (triangles) measured in portal (closed symbols) and peripheral (open symbols) plasma of rats after a bolus oral administration of deuterated water. Results are shown as means ± SE. *P < 0.05 vs. corresponding value in peripheral circulation.

 
Kinetics of IE in plasma and tissues during administration of 2H2O.
KINETICS OF LABELING IN PLASMA WATER AND ALANINE. Stable IE were obtained for water between 2 and 8 h after the initial bolus administration of 2H2O in both portal and peripheral plasma (Table 1). There was a slight increase (0.2–0.4%) in alanine labeling between 2 and 8 h, but this increase represented less than 8% of the value observed at 8 h. Total alanine over water enrichment ratios were 3.8:4.2. Values in portal and peripheral plasma were identical, and comparable results were obtained in fasted and fed rats (Table 1). In fed rats, deuterium enrichment in plasma water and free alanine were also measured in peripheral plasma at 24 and 80 h (Fig. 2); values were identical to those observed from 2 to 8 h. Therefore, reasonable plateau values of labeling for both plasma water and alanine are quickly obtained and maintained in rats, without any difference between portal and peripheral plasma except for the initial 10 min after bolus administration of 2H2O. Moreover, the metabolic status (fed vs. fasted state) did not modify the kinetics of plasma alanine labeling. Experimental points of alanine labeling were fitted to a single exponential rise. The plateau values given by curve fitting were identical to the experimental plateau values and were comparable between portal and peripheral plasma (0–8 h) and between fed and fasted states (0–8 h) (data not shown). Fractional replacement rates of alanine, k, were also identical (1.61–1.93/h).


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  		Table 1. Enrichment (MPE) of plasma water and free alanine in portal and peripheral circulation of rats after ip injection of 2H2O

 

Figure 2
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  		Fig. 2. Deuterium enrichment in plasma water (bullet) and free alanine ({circ}) measured in peripheral circulation of rats for 80 h after an ip bolus injection of deuterated water followed by ad libitum drinking of water enriched with deuterated water. Results are shown as means ± SE. For alanine, the curve shown was obtained by fitting experimental points to a single exponential rise to plateau. The equation obtained was IEt = 5.45(1 – e–1.519t).

 

KINETICS OF FREE ALANINE LABELING IN TISSUES. In all tissues, there was, as in plasma, a slight increase of labeling between 2 and 8 h and stable values thereafter until 80 h (Fig. 3). Again, however, the increase between 2 and 8 h was quite moderate, less than 8% of the plateau value. The kinetics of labeling and the plateau values were comparable in liver, left ventricle, and skeletal muscle and were comparable to values observed in plasma, considering either the values until 8 h or the values until 80 h. We also observed no difference in the values obtained in fasted or fed rats (values obtained until 8 h after the initial bolus; data not shown). Fitting experimental points to a single exponential rise gave plateau values identical to experimental values, without any difference between tissues. k Values were also identical between tissues (liver: 1.274/h; muscle: 1.498/h; heart: 1.598/h) and near to values obtained in peripheral plasma (k = 1.519 for the 0- to 80-h period).


Figure 3
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  		Fig. 3. Deuterium enrichment of free alanine in tissues [liver, skeletal muscle, and heart (left ventricle)] measured in rats for 80 h after an ip bolus injection of deuterated water followed by ad libitum drinking of water enriched with deuterated water. Results are shown as means ± SE. Curves shown were obtained by fitting experimental points to a single exponential rise to plateau.

 

KINETICS OF LABELING IN PROTEIN-BOUND ALANINE. The increase of labeling was, as expected, slower than for free alanine, but near-plateau values were obtained at 80 h for mixed plasma and liver proteins, and the experimental points could be fitted to a single exponential rise (Fig. 4). In liver, the final enrichment values, both experimental (5.50%) and obtained by curve fitting (5.48%), were comparable to the values obtained for free alanine (5.54%); the fractional synthesis rate was much lower than for free alanine (0.0796/h vs. 1.274/h). The rise of labeling was lower in plasma than in liver proteins, with a k value of 0.0473/h. Comparison between fed and fasted states, and between portal and peripheral proteins, was performed only until 8 h after the bolus administration of 2H2O. We observed no differences between peripheral and portal proteins nor between fed and fasted states despite a trend for a faster rise of labeling in the fed state (Fig. 5). In left ventricle and skeletal muscle, the rise in labeling was much slower than in liver or plasma proteins (Fig. 6). Reliable enrichment values could not be measured before 8 h, and values at 80 h were far from plateau values. Therefore, data could not be fitted to a single exponential rise. We estimated k values by linear fitting and using as plateau value the value obtained for the corresponding tissue free alanine enrichment. Values obtained in left ventricle (0.00237/h or 0.40/wk) and skeletal muscle (0.00113/h or 0.19/wk) were much lower (20- to 70-fold) than those in liver or plasma proteins.


Figure 4
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  		Fig. 4. Deuterium enrichment in the alanine of mixed proteins of liver and peripheral plasma of rats for 80 h after an ip bolus injection of deuterated water followed by ad libitum drinking of water enriched with deuterated water. Results are shown as means ± SE. Curves shown were obtained by fitting experimental points to a single exponential rise to plateau.

 

Figure 5
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  		Fig. 5. Comparison of enrichment by deuterium of the alanine of mixed proteins of plasma measured in portal (open symbols) and peripheral (closed symbols) for 8 h following an ip bolus injection of deuterated water followed by ad libitum drinking of water enriched with deuterated water. Rats were studied in the fed (top) or fasted (bottom) state. Results are shown as means ± SE.

 

Figure 6
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  		Fig. 6. Deuterium enrichment of the alanine of mixed proteins of skeletal muscle and heart in rats 8, 24, and 80 h after an ip bolus injection of deuterated water followed by ad libitum drinking of water enriched with deuterated water. Results are shown as means ± SE.

 
Studies in Humans

In the first protocol, we measured deuterium enrichments in plasma water and free alanine during a 12-h period of administration of deuterated water. In fasting subjects, plateau values were obtained from 1 to 12 h (Fig. 7) for both water and alanine, with an alanine-over-water enrichment ratio ~3.8. Stable enrichment in plasma lactate was also obtained, with a lactate-over-water enrichment ratio ~2.8 (data not shown). When subjects ingested glucose during the test, no significant decrease of enrichment in alanine (Fig. 7) or lactate (data not shown) was observed, and the enrichment ratios of alanine over water were unchanged.


Figure 7
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  		Fig. 7. Plasma glucose concentrations and deuterium enrichment in plasma water and in free alanine of healthy subjects during a 12-h deuterated water administration. Studies were initiated in the postabsorptive state. Subjects were fasted (n = 5, bullet) or ingested glucose (n = 5, {circ}) during the study.

 
In the second experiment, we measured labeling in plasma water and total alanine, lactate, and glucose in subjects consuming their usual diet. Stable enrichment level ~0.35% was obtained in plasma water throughout the whole study period. As shown in Fig. 8, enrichment in glucose was lower in the postprandial state than in the postabsorptive state, reflecting, as expected, the dilution of endogenously produced, labeled glucose by exogenous, unlabeled glucose. However, enrichments in lactate and alanine were identical in the postprandial and postabsorptive states. Therefore, feeding did not modify alanine labeling, and plateau values were observed throughout the whole experimental period.


Figure 8
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  		Fig. 8. Deuterium enrichment in plasma glucose (bullet), lactate ({circ}), and alanine ({blacktriangleup}) of healthy subjects during a 60-h administration of deuterated water. Samples were collected in the postabsorptive (12-, 36-, and 60-h) or fed (20- and 44-h) state. Results are shown as means ± SE.

 
In the final study, we compared the kinetics of labeling of apoB100-VLDL by trideuterated leucine (infusion of [5,5,5-2H3]leucine) and deuterated alanine (ingestion of deuterated water). Deuterated water induced only a slight increase in the m + 1 (~0.3%) of plasma leucine. The increase in the m + 1 of leucine in apoB100-VLDL was less than 0.3% and not sufficient to induce any significant increase in the m + 3 of leucine. The final values of enrichments of alanine and leucine in apoB100-VLDL were, respectively, near to the enrichments of plasma alanine and {alpha}-KIC (Fig. 9). The rises of enrichment in leucine and alanine of apoB100-VLDL (Fig. 9) were fitted to a single exponential rise. k Values calculated using leucine or alanine enrichment kinetics were comparable (0.157/h vs. 0.139/h respectively).


Figure 9
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  		Fig. 9. Top: evolution of enrichment of plasma {alpha}-ketoisocaproate ({alpha}-KIC) ({circ}) and apoB100-VLDL leucine in healthy subjects during a 10-h iv infusion of trideuterated leucine. Results are shown as means ± SE. For apoB100-VLDL leucine, the curve shown was obtained by fitting experimental points to a single exponential rise to plateau. Bottom: evolution of enrichment in deuterium of plasma water ({circ}), free alanine (bullet), and alanine of apoB100-VLDL in the same subjects during oral administration of deuterated water. Results are shown as means ± SE. For apoB100-VLDL alanine, the curve shown was obtained by fitting the experimental points to a single exponential rise to plateau.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The use of deuterated water to measure synthetic pathways (7, 10, 16, 24) has recently been extended to the determination of protein synthesis rates (6, 11, 19). In the present report, we examined some aspects of this use of deuterated water to investigate protein metabolism. We show 1) that during 2H2O administration there is rapid equilibrium of deuterium labeling between plasma water and alanine and that stable enrichment values are obtained in both water and alanine without any difference between portal and peripheral plasma, 2) that the kinetics of enrichment and plateau values are comparable between plasma and tissue free alanine, and 3) that these processes are not significantly modified by changes in the nutritional state (fed vs. fasted state).

The oral ingestion or intraperitoneal injection of 2H2O could result in a higher labeling of plasma water, and possibly of free alanine, in portal than in peripheral circulation. We checked, therefore, whether such a gradient in enrichment existed or whether a rapid isotopic equilibrium between portal and peripheral plasma was obtained. We indeed found that 10 min after an oral bolus administration of 2H2O in rats, plasma water and alanine enrichments were moderately higher in portal than in peripheral circulation. However, this difference was transient, since identical enrichment values in portal and peripheral plasma were attained for both water and alanine as early as 20 min after the bolus. In addition, stable enrichment levels were obtained in peripheral, confirming the results of Dufner et al. (11), and portal plasma. When deuterated water was given in rats by intraperitoneal injection followed by ad libitum drinking of water, stable labeling with identical values in portal and peripheral plasma were obtained in the following hours for both water and alanine. Therefore, except for the initial minutes after a loading dose of 2H2O, peripheral enrichments are representative of the portal ones. There is no gradient of enrichment between portal and peripheral circulation that would bear on the measurement of synthesis rates of protein synthesized and secreted by the liver by the deuterated water method. We could not perform such direct comparison of portal and peripheral enrichments during 2H2O administration in humans, but it is highly probable that there is also no enrichment gradient. This is supported by the direct comparison in the same subjects of the kinetics of labeling of apoB100-VLDL by intravenously infused labeled leucine and by alanine deuterated by orally ingested water. From a more general point of view, the finding of comparable turnover rates of apoB100-VLDL when the labeling by one tracer or the other is used for the calculation strongly supports the validity of the use of deuterated water.

Dufner et al. (11) showed that the intravenous bolus injection of unlabeled alanine in rats that received deuterated water induced a transient decrease in plasma alanine enrichment despite stable labeling in water. However, the quantity of alanine injected was largely supraphysiological. We examined whether a physiological entry of unlabeled alanine through intestinal absorption after a meal would modify plasma alanine enrichment. In rats, we observed no difference in peripheral or portal plasma alanine enrichment, nor in tissue free alanine enrichment, between the fasted and the fed states. In humans, we also observed no decrease in plasma alanine, nor in lactate, enrichment after meals despite the clear and expected decrease in total glucose labeling. We also found in humans no decrease in plasma alanine labeling when glycolytic rates were increased by the oral ingestion of glucose. These results show that stable enrichment levels of plasma alanine by deuterated water are indeed maintained in everyday life despite the alternation of feeding and fasting. This further supports the use of deuterated water for long-term studies with measurement of the kinetics of proteins with slow turnover rates.

A major aim was to determine whether, during deuterated water administration, free alanine enrichment by deuterium in tissues is homogenous and whether plasma alanine enrichment can be used as a correct estimate of tissular enrichment for the determination of protein kinetics. We found that the kinetics of free alanine labeling in liver, skeletal muscle, and heart are identical, with near-plateau values at 2 h and identical plateau values. Moreover, these kinetics are comparable to those of free alanine labeling in plasma. When comparing the initial (until 8 h) values obtained in tissues in rats studied in the fed or fasted state, we again observed no differences. Therefore, tissue labeling appears homogenous and, although an absolute demonstration would have needed comparison with the enrichment in tRNA-alanine, our results support the use of plasma free alanine enrichment as a substitute for the intratissular precursor pool enrichment. This is further supported by our finding that the calculated plateau values obtained by fitting experimental points of the labeling of protein-bound alanine were comparable to the plateau values of free alanine measured in tissues and plasma. This comparison could be performed only for mixed liver and plasma proteins. The increase of labeling in alanine of mixed proteins from muscle and heart was too slow to allow such calculation. It is noteworthy that the replacement rates of proteins in these tissues estimated by linear fitting of the rise in alanine enrichment are comparable to the values reported by Bush et al. when nonlinear curve fitting (6) is used. This strongly suggests that, when experimental conditions do not allow one to collect enough data to perform nonlinear curve fitting, the initial, near-linear part of the rise of enrichment in protein-bound alanine can be used to correctly estimate protein dynamics. It should be stressed that what is truly measured by the approach that we used in the present report, modeling of the rise of enrichment in proteins, is protein fractional synthesis rate. This synthesis rate is equal to the replacement rate only if the pool of protein investigated is stable. However, when this pool is changing, 2H2O can be used to determine the fractional synthesis rate and also the fractional degradation rate by modeling the increase and decrease of labeling during 2H2O administration and after its interruption (2, 19).

In conclusion, the present results strongly support the use for determination of protein kinetics during deuterated water administration of enrichment of free alanine in peripheral plasma as a substitute for the enrichment in the tissue precursor pool. They lend further support to the use of deuterated water as a safe, convenient, reliable, and attractive way to investigate protein metabolism in vivo.


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This work was supported, in part, by a grant from the French Ministère de la Recherche (ACI Plate-forme d'exploration fonctionnelle thématisée).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Beylot, INSERM U499, Faculté RTH Laennec, 7 Rue G Paradin, 69008 Lyon, France (e-mail: beylot{at}sant\|[eacute]\|.univ-lyon1.fr)

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.


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