Department of Surgery, State University of New York at Stony Brook, Stony Brook, New York 11794
Submitted 26 August 2003 ; accepted in final form 18 February 2004
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ABSTRACT |
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L-[2H5]phenylalanine; flooding method; constant infusion; ammonium chloride; lymphocytes
Because body proteins are continuously degraded and renewed by de novo synthesis, protein loss can occur as a result of either an elevation of protein degradation or a depression of protein synthesis. The in vivo measurement of whole body protein and amino acid kinetics using labeled amino acids shows that acidosis stimulates protein degradation and amino acid oxidation (30, 34, 35, 41, 42). Reports of the effect of acidosis on whole body protein synthesis have been inconsistent (30, 34, 42). However, the whole body approach may lack the precision and sensitivity to identify changes in protein turnover occurring in muscle or other specific tissues. Nevertheless, studies investigating the effect of acidosis on protein synthesis in specific tissues and cells also produced inconsistent results. Inhibition of protein synthesis by lowering the medium pH has been demonstrated in a variety of cell and tissue culture systems (4, 11, 12, 44, 46). However, when measurements were made in vitro in isolated muscles from acidotic animals, some studies detected an inhibition of protein synthesis by acidosis (20, 25), while others showed no effect (32, 33). Similarly, whereas some studies investigating the in vivo effect of acidosis on muscle protein synthesis have failed to detect a response (13, 29), others have shown a clear inhibitory effect of acidosis on muscle protein and albumin synthesis rates (3, 23).
The aim of this study was to characterize the effect of acidosis on in vivo tissue protein synthesis and to investigate whether individual tissues respond differently. We used an animal model of acute metabolic acidosis, in which groups of rats were made acidotic for 24 h with oral doses of NH4Cl. Protein synthesis rates were then measured in a variety of different tissues, including skeletal muscle, heart, liver, gut, kidney, spleen, and lymphocytes. In vivo protein synthesis was measured with the stable isotope-labeled amino acid L-[2H5]phenylalanine by use of the flooding method. In a separate experiment, the effect of acidosis on muscle and liver protein synthesis was also determined by using a constant infusion of the same labeled amino acid. To exclude the possibility that the agent used to induce acidosis, i.e., NH4Cl, may have specific effects on tissue protein metabolism, metabolic acidosis was also induced using a different approach. In this set of experiments rats were made acidotic by administration of a cation (H+) exchange resin, and tissue protein synthesis was then measured after 24 h.
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METHODS |
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Male Sprague-Dawley rats weighing 300 g (Taconic Farms, Germantown, NY) were used in all experiments. They were singly housed and given water and a standard rodent diet (Purina Mills, Richmond, IN) ad libitum for
7 days before the start of the experiments. All protocols were approved by the Institutional Animal Care Committee of the State University of New York at Stony Brook.
Experimental Protocol
Acidosis induced with NH4Cl All animals were fasted overnight (food removed at 2200) before the experiments began. Metabolic acidosis was induced with one dose of NH4Cl (20 mmol/kg body wt in water) given by gavage, followed 12 h later by a second dose. Similar rats given equimolar amounts of NaCl served as controls. All the measurements were made 24 h after the first gavage.
Experiment 1.
This experiment was planned to check the effectiveness of the treatment with NH4Cl in lowering arterial blood pH. To have access to arterial blood, a carotid arterial catheter was first implanted under anesthesia with ketamine (11.5 mg/kg body wt) and xylazine (11.5 mg/kg body wt). Animals were then allowed to recover for a week after surgery, during which catheters were kept patent with a solution containing heparin. Then rats were randomly assigned to be treated with NH4Cl (acidotic, n = 6) or NaCl (control, n = 6). Arterial blood gas analysis was performed after 24 h. Shorter-term effects of NH4Cl on arterial pH were also assessed in pilot studies on a separate group of similar animals (n = 6).
This was planned as a separate experiment to avoid the possibility that the surgical procedure involved in the placement of carotid arterial catheter might affect tissue protein synthesis.
Experiment 2: flooding method.
Protein synthesis was measured in a variety of different tissues of 24-h-acidotic (n = 8) and control (n = 8) rats with the flooding method (16). L-[2H5]phenylalanine (150 µmol/100 g body wt, 40 moles percent; MassTrace, Woburn, MA) was injected via a tail vein, and after 10 min the animals were killed by decapitation. Hindlimbs, heart, liver, kidney, small intestine, thymus, and spleen were quickly removed and immediately immersed in an ice-water mixture. Cooled-down hindlimbs were then dissected on ice and gastrocnemius, plantaris, and soleus muscles removed. Small intestine (including mucosa and serosa) was extensively washed with ice-cold saline. All cooled tissues were then frozen in liquid N2 and stored at 70°C until further analysis. Tissues were harvested in the same sequence in all experiments, and the time intervals between individual tissue removal and cooling in ice-water mixture were similar for all animals. Lymphocytes were isolated from thymus after tissue disruption through a metal sieve in cold phosphate-buffered saline (PBS) solution containing 0.5 mM cycloheximide (Sigma, St. Louis, MO) to prevent any further incorporation of the isotope in the cell protein (7). Lymphocytes were further purified by density gradient centrifugation onto Ficoll-Paque [Pharmacia, Uppsala, Sweden (6)] and then washed three times with PBS and frozen.
Experiment 3: constant infusion.
In this experiment, the effect of 24-h acidosis on tissue protein synthesis was measured using the constant infusion technique (17). Similarly to experiment 1, two groups of rats were treated with either NH4Cl (acidotic, n = 6) or saline (control, n = 6). After 24 h, a tracer solution containing L-[2H5]phenylalanine (1 µmol·100 g body wt1·h1; MassTrace) was infused for 2 h through a tail vein, after which skeletal muscles (gastrocnemius, plantaris, and soleus), heart, and liver were harvested and frozen, as described in experiment 2.
Experiment 4: Acidosis induced with cation exchange resin.
This experimental protocol was the same as that previously described for experiment 2, with the only exception that acidosis was induced by administration of a cation exchange resin instead of NH4Cl. After an overnight fast, rats were gavaged with 0.2 ml/kg body wt of a cation resin (AG50W-X8; Bio-Rad Laboratories, Hercules, CA) in the hydrogen form (H+) suspended in distilled water, followed by a second dose of 0.12 ml/kg body wt 12 h later. Control animals were treated with a similar dose of resin slurry but in the Na+ form. The resin suspensions were prepared by mixing the dry resin with distilled water and then by washing it with 1 N HCl (for the H+ form) or 1 N NaCl (for the Na+ form). The resin was then extensively washed with several volumes of distilled water and then resuspended with an equal volume of distilled water.
Protein synthesis in skeletal muscle (gastrocnemius, plantaris, and soleus) and liver was measured 24 h after the first gavage by using the flooding method with L-[2H5]phenylalanine, as described for experiment 2.
Analytical Methods
Enrichment of L-[2H5]phenylalanine in tissue protein. Tissues were powdered in liquid N2, and protein was separated from tissue free amino acids by precipitation with cold 0.2 M perchloric acid. Protein pellets were solubilized in 0.3 M NaOH at 37°C for 1 h, extensively washed with 0.2 M perchloric acid, and then hydrolyzed with 6 N HCl (16). Lymphocyte protein was first precipitated with cold 0.2 M perchloric acid and then processed similarly to tissue protein (8).
The enrichment of L-[2H5]phenylalanine in protein hydrolysates was measured with a MD800 gas chromatograph-mass spectrometer (Fisons Instruments, Beverly, MA) after enzymatic conversion to phenylethylamine and solvent extraction, as previously described (36). The ions at mass-to-charge ratio (m/z) 106 (m + 2) and 109 (m + 5) of the n-heptafluorobutyryl derivative were monitored under electron impact condition and in splitless mode.
Enrichment of tissue fluid free L-[2H5]phenylalanine. Tissue free amino acids obtained after precipitation of tissue protein with perchloric acid were purified by cation exchange chromatography (AG Resin, 100200 mesh, hydrogen form; Bio-Rad Laboratories, Richmond, CA). Phenylalanine was derivatized as its tertiary butyldimethylsilyl derivative, and the enrichment was measured by monitoring the ions at m/z 336 and 341 on an MD800 gas chromatograph-mass spectrometer (Fisons Instruments) (36).
Amino acid, protein, and RNA analysis. Plasma amino acid concentration was determined on blood collected at death on a Waters 2696 HPLC system (Waters, Milford, MA) after precipitation of plasma proteins with acetonitrile and derivatization with o-phthaldehyde. 6-Amino-n-caproic acid was used as internal standard. Protein concentration in tissue samples was determined with a modification of the method of Lowry et al. (27). RNA concentration was measured spectrophotometrically from its absorbances at 260 and 232 nm, as previously described (37).
Calculations
Tissue protein synthesis was calculated from the enrichment of L-[2H5]phenylalanine in protein and in tissue free amino acids (precursor pool). For the measurements with the flooding method (experiments 2 and 5), the fractional rates of protein synthesis (FSR) were calculated using the formula described by Garlick et al. (16):
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For the constant infusion method (experiment 3), the rates of protein synthesis (Ks) were calculated in skeletal muscle and heart with the formula (17):
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Protein synthesis rates (%/day) were obtained by solving the equation for Ks graphically by plotting curves of Ks and EB/Ei at fixed RP or p values. The values of Ks corresponding to the measured EB/Ei values were calculated off the curve. The
p value was experimentally estimated in a separate group of rats by measuring the time course of the rise of free phenylalanine enrichment in plasma during a constant infusion of L-[2H5]phenylalanine, similarly to experiment 3.
Statistics
All values are presented as means ± SE. Two-tailed t-tests for unpaired data were used for comparisons of groups within the same experiment and between separate experiments. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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Treatment with NH4Cl induced a prompt decline of blood pH. Thirty minutes after the administration of NH4Cl, arterial pH had dropped from 7.50 ± 0.009 to 7.28 ± 0.013 (P < 0.0001). pH values were 7.26 ± 0.027 and 7.24 ± 0.034, respectively, after 2 and 3 h (P < 0.0001) and remained stable thereafter. At 24 h, arterial pH values were 7.47 ± 0.01 in the control and 7.22 ± 0.02 in the acidotic group (P < 0.0001; experiment 1). This was accompanied by a significant decrease in blood HCO3 concentration (28.1 ± 0.8 vs. 11.5 ± 1.3 mmol/l, P < 0.0001) and base excess (4.5 ± 0.5 vs. 15.3 ± 1.7 mmol/l, P < 0.0001).
Administration of the cation exchange resin induced a decrease in blood pH comparable to that obtained with NH4Cl. In preliminary experiments in a group of rats in which an arterial carotid catheter had been previously implanted (n = 4), pH dropped from 7.51 ± 0.005 to 7.34 ± 0.01 (P < 0.0001) by 30 min, and to 7.26 ± 0.03 (P < 0.001) 1 h after the administration of the cation exchange resin in the H+ form. The pH then remained stable and was 7.27 ± 0.03 (P < 0.001) 24 h after the first gavage. The blood gas analysis performed on mixed arterial-venous blood collected at death in animals without arterial catheters in experiment 4 confirmed that the treatment with the cation exchange resin was effective in maintaining acidosis. The pH values of mixed blood were 7.50 ± 0.02 and 7.30 ± 0.18 (P < 0.0001), respectively, in the control and acidotic groups. Blood bicarbonate (27.7 ± 1.4 vs. 18.2 ± 1.5 mmol/l, P < 0.01) and base excess (4.4 ± 1.2 vs. 7.1 ± 1.5 mmol/l, P < 0.01) concentrations were also lower in the acidotic group.
Plasma Amino Acids
Table 1 shows plasma amino acid concentrations measured in experiment 2. The plasma levels of glutamic acid, isoleucine, and tryptophan were significantly lower during acidosis (P < 0.05; Table 1), although the concentrations of most amino acids were increased in acidotic animals.
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The rates of protein synthesis in skeletal muscles were all significantly depressed in acidotic animals in experiments 2 and 3, as shown in Tables 2 and 3. When measured with the flooding method (experiment 2), acidosis depressed the protein FSR by 29% in gastrocnemius muscle (P = 0.0005), by 23% in plantaris (P = 0.0004), and by 17% in soleus (P = 0.006; Table 2). The same results were obtained with the constant infusion method (experiment 3), showing a reduction of muscle protein FSR by 28% in gastrocnemius (P < 0.001), 19% in plantaris (P < 0.02), and 6% [P = not significant (NS)] in soleus in acidotic animals (Table 3). The decreases in protein synthesis were not associated with detectable changes in tissue RNA content, whereas the rate of synthesis per milligram of RNA (KRNA) was depressed significantly in each muscle (P < 0.01; Table 2). In contrast to skeletal muscles, the protein FSR of cardiac muscle was not affected by acidosis with either method used for measurement (Tables 3 and 4). Also, no effect of acidosis could be detected on protein synthesis of visceral organs such as liver, small intestine, kidney, or spleen (Tables 3 and 4). However, lymphocyte protein FSR was decreased by 8% in acidotic animals (P = 0.04).
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DISCUSSION |
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Several studies in vitro have demonstrated a direct effect of pH on protein synthesis in tissues and cells. Lowering the pH of the culture medium or perfusate reduced protein synthesis in myocytes (11), hepatocytes (4, 46), and perfused heart (44). However, when protein synthesis was measured in perfused muscle preparations from chronically uremic and acidotic rats, it was found depressed in some (20, 25), but not all, studies (32, 33), although there was a consistent increase in protein degradation. Only a few studies have investigated the effect of acidosis on individual tissues in vivo. Maniar et al. (29) compared both muscle protein synthesis and degradation in a set of rats that were uremic and acidotic with those of similar pair-fed animals that were uremic but not acidotic because of receiving NaHCO3 and with those of ad libitum-fed controls. They showed that rates of protein synthesis in muscle, measured in vivo with a flooding amount of L-[3H]phenylalanine, were lower in uremic animals than in controls. However, the reduction in muscle protein synthesis appeared to be mainly the result of reduced food intake, and no independent effect of acidosis on muscle protein synthesis could be detected. The conclusion of Maniar et al. that metabolic acidosis does not affect protein synthesis differs from the results of the present study. Although in both studies muscle protein synthesis was measured with the flooding method, the experimental design and the age of the animals were different. In the study by Maniar et al., young, growing rats (60 g) were made acidotic for 15 days, and the nonacidotic animals were pair-fed to the acidotic ones, with the result that they ate substantially less and grew less rapidly than normal. Because protein synthesis in muscle is sensitive to food intake in the young, growing rat (1, 15), protein synthesis in both uremic acidotic and nonacidotic rats was significantly lower compared with nonuremic ad libitum-fed controls. It is therefore possible that the underlying depression of muscle protein synthesis in uremic animals, caused by limited food intake, prevented any possible further inhibition by metabolic acidosis. In our study, animals were close to adulthood and therefore less sensitive to the effect of feeding (2). Also they were made acidotic for a period of only 24 h, in which both treated and control animals had no access to food, eliminating any possible interference due to disparity in food intake. However, it is not known whether the inhibitory effect of acidosis on muscle protein synthesis depends on the feeding state of the animal and whether it is abolished or exacerbated in the fed state.
The direct effect of acidosis on muscle protein synthesis in vivo has also been investigated in two human studies. Garibotto et al. (13) compared forearm protein turnover in controls and patients with chronic renal failure (CRF) by use of the arteriovenous difference technique and a constant infusion of L-[3H]phenylalanine. Because muscle is the most abundant tissue in the forearm and receives the majority of the blood supply, it is generally assumed that forearm protein turnover mainly reflects that of muscle tissue (18, 26). That study (13) showed that CRF patients have significantly higher rates of both synthesis and degradation of protein in the forearm, which is in apparent contrast with the findings of our study. However, data from that study are difficult to interpret because, although both protein synthesis and degradation were increased, no difference in amino acid balance across the forearm was detected in patients with CRF, leading to the conclusion that the increase in protein synthesis was entirely balanced by the increase in degradation. Hence, patients were not more catabolic than controls at the time of measurement. Even though pH values were not reported, it is likely that CRF patients participating in the study were only moderately acidotic, as estimated by the mean arterial bicarbonate values of 20 mmol/l, in contrast with the model of metabolic acidosis used in our study, in which blood pH was 0.25 units lower than controls and plasma bicarbonate concentration was severely depressed (see RESULTS).
In another human study, Kleger et al. (23), using the flooding method with L-[2H5]phenylalanine, measured muscle protein synthesis before and after oral treatment for 48 h with NH4Cl in healthy human volunteers. The results showed that acidosis (pH 7.32 ± 0.04 vs. 7.43 ± 0.02) depressed muscle protein synthesis by 33% (23). That study, which can be closely compared in terms of experimental design and method for measurement of muscle protein synthesis to the present work, is in excellent agreement with our findings, not only because it clearly shows an inhibitory effect of acidosis on muscle protein synthesis, but also for the similarity in the magnitude of this effect despite the less pronounced acidosis in the humans.
Administration of NH4Cl has been widely used as a model of metabolic acidosis in both humans and animals (e.g., Refs. 3, 9, 23, 29, 31, 32, 42). NH4Cl is taken up by the liver with formation of urea and net release of HCl, which is ultimately responsible for lowering the body's acid-buffering capacity and induction of acidosis. To confirm that the observed inhibition of muscle protein synthesis was a consequence of acidosis and not a specific effect of NH4Cl, i.e., through urea production, a different model of acute metabolic acidosis was also investigated. In experiment 4, tissue protein synthesis was measured after the animals were treated with a cation exchange resin. When the resin is administered in the H+ form, since its affinity for hydrogen ions is lower than for other cations, it induces acidosis by releasing hydrogen ions in exchange for other cations in the gastrointestinal tract, thus lowering the systemic pH. The administration of the same resin in the Na+ form, on the contrary, does not modify blood pH, representing an optimal control treatment. The results of experiment 4, showing an inhibition of muscle protein synthesis in skeletal muscle and no effect on liver in acidotic rats, are comparable to those obtained in experiments 2 and 3, in which acidosis was induced with NH4Cl (Tables 25). The findings demonstrate that the inhibitory effect on muscle protein metabolism can be reproduced using different models of acidosis and that they therefore are not specific to NH4Cl.
The inhibition of protein synthesis by acute metabolic acidosis is limited to skeletal muscle, and no effect on cardiac muscle or other visceral organs, such as liver, kidney, spleen, and gut could be detected after 24 h (Tables 3 and 4). Among skeletal muscles, soleus showed a smaller and less consistent inhibitory effect by acidosis compared with gastrocnemius and plantaris muscles (the small difference in protein synthesis rates between control and acidotic animals did not reach statistical significance in experiments 3 and 4; Tables 2, 3, and 5). Soleus is a mainly oxidative muscle formed predominantly by slow-twitch fibers, compared with gastrocnemius and plantaris, which have a mixed-fiber composition, and has been shown to be less responsive to several nutritional and pharmacological treatments (e.g., Refs. 1, 21, 40). The different response of individual skeletal muscles to acidosis may therefore be dependent on their fiber type composition, with oxidative slow-twitch muscles being less sensitive to the inhibitory effect of acidosis.
Because muscle contributes to only 20% of whole body protein turnover (14), this may explain why any effect of acidosis on protein synthesis could not be consistently detected in previous studies in which whole body protein turnover was measured in humans. Ballmer et al. (3) observed a significant drop in the synthesis rate of the major liver export protein albumin after 7 days of metabolic acidosis in healthy volunteers. However, it is not known whether that inhibition is specific to albumin or whether it represents a general response of liver protein to acidosis. When a measurement of albumin synthesis rate was made after only 2 days of acidosis, synthesis rates were not altered (23), in agreement with the results of our study showing no effect of 24-h metabolic acidosis on total liver protein synthesis rates (Tables 3, 4, and 5). This suggests that liver albumin synthesis may not be affected acutely but only after longer periods of acidosis.
Apart from skeletal muscle, the only other cells that were shown to be sensitive to acidosis were lymphocytes. Lymphocyte protein synthesis rates were lower in acidotic animals compared with controls (Table 4), although the effect of acidosis was quite small (8%, P < 0.05) compared with muscle. Lymphocyte protein synthesis is a potential marker of lymphocyte in vivo metabolic activity, reflecting the amount of protein synthesized for the production of new cells and for secretion of antibody as well as the basal cellular protein. Lymphocyte protein synthesis has been shown to be stimulated in conditions in which immune cells are activated and proliferate (22, 39), and it therefore also represents an important marker of the prevailing immune activation in vivo (8, 28, 39). The findings of a lower lymphocyte protein synthesis in acidotic animals therefore indicates that lymphocyte metabolic activity may be depressed by acidosis and that lymphocyte activation and responsiveness to immune challenges may also be affected by lower pH. This is in line with previous observations of impaired immune function at acidic pH (24) and suggests that acidosis itself may be one of the factors contributing to immunosuppression and/or immune dysfunction often observed in clinical conditions associated with acidosis (10, 24).
Inhibition of protein synthesis can be achieved through one or more distinct mechanisms that alter the number of ribosomes in the cell, the translational efficiency of ribosomes, and/or the concentration of translatable mRNA. In the present study, we found that the total muscle RNA content was not different in acidotic and control animals (Table 2). Because the majority of cellular RNA is ribosomal (47), the findings suggest that the observed inhibition of muscle protein synthesis by acidosis is achieved through a reduction of ribosomal translational efficiency, without any major changes in the total RNA cell content (Table 2). Acute acidosis appears, therefore, to downregulate the synthesis of muscle protein at the level of translation. However, further investigation is required to elucidate which step in the translation process is affected and whether the effect of pH is direct or mediated via secondary factors such as hormones. It is also not clear whether extracellular pH acts directly on cell membrane to influence protein synthesis [i.e., through specific receptors for pH or by influencing receptors for other substances (e.g., Refs. 5 and 45)] or whether the effects on protein synthesis are dependent on changes of intracellular pH.
The plasma concentrations of several amino acids were found to be elevated in acidotic animals (Table 1). Because in the fasting state plasma amino acids are supplied mainly by degradation of endogenous protein, increased plasma amino acid levels suggest that the net catabolism of body protein was enhanced by acute acidosis. May et al. (31) observed depressed plasma levels of branched-chain amino acids (BCAA) in rats made acidotic with NH4Cl and suggested that the drop in the plasma concentration of BCAA may be a consequence of the stimulation in the activity of muscle and liver branched-chain keto acid dehydrogenase, the rate-limiting enzyme of BCAA catabolism, by acidosis (31, 34). However, other studies in humans and animals have not shown a specific depression of BCAA plasma concentration in acidosis (42, 43). In the present study, we detected a drop in plasma concentration of isoleucine and no changes in valine, whereas leucine concentration tended to be higher in acidotic animals (Table 1). The apparent discrepancy with the findings of May et al. may in part be explained by the difference in the study design and in particular in the duration of acidosis, which was 5 days in the study of May et al. However, there is no evidence from the present results to suggest that the fall in muscle protein synthesis is mediated by depressed plasma levels of BCAA or other amino acids.
In conclusion, this study demonstrates that acute metabolic acidosis depresses protein synthesis in skeletal muscle but does not affect protein synthesis in heart, liver, kidney, and intestine in rats. The results indicate that a direct inhibition of muscle protein synthesis might be another mechanism contributing to the loss of muscle tissue observed in clinical conditions associated with acidosis. A small decrease in protein synthesis was also observed in lymphocytes, suggesting that acidosis itself might affect the metabolism of immune system cells and contribute to impaired immune function in clinical conditions accompanied by acidosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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