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Nutritional Methodology

Fractional Protein Synthesis Rates Measured by an Intraperitoneal Injection of a Flooding Dose of L-[ring-²H₅]Phenylalanine in Pigs^1,2

Kristjan Bregendahl^*,3, Lijuan Liu^*, John P. Cant^*, Henry S. Bayley, Brian W. McBride^*, Larry P. Milligan^*, Jong-Tseng Yen^** and Ming Z. Fan^*,4

Departments of ^* Animal and Poultry Science and Human Biology and Nutritional Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 and ^** U.S. Department of Agriculture/Agricultural Research Service, The United States Meat Animal Research Center, Clay Center, NE 68933

⁴To whom correspondence should be addressed. E-mail: mfan{at}uoguelph.ca.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our objectives were to examine the effect of an i.p. injection of a flooding dose of L-phenylalanine (Phe) containing L-[ring-²H₅]Phe on time courses of physiologic responses, the tracer Phe enrichments, and fractional protein synthesis rates (FSR) in plasma, visceral organs, and muscles. In a randomized complete block design, 5 blocks of 5 littermate piglets were weaned at 16 d of age and injected i.p. with a flooding dose of L-Phe (1.5 mmol/kg body weight) on d 8 postweaning under fed conditions. Tissues were collected at 15, 30, 45, 60, and 75 min postinjection. Plasma glucose concentration increased (cubic effect, P < 0.05) from 4.8 preinjection to 5.8 mmol/L 15 min postinjection and returned to preinjection levels thereafter. Plasma insulin concentration did not change (P > 0.05) over time. Plasma Phe concentration increased logarithmically (P < 0.05) from 85 to 711 µmol/L and reached 95% of the maximum concentration 48 min postinjection, but no changes (P > 0.05) in tissue contents of other free amino acids were observed. The Phe free pools in plasma, visceral organs, and muscles were evenly enriched (32.3 ± 1.4 mol%) with L-[²H₅]Phe 15 min after the i.p. injection. The FSR in visceral organs did not change (P > 0.05), whereas plasma and muscle protein FSR decreased (P < 0.05) over time. We conclude that the i.p. injected tracer Phe rapidly distributed into plasma and intra- and extracellular spaces, and was effective for measuring FSR in visceral organs, but not in plasma and muscles of pigs.

KEY WORDS: • route of tracer delivery • stable isotopes • isotopic enrichment • piglets

Protein synthesis accounts for 20% or more of daily energy expenditure in growing animals (1). It is affected by the growth or developmental stage (2,3), plane of nutrition (4,5), immune status (6–8), activity level (9), hormones (3,10), and stress (11) of the animals. The study of protein metabolism, therefore, is integral to understanding animal biology and the effects of dietary and/or medicinal treatments.

Fractional protein synthesis rates (FSR)⁵ can be measured by the continuous-infusion or the flooding-dose methods, both of which rely on the administration of a labeled amino acid tracer and its subsequent incorporation into protein (12–14). For the continuous-infusion method, several hours of constant tracer infusion are required to label the precursor pool, during which the study subject must remain in steady state. Furthermore, a potential problem with the continuous-infusion method is a nonuniform labeling of the plasma free, intracellular free, and tRNA-bound precursor pools (15). This problem can be minimized by administering a substantially larger dose of a labeled amino acid to flood amino acid free pools in all tissues, minimizing the difference between tracer amino acid enrichments or specific activities of intra- and extracellular free amino acid pools and the true protein precursor, amino-acyl tRNA (16–18). The flooding-dose method also shortens the time required to reach uniform precursor tracer enrichment or specific activities from hours to minutes after administration, making the method more suitable to nonsteady-state situations and for proteins with a relatively high FSR (18–20). Although the flooding dose can be delivered i.v. using a syringe and needle, surgical placement of at least 1 catheter under anesthesia and an ensuing recovery period are required. The trauma inflicted by surgery affects protein metabolism (19,21), whereas the postsurgical period carries with it risks of infection and catheter occlusion.

Using i.p. injection of a flooding dose of a labeled tracer to measure FSR would eliminate the need for surgery and postsurgical care of the animals. When injected i.p., amino acids were detected in plasma within minutes after injection (22–24). When injected i.p., L-lysine · HCl was used for body weight (BW) gain with the same efficiency as orally ingested L-lysine · HCl (25). Amino acid tracers were injected i.p. to measure FSR in mice (8,26), rats (27,28–30), and poultry (31,32). Nevertheless, tracer i.p. administration is not commonly used in measurements of FSR, perhaps due to limited information on physiologic responses and the behavior of tracer amino acid isotopic enrichments or specific radioactivities in the free and bound pools of target tissues (26,28–31).

To our knowledge, no published information exists on overt physiologic responses to i.p. injection of an amino acid tracer, the behavior of the free and bound pool tracer enrichments, and the measurement of FSR beyond 30 min postinjection. Therefore, the objectives of the present study were to investigate the time course of physiologic responses, isotopic enrichment of the free and bound tracer pools in the target tissues, and FSR estimates after an i.p. injection of a flooding dose of L-Phe containing a stable-isotope Phe tracer in piglets.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and design. Five blocks of 5 littermate purebred Yorkshire gilts from the University of Guelph Arkell Swine Research Station were weaned at 16 d of age and given free access to a commercial-type pelleted diet for 8 d. The diet, containing 14.0 MJ/kg of metabolizable energy and 1.2% of true ileal digestible lysine, was formulated to meet or exceed nutrient recommendations by the NRC (33). The use and care of the animals conformed to the guidelines of the Canadian Council on Animal Care (34). The procedures were approved by the Animal Care Committee at the University of Guelph.

    Infusion protocol and sample collection. On d 8 postweaning, littermate pigs with free access to the diet and water were randomly assigned to 1 of 5 treatments (i.e., labeling for 15, 30, 45, 60, or 75 min, respectively) and injected i.p. with a flooding dose of L-Phe (1.50 mmol/kg BW) containing L-[ring-²H₅]Phe at 40 mol% (0.60 mmol/kg BW; Cambridge Isotopes) dissolved in sterile saline (154 mmol/L). The i.p. injection was completed in 5–10 s. Venous blood samples (10 mL) were collected by puncture of the orbital sinus into prechilled centrifuge tubes containing Na₂-EDTA (Sigma) immediately before injection and 15, 30, 45, 60, or 75 min postinjection, depending on the treatment. To better define the plasma free pool tracer enrichment curve, an additional blood sample was collected from each pig 7 min before killing, resulting in a collection of 3 blood samples from each pig. The exact time (min) of tracer labeling measured from the end of i.p. tracer injection to the time the blood samples were placed on ice was recorded. Immediately after the last blood sample was collected, pigs were killed with an intracardial injection of sodium pentobarbital (50 mg/kg BW; Schering Canada) and bled by exsanguination. After opening the abdomen, organs (i.e., samples of proximal small intestine, samples of distal small intestine, samples of colon, cecum, pancreas, spleen, heart, lungs, kidneys), and samples of longissimus dorsi and biceps femoris were quickly removed, thoroughly rinsed in ice-cold saline (154 mmol/L, pH 7.4) containing a protease inhibitor (0.1 mmol/L phenylmethylsulfonyl fluoride; Sigma), and snap-frozen in liquid nitrogen. The exact time (min) of labeling measured from the end of i.p. tracer injection to the time all of the tissue samples were frozen in liquid nitrogen was recorded.

    Sample preparation and GC-MS. After collection, blood was stored on ice until centrifugation (2000 x g at 4°C for 20 min) and the plasma frozen at –70°C until analysis. Tissue samples, stored at –70°C, were subsampled and pulverized under liquid nitrogen using a mortar and pestle. Duplicate plasma (0.75 mL) and pulverized tissue samples (0.5 g) were homogenized (PowerGen 700D, Fisher Scientific) on ice with 200 µL L-norleucine solution (2.5 mmol/L; Sigma) in 4 mL of trichloroacetic acid (TCA) solution (2 mol/L; Sigma), and subsequently centrifuged (2000 x g at 4°C for 20 min). The supernatant containing the free amino acid tracer pool was applied to a cation-exchange column (Dowex 50W x 8 H⁺-form; Bio-Rad), washed to neutrality with deionized water, and the free amino acids eluted with 2 mL of NH₄OH solution (4 mol/L; Sigma). The pellet containing the bound pool was washed 3 times in 4 mL of the TCA solution, and hydrolyzed in 3 mL of an HCl solution (6 mol/L; Sigma) at 110°C for 24 h in screw-capped tubes purged with nitrogen gas. After the hydrolysis, 3 mL of the hydrolyzed samples was centrifuged (2000 x g at 4°C for 20 min) and the resulting supernatant was further cleaned on the cation-exchange column as described above for the free amino acid tracer pool samples.

The isotopic enrichment of L-[²H₅]Phe in plasma and tissue free and bound pools was determined in duplicate by GC-MS as the n-propyl heptafluorobutyrate derivative (35,36) with a model 6890 GC linked to a 5973N quadrupole MS (Agilent Technologies) operating in the electron ionization mode. Ions with mass-to-charge ratios of 91 and 96 were monitored and converted to percentage molar enrichment (mol%) using calibration curves prepared with purified L-[ring-²H₅]Phe standards (Cambridge Isotopes). Concentrations of free amino acids in plasma and the contents of free amino acids in tissues were obtained from the chromatographs using L-norleucine as an internal standard (36). The plasma concentration of glucose was determined in duplicate using a glucose-oxidase kit (Sigma Diagnostics #510-DA), and the plasma concentration of insulin was determined in duplicate by RIA (Coat-A-Count Insulin; Diagnostic Product).

    Calculations and statistical analyses. The change in isotopic enrichment of L-[ring-²H₅]Phe in plasma over time was described according to Wolfe (14) as

(1)

where E_{Free t} is the predicted isotopic enrichment of L-[ring-²H₅]Phe (mol%) in the plasma free pool at time t, E_Max is the L-[ring-²H₅]Phe isotopic equilibrium (mol%) in plasma, k is the rate constant (1/min) for elimination of the tracer, and t is the exact time (min) of tracer labeling measured from the end of i.p. tracer injection to the time the blood samples were placed on ice. The time to reach 99% of the maximal isotopic enrichment of L-[ring-²H₅]Phe was calculated from the integral of Eq. (1):

(2)

where t_{99% of EMax} is the time (min) to reach 99% of the tracer isotopic equilibrium.

The time course data of both the tracer free and bound pool enrichments allowed us to simultaneously compare 2 methods of calculating FSR for different tissues and organs. The FSR was calculated according to Garlick et al. (37) as

(3)

where FSR₃ is the fractional protein synthesis rate (%/d) defined as the percentage of protein renewed in a day, E_{Bound t} is the isotopic enrichment (mol%) of L-[ring-²H₅]Phe in the bound tissue pool at time t, E_{Free t} is the observed isotopic enrichment (mol%) of L-[ring-²H₅]Phe in the free tissue pool at time t, and t is the exact time (min) of labeling measured from the end of the i.p. tracer injection to the time the tissue sample was frozen in liquid nitrogen. In addition, the area under the plasma tracer enrichment curve (Eq. 1) between the time of injection and t min postinjection (A_{Free t}; % x min) was used to estimate the precursor tracer enrichment for calculations of tissue FSR according to Wolfe (14) as

(4)

The constants E_Max and k in Eq. 1 were estimated with the nonlinear modeling option in JMP 5.0 (SAS Institute). For the purpose of this calculation only, the time for collecting blood samples before tracer injection was designated to be zero.

Data were subjected to ANOVA according to a randomized complete block design with block (i.e., litter) and duration of labeling (i.e., 15, 30, 45, 60, or 75 min) as the sources of variation. The main effect of time after tracer injection on the endpoints was evaluated using orthogonal contrasts. The FSRs, calculated using Eqs. 3 and 4 at different time points, were compared by paired t tests for each of the 5 time points. The concentrations of glucose and insulin in plasma 15, 30, 45, 60, and 75 min after tracer injection were compared to the basal value using Dunnett’s t test (38). The free pool enrichment in individual organs and tissues at different time points after i.p. injection was compared using Fisher’s protected least significant difference (StatView 5.0.1, SAS Institute). Individual pigs were the experimental units and P-values < 0.05 were considered significant in all comparisons. Where appropriate, means and associated SE are reported in the text.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The BW at weaning (5.3 ± 0.2 kg) and at i.p. tracer injection (5.7 ± 0.2 kg) did not differ among treatments. No effects of the i.p. injection were apparent on the behavior of the pigs postinjection or on the gross anatomy of the visceral organs upon opening the abdomen.

The i.p. injection of a flooding dose of Phe did not (P = 0.29) change the plasma insulin concentration over time (data not shown). The plasma glucose concentration increased (P < 0.05) at 15 min postinjection, but did not differ from the basal concentration at subsequent time points (Fig. 1). The concentration of free Phe in plasma increased logarithmically (P 0.001) over time (Table 1), following a curve similar to that of the tracer Phe isotopic enrichment (Fig. 2). The predicted maximal Phe concentration was 711 µmol/L, 95% of which was reached 48 min postinjection. Additionally, concentrations of other free amino acids in plasma did not vary from basal values over time, with the exceptions of aspartate + asparagine, serine, glutamate + glutamine, tyrosine, and lysine (Table 1), which were lower (P < 0.05) than their respective basal values at 60 min postinjection.

View larger version (14K): [in this window] [in a new window]   FIGURE 1 Responses in plasma concentrations of glucose after an i.p. injection of a flooding dose of phenylalanine in fed weaned pigs. Values are means ± pooled SE, n = 5. There were cubic effects (P < 0.05) of time on the plasma glucose concentration. aDifferent from the baseline mean (Dunnett’s t test), P < 0.05.

View larger version (16K): [in this window] [in a new window]   FIGURE 2 Quantitative relationship between isotopic enrichment (Y: mol%) of L-[ring-2H5]phenylalanine in the plasma free pool and time (x: min) after an i.p. injection of a flooding dose of phenylalanine in fed weaned pigs. Values are means ± pooled SE, n = 5. The increase in isotopic enrichment of L-[ring-2H5]phenylalanine was described according to a previously established model (14).

The tracer Phe enrichment of the plasma free pool increased logarithmically (P < 0.05) to a plateau of 32.8 mol%, 99% of which was reached 15 min postinjection (Fig. 2). The free pool tracer Phe enrichment in plasma and various tissues and organs did not change beyond 15 min after injection (Table 2), indicating that the maximal tissue free-pool tracer Phe enrichment was reached after 15 min of administration of the label. The constant free pool tracer Phe enrichment allowed a comparison of the overall means in individual tissues with that of plasma, which, with the exception of cecum, colon, and pancreas, did not differ (Table 2). This lack of difference indicated that "flooding" of all tissues occurred 15 min after the i.p. tracer injection.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In experiments in which a bolus dose of Phe was injected i.v., the maximal plasma free Phe concentration was achieved at the end of the infusion period or shortly thereafter, after which it declined (41,43,44). In the present study with similar doses of Phe, the plasma free Phe concentration reached 95% of its maximal value 48 min postinjection and it was therefore expected that i.p. injection would elicit relatively smaller physiologic responses than a corresponding i.v. injection. Although the plasma free Phe concentration was increased by 791%, small changes were observed in plasma concentrations of some other free amino acids and glucose (Table 1). It is difficult to explain the transient decline in the plasma concentrations of some free amino acids at the 60-min time point. No effects of the i.p. injection on the plasma insulin concentration were observed; therefore, plasma insulin was not likely responsible for the transient plasma glucose surge reported in Figure 1. It was reported that acute human-pig interactions, e.g., handling piglets during sampling, caused transient plasma surges of glucose (45) and glucocorticoids (46). Although, plasma glucocorticoid levels were not measured in this experiment, it was possible that a transient plasma glucocorticoid surge was responsible for the transient plasma glucose elevation observed in this study.

The FSR calculated using Eq. 4 takes into account the rise in the precursor tracer enrichment immediately after tracer injection (14). However, repeated samples from various time points after tracer administration are required for accurate calculation of the area under the free pool enrichment curve. In the classical studies by McNurlan et al. (47) and Garlick et al. (17), the free-pool specific activities⁶ reached maximal values within 2 min postinjection and declined linearly thereafter. It was therefore possible to estimate the area under the curve by averaging the precursor specific activity 2 and 10 min postinjection. In a subsequent paper, Garlick et al. (37) argued that the decline in free-pool specific activity over 10 min was small and the error associated with assuming constant free-pool specific activity from zero to 10 min postinjection was <5%. Consequently, FSR could be calculated using Eq. 3, in turn requiring only 1 measurement of the free-pool specific activity. However, values for FSR₃ are underestimated at time points close to injection because of the rise in free-pool specific activity immediately after tracer injection. The FSR₃ values become closer to FSR₄ values at later time points as the error in the estimation of the free-pool specific activity becomes proportionally smaller (44). The error is especially evident in situations in which the rise in free-pool tracer specific activity is relatively slow as observed in the present and other experiments (41,44). As expected, the FSR values calculated using Eq. 3 in the present experiment were numerically lower than those calculated using Eq. 4, but differed significantly only at the 15-min time point for half of the tissues examined (Table 4). Thus, tissues can be harvested 30 min after the i.p. tracer injection and the FSR calculated using Eq. 3 without the need to define the tracer enrichment time course.

Because there was no effect of time on the FSR, the visceral organs can be sampled as early as 15 min postinjection when Eqs. 3 and 4 are employed for calculations. However, sampling at 30 min postinjection minimized the effect of the rise in the free-pool tracer Phe enrichment. For plasma as well as cardiac and skeletal muscles, however, the FSR decreased linearly over time when calculated using Eqs. 3 and 4, (Table 4). Similar observations were made in visceral organs of sheep (44) and in skeletal muscle of dogs (48) after an i.v. flooding dose of amino acid tracers. Insulin was shown to affect the FSR of cardiac and skeletal muscles, but not of visceral organs (12). However, an effect of the i.p. injection on the plasma insulin concentration was not detected in the present experiment. The use of surrogate measures, i.e., the free-pool tracer Phe enrichments, rather than amino acyl-tRNA as the true precursor pool in this study is the first potential cause to be explored. Surrogate measures of precursor pools were shown to be similar to the true amino acyl-tRNA precursor pool in muscles when an i.v.-flooding dose of Phe was given to dogs and pigs (16,49). However, surrogate measures of precursor pools were not ideal for muscles compared with the true precursor amino acyl-tRNA when continuous infusion was used in humans (50). Therefore, the use of i.p. injection as a route for tracer delivery for measuring FSR in plasma and muscles is questionable at present. Future research must be conducted to elucidate the cause of reduced muscle FSR in response to an i.p. flooding dose of Phe.

In conclusion, the i.p.-injected Phe was quickly distributed into intra- and extracellular pools and was incorporated into plasma, organ, and tissue proteins. The concentration of free Phe in plasma increased by 800%, but no effect was observed on plasma insulin concentration, and only transient changes were observed in plasma concentrations of glucose and some free amino acids. The FSR of visceral organs, but not of plasma and muscles in pigs can be measured by a flooding dose of Phe tracer injected i.p. and with tissue harvest 30 min after injection.

	ACKNOWLEDGMENTS

 
The authors are grateful to A. Ajakaiye, A. Holt, T. Rideout, S. Shen, E. Wallace, N. Wedel, D. Wey, V. Wideman, and X. Yang (University of Guelph) for invaluable help during tissue collection, and to G. Werchola (University of Guelph) for assistance with the hormonal assay.

	FOOTNOTES

 
¹ Presented in part at the 9th International Symposium on the Digestive Physiology of Pigs, May 2003, Banff, AB, Canada [Bregendahl, K., Liu, L., Fan, M. Z., Cant, J. P., Bayley, H. S., McBride, B. W., Milligan, L. P. & Yen, J. T. (2003). Intraperitoneal injection of a flooding dose of L-[ring-²H₅]-phenylalanine is effective in measuring the fractional protein synthesis rate in visceral organs of weanling pigs. In: Digestive Physiology of Pigs–Proceedings of the 9th International Symposium (Ball, R., ed.), pp. 43–45. University of Alberta, Banff, AB, Canada].

² Funded by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada Research Partnerships’ New Faculty and the Discovery Programs, and by the University of Guelph–Ontario Ministry of Agriculture and Food (OMAF) Animal and Resources Management and Environment Research Programs (to M.Z.F.).

³ Present address: 201 Kildee Hall, Department of Animal Science, Iowa State University, Ames, IA 50011-3150 (E-mail: kristjan{at}iastate.edu).

⁵ Abbreviations used: BW, body weight; FSR, fractional protein synthesis rates; TCA, trichloroacetic acid.

⁶ The terms "isotopic enrichment" and "specific activity" are used interchangeably in the discussion and refer to the use of stable or radioactive isotopes, respectively.

Manuscript received 19 March 2004. Initial review completed 29 April 2004. Revision accepted 6 July 2004.

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