1 United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; 2 United States Department of Agriculture/Agricultural Research Service Growth Biology Laboratory, Beltsville, Maryland 20705; and 3 Department of Animal and Dairy Science, Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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Somatotropin (ST) administration enhances
protein deposition in well-nourished, growing animals. To determine
whether the anabolic effect is due to an increase in protein synthesis
or a decrease in proteolysis, pair-fed, weight-matched (~20 kg)
growing swine were treated with porcine ST (150 µg · kg1 · day
1,
n = 6) or diluent (n = 6) for 7 days. Whole body
leucine appearance (Ra), nonoxidative leucine disposal
(NOLD), urea production, and leucine oxidation, as well as tissue
protein synthesis (Ks), were determined in the fed
steady state using primed continuous infusions of
[13C]leucine,
[13C]bicarbonate, and
[15N2]urea. ST treatment increased
the efficiency with which the diet was used for growth. ST treatment
also increased plasma insulin-like growth factor I (+100%) and insulin
(+125%) concentrations and decreased plasma urea nitrogen
concentrations (
53%). ST-treated pigs had lower leucine
Ra (
33%), leucine oxidation (
63%), and urea
production (
70%). However, ST treatment altered neither NOLD
nor Ks in the longissimus dorsi, semitendinosus, or
gastrocnemius muscles, liver, or jejunum. The results suggest that in
the fed state, ST treatment of growing swine increases protein
deposition primarily through a suppression of protein degradation and
amino acid catabolism rather than a stimulation of protein synthesis.
protein synthesis; insulin-like growth factor I; insulin; growth hormone; muscle
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INTRODUCTION |
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THE QUEST FOR INCREASED LEAN muscle production has been a focus of many studies involving somatotropin (ST) administration and other anabolic growth promoters. Exogenous administration of ST to domestic animals increases both weight gain and the efficiency with which dietary amino acids are utilized for protein deposition (16, 17). In swine, ST administration increases protein deposition (4, 5) and decreases fat accretion, while simultaneously lowering feed intake (1). The increase in growth that occurs with ST administration includes not only that of skeletal muscle, but also that of skin, intestine, and liver (5, 6).
The increase in protein deposition with ST treatment is accompanied by a reduction in blood urea nitrogen (PUN) concentrations (3, 12, 33, 36), suggesting that ST treatment reduces the catabolism of amino acids. This possibility is supported by direct measurement of amino acid oxidation in adult humans (19, 24). However, the role of amino acid catabolism in the enhanced protein deposition that occurs with ST treatment of domestic animals is not completely understood.
It is generally held that the increase in protein deposition with ST treatment is due to an increase in protein synthesis rather than to a reduction in protein degradation. This premise is based on studies that were performed either in adult humans or in domestic animals approaching maturity (3, 14, 18, 19, 24, 31, 33). On the other hand, our recent study in young pigs (34) suggests that ST treatment has little effect on protein synthesis, despite an enhancement in the gain of tissue mass. Rates of protein degradation were not determined. There is also considerable variability in the protein synthetic responses of different tissues to ST treatment. For example, ST treatment of mature steers increases protein synthesis in some, but not all, skeletal muscles, and it has no effect on protein synthesis in liver and intestine, despite an increase in tissue mass (14). Thus the mechanisms that regulate the enhanced rate of protein deposition in growing animals treated with ST remain unclear.
The objectives of this study were 1) to determine whether the increase in protein deposition after ST treatment is due to a suppression in whole body proteolysis and/or a stimulation in whole body protein synthesis, 2) to identify which tissues respond to ST with an increase in protein synthesis, and 3) to determine whether the reduction in PUN with ST administration reflects a lower rate of urea production and amino acid oxidation. Studies were performed in rapidly growing young swine (~20 kg) in which the inherent rate of protein accretion was about threefold higher than that of the mature animals used in most other studies that investigated the effects of ST on protein metabolism (e.g., 50- to 100-kg swine, Ref. 15). In addition, energy and protein intakes were matched in ST and control pigs during the 7 days of treatment and were rigorously controlled during the 8-h isotope infusion study to ensure a fed steady-state status.
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MATERIALS AND METHODS |
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Animals and design.
Twelve crossbred (Landrace × Yorkshire × Hampshire × Duroc) barrows (castrated males, Agriculture Headquarters, Texas
Department of Criminal Justice, Huntsville, TX) weighing 15 kg (~10
wk old) were housed in individual cages. Because relatively high
protein intakes are required to obtain the maximum growth-promoting
effects of ST (4, 5), pigs were fed a 24% protein diet (Producers Cooperative Association, Bryan, TX). During an initial 7-day adjustment period, the diet was provided at 6% body wt, a level which is ~90%
of the ad libitum intake of pigs of this age. Water was provided ad
libitum. After the 7-day adjustment period, pigs were assigned randomly
to one of two treatment groups, either diluent (saline; control,
n = 6) or recombinant porcine ST (n = 6, Southern Cross Biochemicals, Melbourne, Australia) at a rate of 150 µg · kg body wt1 · day
1
for 7 days. This dose has been demonstrated to be slightly greater than
that required to maximally stimulate protein accretion in swine (100 µg · kg body
wt
1 · day
1)
(15). The dose of saline or ST was divided into two daily injections
(75 µg/kg body wt) administered into the neck region in alternating
sides. Body weights were measured daily, and the dietary intake and
treatment doses were adjusted accordingly. The ST-treated pigs were
offered the diet at 6% body wt/day, and the control pigs were pair-fed
to the intake level of the ST-treated pigs to minimize confounding
effects of differences in feed intake. The daily feed allowance was
divided into two meals to coincide with the injection times. Pigs
generally consumed all feed presented to them, and if not, any
unconsumed or spilled food was accounted for in the estimate of feed intake.
Infusions.
Pigs were placed in individual cages and maintained in the fed state
throughout the 8-h infusion period by being fed hourly meals. The daily
allowance of feed was partitioned into hourly meals based on a rate of
6% body wt/day (i.e., 2% body wt over the 8 h of study). On the day
of stable isotope infusions, the pigs were administered their daily
dose of ST (150 µg/kg body wt) or diluent and fed their first meal at
60 min before infusion. To measure whole body protein turnover, tissue
protein synthesis, and urea production, pigs were infused for 8 h. Pigs
were administered a primed (7.5 µmol/kg body wt), continuous (10 µmol · kg body wt1 · h
1)
infusion of [13C]bicarbonate from 0 to 120 min
to determine CO2 production rate. A primed (10 µmol/kg
body wt), continuous (10 µmol · kg body
wt
1 · h
1)
infusion of [1-13C]leucine lasting from 120 to
480 min was given to determine whole body leucine flux, leucine
disposal into protein, leucine oxidation, and tissue fractional protein
synthesis rates. A primed (242 µmol/kg body wt), continuous (24.2 µmol · kg body
wt
1 · h
1)
infusion of [15N2]urea lasting from
0 to 480 min (Cambridge Isotope Laboratories, Andover, MA) was
administered to quantify amino acid catabolism. Blood and breath
samples were collected at
10, 0, 90, 100, 110, 120, 420, 440, 460 and 480 min for measurement of 13C and
15N2 enrichment. Additional blood samples were
collected every 30 min throughout the 480-min infusion for measurement
of glucose and insulin concentrations. Concentrations of insulin-like
growth factor I (IGF-I), glucagon, amino acids, ST, and PUN were
measured at 0, 240, and 480 min. Pigs were killed at 480 min by
exsanguination under anesthesia (pentobarbital sodium). Immediately
after exsanguination, tissue samples from the longissimus dorsi,
semitendinosus, and gastrocnemius muscles, liver, and jejunum were
rapidly removed and frozen in liquid nitrogen.
Analysis of tracer enrichment. Tissues were homogenized in 0.2 M perchloric acid (PCA) as previously described (8). Briefly, the homogenate supernatant containing the tissue free amino acid pool was separated from the PCA-insoluble precipitate. The PCA-insoluble precipitate was washed and solubilized. An aliquot of the solubilized pellet was assayed for protein with the use of bicinchoninic acid (Pierce, Rockford, IL) by the method of Smith et al. (32). The remaining solution was reacidified, and the acid-soluble fraction was assayed for RNA by the method of Munro and Fleck (27). The protein pellet was washed (4 times) and hydrolyzed for 24 h with 6 N HCl. The protein and blood supernatants were analyzed for [1-13C]leucine enrichment.
Leucine was isolated by cation exchange chromatography (AG-50W resin, Bio-Rad, Hercules, CA). Leucine was derivatized with a trifluoroacetyl-methyl ester (F3NAM) derivative (26) and prepared for measurement of isotopic leucine enrichment with continuous flow gas chromatography combustion isotope ratio mass spectrometry (GIRMS) (Hewlett-Packard 5890 Series II GC equipped with a Europa Orchid 20/20 stable isotope analyzer; Hewlett-Packard, Palo Alto, CA). In addition, the isotopic enrichment of free [13C]leucine was determined by electron impact ionization (EI) gas chromatography mass spectrometry (GC-MS) by monitoring ions at mass-to-charge ratios (m/z) 153 and 154. The enrichment of 13CO2 from the oxidation of [1-13C]leucine was measured by Europa RoboPrep-G GC-MS analysis (Hewlett-Packard). PlasmaPlasma hormones and amino acids.
Heparinized blood samples were obtained and centrifuged, and the plasma
was frozen at 70°C. Plasma insulin concentrations were
measured with a porcine insulin RIA kit (Linco, St. Charles, MO) with
the use of porcine insulin antibody and human insulin standards. Blood
glucose concentrations were rapidly analyzed during the infusion period
by means of a glucose oxidase reaction (YSI 2300 STAT Plus, Yellow
Springs Instruments, Yellow Springs, OH). Plasma glucagon
concentrations were measured with a glucagon RIA kit (Linco). Plasma
total IGF-I concentrations were measured by means of a two-site
immunoradiometric assay for human IGF-I (Diagnostic System
Laboratories, Webster, TX). Porcine ST (pST) was assayed by use of a
previously described method with minor modifications (6). Briefly,
purified pST was iodinated using IODO-GEN [Pierce Chemical,
Rockville, IL (10)]. To improve sensitivity, a nonequilibrium
assay format was used. Standard (pST-I-1, range 0.2-20 ng/tube) or
sample and baboon antiporcine ST were incubated for ~18 h. Then
~12,000 cpm 125I-labeled pST were added and tubes
incubated for an additional 24 h. The immune complex was precipitated
with goat anti-monkey serum and cold 10% polyethylene glycol(PEG).
Assay sensitivity was ~3 ng/ml, and the intra-assay and interassay
coefficients of variation were <10%. PUN concentrations were
measured with the use of an end-point enzyme assay (Roche, Somerville, NJ).
Calculations.
Whole body protein turnover was calculated by means of the following
standardized steady-state equations
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Statistics. ANOVA for repeated measures (SPSS) was used to test for changes with treatment during intensive sampling during the 8-h infusion for plasma insulin, glucose, IGF-I, ST, PUN, glucagon concentrations, and [1-13C]leucine and [1-13C]KICA enrichment. Two-way ANOVA (SPSS) was used to test for changes with treatment for body weight, daily weight gain, gain-to-feed ratio, fractional rate of protein synthesis, whole body protein turnover, and urea synthesis. Results are presented as means ± SD. Probability values of <0.05 were considered statistically significant and are not reported in the text.
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RESULTS |
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Animal weights. Body weight did not differ significantly between control and ST-treated pigs at the initiation of the treatment period (18.6 ± 0.74 vs. 17.7 ± 1.68 kg, respectively) and at the end of the 7 days of treatment (24.0 ± 1.82 vs. 24.2 ± 1.91 kg, respectively). However, the daily gain in body weight for the treatment period tended (P < 0.07) to be higher in the ST-treated pigs compared with controls (0.91 ± 0.14 vs. 0.78 ± 0.23 kg, respectively). The efficiency with which feed was used for growth was greater in ST-treated pigs than in controls, as reflected by their higher gain-to-feed ratios (0.75 ± 0.11 vs. 0.61 ± 0.15 kg gain/kg feed intake).
Plasma hormone and substrate concentrations.
The plasma hormone and substrate concentrations shown in Table
1 are mean values over the 8-h infusion
period. Plasma ST concentrations were elevated in ST-treated pigs
compared with negligible levels in controls. Plasma IGF-I and insulin
concentrations were twofold higher in ST-treated pigs than in controls,
and glucose concentrations were also elevated (+22%) in ST-treated
pigs compared with controls. Glucagon concentrations were lower
(25%) in ST-treated than in control pigs; PUN concentrations
were decreased by >50% in ST-treated pigs compared with controls.
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Whole body protein turnover.
Plasma leucine, -KICA, urea, and breath carbon dioxide had reached
substrate and isotopic steady state during the last 3 h of infusion
(data not shown). ST treatment decreased leucine flux (
18%),
endogenous Ra (
34%), and leucine oxidation
(
63%) in comparison with controls (Fig.
2). NOLD was virtually identical in the two
treatment groups.
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Tissue protein synthesis.
The fractional rates of protein synthesis (Ks) were
determined with several precursor pool models (intracellular free
leucine pool, plasma leucine, and plasma -KICA). Also, two different methods (GIRMS and GC-MS) were used to analyze isotopic enrichment values. Each precursor pool model and analytical method gave similar results (data not shown); therefore, the
-KICA precursor pool model
was used to calculate Ks for all tissues. The
Ks in the longissimus dorsi, semitendinosus, and
gastrocnemius muscles, and in the liver and jejunum were similar in
ST-treated and control pigs (Table 3).
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DISCUSSION |
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The results of the present study demonstrate that a week of ST treatment in rapidly growing swine increases protein balance and the efficiency with which feed is used for growth. This improvement in protein anabolism can be attributed to a suppression in both whole body proteolysis and amino acid catabolism in the fed, steady state. This is evident from the reduction in total leucine flux and leucine appearance from protein (endogenous Ra), an indicator of whole body protein proteolysis, in ST-treated pigs. In addition, leucine oxidation and the rate of urea synthesis were markedly lower in ST-treated pigs. However, neither the rates of whole body protein synthesis nor the individual tissue fractional rates of protein synthesis were altered by ST treatment. It is critical to emphasize that the amino acid and energy intakes both before and during the isotope infusions were the same for both treatment groups. Despite the carefully maintained pair-feeding design, plasma amino acid concentrations were lower in ST-treated pigs, likely reflecting the altered balance between protein synthesis and proteolysis.
Growth and metabolic effects of ST. The results of the present study indicate that 7 days of administration of ST to rapidly growing swine successfully elicited the classic metabolic responses that are characteristic of ST treatment in domestic animals. Two of the fundamental anabolic effects of ST treatment in domestic animals are increased body weight gain and an increase in the efficiency with which the diet is used for growth (16, 17, 31, 34). In the present study, administration of ST to rapidly growing swine improved feed efficiency, and after only 7 days of treatment, differences in weight gain were already emerging. Well established metabolic effects of ST administration include the characteristic stimulation of the somatotropic axis, indicated by an increase in circulating IGF-I concentrations (5, 12, 34, 36), and the diabetogenic effect, indicated by an increase in circulating concentrations of both insulin and glucose (6, 12, 35, 36). In the present study, a 22-fold elevation in plasma ST concentrations resulted in a twofold increase in plasma IGF-I and insulin concentrations and a 22% increase in plasma glucose concentrations. Plasma glucagon levels were lower in ST-treated pigs compared with controls, a response which has been reported previously in the fed but not the fasting state (25, 35).
Another characteristic metabolic effect of ST treatment is a reduction in PUN concentrations (3, 12, 28, 36). In this study, PUN concentrations were decreased by 53% in ST-treated pigs. These results, in combination with the observed improvement in total feed efficiency, suggest that ST treatment of young growing animals results in a more efficient use of dietary amino acids for growth. Reports of the effect of ST treatment on plasma amino acid concentrations are limited and variable (3, 11, 24). In this study, total plasma amino acid concentrations, as well as essential and branched-chain amino acid concentrations, were lower in ST-treated pigs compared with controls, despite the similar intakes of both protein and energy in the two groups of animals. This reduction in plasma amino acid concentrations could reflect either an increase in removal from, and/or a decrease in entry to, the plasma amino acid pool in response to ST treatment.Amino acid catabolism. Our results indicate that ST treatment reduces amino acid catabolism. This reduction in amino acid catabolism by ST treatment was demonstrated by the reduction in leucine oxidation, urea synthesis, and plasma urea nitrogen concentrations. Moreover, the percentages of reduction by ST treatment in all three measures of amino acid catabolism were similar, i.e., 63, 70, and 53%, respectively.
Two possible mechanisms can explain the ST-induced decrease in leucine oxidation and urea production. First, ST treatment lowered circulating amino acid concentrations. Because the rate of catabolism of amino acids is a direct function of the circulating level of those amino acids (29), the lower circulating leucine concentrations may well be responsible for the lower rate of amino acid catabolism. Second, lower rates of amino acid oxidation and urea production may reflect a direct effect of ST on the pathways of amino acid catabolism. This hypothesis is supported by the reduction in leucine oxidation in ST-treated heifers (13), the reduction in lysine, methionine, and valine oxidation in livers of ST-treated rats (2), and the reduced urea nitrogen synthesis capacity and mRNA levels of urea cycle enzymes in ST-treated rats (22).Proteolysis. The regulation of protein deposition occurs through changes in the balance between the rates of protein synthesis and proteolysis. Based on previous reports in the literature (3, 14, 33), we had hypothesized that ST treatment would increase protein deposition through an increase in protein synthesis rather than by a reduction in protein degradation. For example, studies in mature 65-kg pigs (33) and 240-kg cattle (3) found either an increase (33) or no change (3) in proteolysis with ST treatment. However, a recent study by Dawson et al. (11) demonstrated that ST treatment of 168-kg steers reduced leucine flux in cattle that were fed an energy- and protein-rich diet, but not in those fed at a lower plane of nutrition.
The results of the present study demonstrate that in the fed steady state, ST increases protein deposition in rapidly growing swine through a suppression of protein degradation rather than by a stimulation of protein synthesis. This is evident from the reduction in total leucine flux and leucine appearance from protein (endogenous Ra), an indication of whole body proteolysis, in ST-treated pigs compared with controls. The reduction of protein degradation in ST-treated pigs resulted in a dramatic increase in protein balance (177%), which suggests an increase in the overall metabolic efficiency of ST-treated pigs. However, the current study does not indicate in which tissues the reductions in proteolysis occurred.Protein synthesis. We observed no effect of seven days of ST treatment on protein synthesis in rapidly growing swine when this was measured under fed steady-state conditions. This lack of effect of ST treatment on protein synthesis in individual tissues was consistent with our finding on a whole body basis, as indicated by the lack of change in the rate of total leucine disposal into protein. In addition, the fractional rates of protein synthesis in three muscles, the longissimus dorsi, semitendinosus, and gastrocnemius, and in two visceral tissues, liver and jejunum, were unaltered by ST treatment. This lack of effect of ST treatment on the fractional rates of tissue protein synthesis contrasts with that of other studies in mature 52-kg swine (31), 289-kg steers (14), and adult humans (19). On the other hand, the protein synthetic capacity (RNA-to-protein ratio) of one of the three muscles and the liver was increased by ST treatment in the present study, suggesting that there is the potential for an elevation in protein synthesis in growing pigs treated with ST.
We hypothesize that protein synthesis was not stimulated by ST treatment in the present experiment because the inherent rate of protein synthesis in these rapidly growing, fully fed pigs was already elevated and, theoretically, near maximum. There are several lines of evidence to support this hypothesis. First, fractional rates of protein synthesis, particularly in skeletal muscle, are quite elevated in young, growing animals, and they decrease with age (7, 8, 21). In this study, muscle protein synthesis rates were almost as high as those of neonatal pigs (7, 34) and were substantially higher than those in mature swine (31), cattle (14), and humans (19). In our previous study with neonatal pigs, ST treatment had no effect on tissue protein synthesis, despite an increase in body weight and tissue protein mass (34). This suggests that an enhancement of protein synthesis rates in young, rapidly growing animals, which have inherently high rates of protein synthesis, may be difficult to attain. Second, one of the most potent stimulators of protein synthesis is eating (7, 9, 20, 30). In the current study, feed intake was rigorously controlled during the 8-h infusion to ensure the fed steady-state status; thus it is likely that protein synthesis rates were fully stimulated. However, most previous studies that have examined the effects of ST on protein turnover were not performed in the fully fed state (14, 18, 19, 31, 33). This suggests that in fully fed animals with elevated rates of protein synthesis, the potential for ST administration to further stimulate the already high rate of protein synthesis may be minimal. On the other hand, the study does not exclude the possibility that ST administration can enhance protein synthesis in the postabsorptive state in young swine or even in the postprandial state in older animals. Moreover, the elevation in protein synthetic capacity in muscle and liver of ST-treated pigs suggests that the potential for stimulation of protein synthesis by ST can be realized if protein synthesis rates are reduced by fasting or when the inherent rate of protein synthesis declines as the animal matures.Perspectives. The results of this study suggest that, during meal absorption, ST treatment can enhance protein deposition in rapidly growing swine by suppressing protein degradation and amino acid catabolism rather than by stimulating protein synthesis. This inability of ST to stimulate protein synthesis may be due to the inherently high rate of protein synthesis, which is characteristic of the fully fed pig during early stages of development. Thus the more efficient use of amino acids for protein deposition, which is characteristic of ST treatment, is attained by the suppression of proteolysis, rather than by a stimulation of protein synthesis, during meal absorption in growing pigs administered ST. Whether ST treatment of young pigs can enhance protein synthesis in conditions when protein synthesis is submaximal, e.g., postabsorption, remains to be established.
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ACKNOWLEDGEMENTS |
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The authors thank M. Haymond and F. Jahoor for their helpful suggestions. We also express appreciation for the technical assistance of J. Wen, R. Parsons, J. Cunningham, M. Stubblefield, and F. Biggs. We thank E. O. Smith for statistical assistance, L. Loddeke for editorial review, A. Gillum for graphics, and M. Alejandro for secretarial assistance.
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FOOTNOTES |
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This project has been funded by the US Department of Agriculture, National Research Initiative Grant 96-35206-3657, the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 58-6250-6-001, and National Institute of Child Health and Human Development Training Grant T32-HD-07445.
This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).
Received 16 July 1999; accepted in final form 27 October 1999.
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