Journal of Nutrition LabDiet, Your World of Nutritional Answers

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bregendahl, K.
Right arrow Articles by Fan, M. Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bregendahl, K.
Right arrow Articles by Fan, M. Z.
© 2008 American Society for Nutrition J. Nutr. 138:1976-1981, October 2008

Methodology and Mathematical Modeling

Fractional Protein Synthesis Rates Are Similar When Measured by Intraperitoneal or Intravenous Flooding Doses of L-[ring-2H5]Phenylalanine in Combination with a Rapid Regimen of Sampling in Piglets1,2

Kristjan Bregendahl3,5, Xiaojian Yang3, Lijuan Liu3, Jong-Tseng Yen4,6, Todd C. Rideout3, Yingran Shen3, George Werchola3 and Ming Z. Fan3,*

3 Center for Nutrition Modeling, Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1 and 4 USDA/Agricultural Research Service U.S. Meat Animal Research Center, Clay Center, NE 68933

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


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Fractional protein synthesis rates (FSR) are widely measured by the flooding dose technique via either an i.g. or an i.v. route. This study was conducted to compare differences in tracer incorporation and FSR in organs and tissues of fed piglets. The piglets were surgically implanted with catheters and randomly assigned to receive a flooding dose of Phe (1.5 mmol/kg body weight, 40 percent molar enrichment with [2H5]Phe) in saline administered via an i.p. or an i.v. route. [2H5]Phe free-pool enrichment in plasma increased logarithmically (P < 0.05) from 0 to 25% in the i.p. group, whereas it rose to a peak level within 3 min of the tracer injection and then decreased linearly (P < 0.05) in the i.v. group. Intracellular free-pool tracer enrichments in organs and tissues were within the range of the values measured for the plasma-free pool (25–27%), reaching the flooding status. Administration of the tracer via the i.p. and i.v. routes induced a logarithmical pattern (P < 0.05) of a surge in plasma cortisol concentrations within 30 min. Measurements of FSR in plasma, cardiac muscle, and skeletal muscles were lower (P < 0.05) in the i.p. than in the i.v. group due to the adverse effect of cortisol surge being more dramatic (P < 0.05) in the i.p. than in the i.v. group at 30 min of the post-tracer administration. We conclude that FSR may be measured by the flooding dose through an i.p. or an i.v. route and the i.p. route may underestimate FSR by the flooding dose for plasma, cardiac muscle, and skeletal muscles. This concern may be addressed by a fast regimen of sampling to be completed within 12–20 min after an i.p. route of tracer injection.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The flooding dose technique for the measurement of fractional protein synthesis rates (FSR)7 was first described by Henshaw et al. (1) and since modified by McNurlan et al. (2) and Garlick et al. (3,4). Routes of tracer delivery, including an i.g. compared with an i.v. route, are an important methodological consideration for application of the flooding dose technique for measurements of FSR (5,6). Measurements of FSR are widely carried out by both an i.p. route (1,7–15) and an i.v. route (2–4,16–21). However, there is a scarcity of information regarding a systemic comparison of the i.p. and i.v. routes for measurements of FSR by the flooding dose technique.

Time courses of isotopic enrichments of free and bound tracer pools and corresponding FSR in plasma, visceral organs, cardiac muscle, and skeletal muscle after an i.p. route of injection of a flooding dose of Phe containing L-[2H5]Phe was investigated between 15 and 75 min of post-tracer injection in studies with pigs (15). Furthermore, FSR measured in visceral organs did not change, whereas FSR determined in plasma, cardiac muscle, and skeletal muscle decreased significantly over the time (15). Thus, it is essential to understand factors responsible for the reduction in FSR associated with plasma and the muscles measured by the flooding dose via an i.p. route compared with an i.v. route.

Therefore, the objectives of this study were to systematically compare differences in tracer incorporation kinetics and FSR in organs and tissues measured by the flooding dose technique between an i.p. and an i.v. route and to further reveal the factors responsible for potential differences in FSR associated with these 2 routes of tracer delivery.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Pigs, diets, surgery, and experimental design. Five blocks of 2 littermate purebred Yorkshire gilts from the University of Guelph Arkell Swine Research Station were weaned at 16 d of age in 2 groups of 5 pigs each. On the day before weaning, pigs were treated with 40 mg of trimethoprim and 200 mg of sulfadoxine per kg of body weight (BW) (Intervet Canada). Immediately upon weaning, the pigs were transferred into the animal wing in the Department of Animal and Poultry Science at the university and given free access to water and a pelleted commercial starter diet. A commercial source of cereal grain, soybean meal, fish meal, and whey powder-based starter diet was formulated to meet or exceed nutrient recommendations by the NRC (22) and contained 20% crude protein, 14.0 MJ/kg of metabolizable energy, and 1.2% of true ileal digestible lysine.

Five days after weaning, Tygon catheters (1.78-mm o.d.) were surgically placed in the jugular vein and carotid artery of all pigs under isoflurane inhalation anesthesia and strict aseptic conditions by following the procedure established previously (23). The catheters were filled with heparinized saline (200 kIU/L) to ensure patency and protected by bandages. After 3 d of recovery, littermate gilts were randomly assigned to receive the tracer by either an i.p. or an i.v. route through the jugular vein catheter. The use and care of the animals conformed to the guidelines of the Canadian Council on Animal Care (24) and all procedures were approved by the Animal Care Committee at the University of Guelph.

    Infusion protocols and sample collections. The pigs were food deprived for 1.5 h and subsequently given free access to feed for 1 h before the tracer administration in an attempt to standardize the feed intake. During and after the tracer administration, pigs were held in the lap of a researcher and, therefore, had no access to feed and water for the duration of the 30-min tracer administration and sampling protocols.

The flooding dose (10 mL/kg BW), administered via either an i.p. or an i.v. route, consisted of L-Phe (1.5 mmol/kg BW; Sigma/Aldrich) containing 0.60 mol/kg BW of L-[ring-2H5]Phe (Cambridge Isotopes) in sterile saline (154 mmol NaCl/L) and was prewarmed in a water bath to 37°C before its administration. The i.p. route of tracer administration was performed in 5–10 s with a sterile 60-mL syringe and a 16-gauge needle, whereas a syringe pump (Harvard Apparatus) was used to administer the flooding dose via the i.v. route. The syringe pump was programmed to deliver the flooding dose via the i.v. route during a 15-min period, because data from our previous experiment showed that 99% of the maximal tracer Phe isotopic enrichment of free Phe in plasma was reached 15 min after the i.p. tracer injection (15). Moreover, Southorn et al. (16) recommended a relatively long tracer infusion period (12 min) via an i.v. route to eliminate physiological effects potentially caused by the flooding dose itself. After the i.v. route of tracer infusion, the jugular catheter was flushed with 4 mL of saline. To avoid potential differences in FSR measurements caused by handling of the pigs, sham-i.v. and sham-i.p. routes of tracer administration were performed on the pigs receiving the i.p. or the i.v. treatment, respectively, as the following. The jugular catheter of the pigs in the i.p. route group was freed from the bandages and briefly manipulated to simulate the i.v. route of infusion. Similarly, a sterile 16-gauge needle was introduced via the i.p. route for a few seconds to simulate the i.p. route of the tracer administration in the pigs receiving the tracer via the i.v. route. Saline was not administered as a part of the sham injections, because a considerable volume of saline (10 mL/kg BW) was injected as a part of the flooding dose treatment itself for both groups.

Blood samples (4 mL) were collected into heparinized tubes (Vacutainer, 72 IU/tube; Fisher Scientific) from the carotid artery catheter immediately before and 3, 6, 9, 15, and 30 min after the beginning of the flooding dose administration and placed on ice. The catheter was flushed with 4 mL of the heparinized saline between samplings to ensure patency of the catheter and to maintain blood volume. Immediately after the last blood sampling, the pigs were killed with sodium pentobarbital (50 mg/kg BW; Schering Canada) administered through the jugular catheter. Visceral organs were then quickly removed, rinsed thoroughly in ice-cold saline (154 mmol NaCl/L at pH 7.4) containing a protease inhibitor (0.1 mmol/L phenylmethysulfonyl fluoride; Sigma), snap-frozen in liquid nitrogen, and stored at –70°C until analysis. We recorded the exact time (min) of tracer labeling measured from the end of the i.p. route of tracer injection or the start of the i.v. route of tracer infusion to the time the blood samples were placed on ice or tissue samples were placed into liquid nitrogen.

    Sample preparations and chemical analyses. After collection, blood was stored on ice until centrifugation (2000 x g; 20 min at 4°C) and the plasma frozen at –70°C until analysis. Tissue samples, stored at –70°C, were pulverized under liquid nitrogen using a mortar and pestle. Plasma cortisol concentrations were measured with solid phase (antibody-coated tubes) 125I-cortisol radioimmunoassay (Clinical Assays GammaCoat Cortisol 125I-RIA kit, CA-1549, DiaSorin).

Duplicate plasma (0.75 mL) and pulverized tissue samples (0.5 g) were homogenized (PowerGen 700D; Fisher Scientific) on ice in 4 mL of trichloroacetic acid solution (2 mol/L; Sigma) and subsequently centrifuged (2000 x g; 20 min at 4°C). The free and bound amino acid pools in samples were prepared as described in our previous studies (15).

The isotopic enrichment of L-[ring-2H5]Phe in plasma and tissue free and bound pools was determined in duplicate by GC-MS as the n-propyl hepta-fluorobutyrate derivative (25,26) 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 percent molar enrichment (mol%) using calibration curves prepared with L-[ring-2H5]Phe standards (6,15).

    Calculations and statistical analyses. In the i.p. route group of pigs, the increase in plasma free-pool tracer Phe isotopic enrichment over time was described according to Wolfe (27) as:

Formula

where EFree is the predicted isotopic enrichment (mol%) of L-[ring-2H5]Phe in the plasma free-pool at time t, EMax is the L-[ring-2H5]Phe isotopic equilibrium (mol%) in plasma, k is the rate constant (1/min), and t is the exact time (min) of the tracer labeling. The constants EMax and k were estimated with the nonlinear modeling option in JMP 5.1.2 (SAS Institute). For the purpose of this calculation only, the time (t) for collecting blood samples before the tracer administration was designated to be 0 (rather than the observed – 3). Repeated measurement analysis was used to examine the effects of time, tracer administration routes (i.p. vs. i.v.) and their interactions, and differences in plasma cortisol concentrations between the i.p. and the i.v. groups at 0, 3, 6, 9, 15, and 30 min postadministration, respectively. The time to reach 99% of the maximal isotopic enrichment of L-[ring-2H5]Phe was determined by derivatization described previously (15). Curve fitting of the time course of plasma cortisol concentrations was conducted by using the nonlinear procedure of SAS.

In the i.v. route group of pigs, the change in plasma free-pool enrichment over time was described linearly as:

Formula

where a is the slope (mol%/min) and b is the intercept (mol%). The constants a and b were determined using the Fit-Y-by-X option of the JMP.

Regardless of the treatment groups, the FSR in organs or tissues was calculated as:

Formula

where FSR is the fractional protein synthesis rate (%/d) defined as the percentage of protein renewed in a day in a target organ or tissue, EBound-t is the isotopic enrichment (mol%) of L-[ring-2H5]Phe in the bound-pool at time t in a target organ or tissue, EFree-t is the observed isotopic enrichment (mol%) of L-[ring-2H5]Phe in the free-pool at time t in a target organ or tissue, and t is the exact amount of time (min) of the tracer labeling (4,15).

Data were subjected to ANOVA according to a randomized complete block design with the tracer administration routes as the main effect and litter as the block factor using the mixed model of SAS. Differences between free-pool tracer enrichments of L-[ring-2H5]Phe in individual organs or tissues and plasma at 30 min postinjection within the i.p. group were compared using the pooled t test (28). Individual pigs were the experimental units and P-values < 0.05 were considered significant in all comparisons. Where appropriate, means and associated SE values are reported in the text and tables.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
The BW of the piglets did not differ between the 2 groups at the time of weaning (5.9 ± 0.3 kg), surgery (6.6 ± 0.4 kg), or the tracer administration (7.2 ± 0.5 kg). After tracer injection, the tracer isotopic enrichment in the plasma free-pool increased logarithmically over time (P < 0.05) to 24.6 mol%, 99% of which was reached at 11 min postinjection in the i.p. group (Fig. 1). The tracer isotopic enrichment in the plasma free-pool reached its highest level within 3 min of the flooding dose of Phe and then decreased linearly (P < 0.05) at a very slow rate after i.v. tracer administration (Fig. 1).


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 1  Quantitative relationships between isotopic enrichment of L-[ring-2H5]Phe in the plasma free-pool and time after an i.p. or an i.v. administration of a flooding dose of Phe containing L-[ring-2H5]Phe in fed weanling piglets. Values are means ± SEM, n = 5, except at 6, 9, and 15 min in the i.v. group, n = 4. For the i.p. group, Y = 24.60[1 – e(-0.403x)], R2 = 0.99, P < 0.05, n = 30; and for the i.v. group, Y = 26.90 – 0.01x, R2 = 0.54, P < 0.05, n = 22.

 
Albeit of a small magnitude, the tracer isotopic enrichment in the plasma free-pool at 30 min was lower in the i.p. group (P < 0.05) than in the i.v. group (Table 1). Furthermore, the tissue intracellular free-pool tracer isotopic enrichment differed (P < 0.05) between the i.p. and i.v. groups in heart, biceps femoris, longissimus dorsi, liver, lungs, kidney, cecum, and colon, while the free-pool enrichment did not differ between the i.p. and i.v. groups at 30 min after tracer administration in other visceral organs, including pancreas, spleen, stomach, and jejunum. On the other hand, the tissue intracellular free-pool tracer isotopic enrichments were higher (P < 0.05) in pancreas, cecum, and colon than in the plasma within the i.p. group.


View this table:
[in this window]
[in a new window]

  		TABLE 1 Free-pool tracer Phe enrichment in organ and tissue proteins of fed weanling piglets 30 min after an i.p. or i.v. administration of a flooding dose of Phe containing L-[ring-2H5]Phe1

 
The bound-pool tracer enrichments in plasma, cardiac muscle, and skeletal muscle were lower (P < 0.05) in the i.p. group than in the i.v. group (Table 2). Furthermore, the bound-pool tracer enrichments were lower (P < 0.05) in the i.p. group than in the i.v. group in liver, spleen, kidney, and stomach (Table 2). However, the bound-pool tracer enrichments did not differ between the i.p. and the i.v. groups in the other visceral organs.


View this table:
[in this window]
[in a new window]

  		TABLE 2 Bound-pool tracer Phe enrichment in organ and tissue proteins of fed weanling piglets 30 min after an i.p. or i.v. administration of a flooding dose of Phe containing L-[ring-2H5]Phe1

 
The FSR measured in the plasma was lower (P < 0.05) in the i.p. group than in the i.v. group (Table 3). The FSR measured in both cardiac and skeletal muscles were lower (P < 0.05) in the i.p. group than in the i.v. group. Furthermore, FSR measured for spleen and stomach was considerably lower (P < 0.05) in the i.p. group than in the i.v. group. However, FSR measured in the other visceral organs did not differ between the i.p. and the i.v. groups (Table 3).


View this table:
[in this window]
[in a new window]

  		TABLE 3 Fractional protein synthesis rates in organ and tissue proteins of fed weanling piglets 30 min after an i.p. or i.v. administration of a flooding dose of Phe containing L-[ring-2H5]Phe1

 
Changes in plasma cortisol concentrations were investigated in association with the routes of tracer administration for the measurement of FSR in organs or tissues. Both routes of tracer administration induced a similar logarithmical pattern of cortisol concentration surge (P < 0.05) in the blood circulation within 30 min of the tracer administration (Fig. 2). The surge was much greater in the i.p. group than in the i.v. group (P < 0.05) at 30 min of the post-tracer administration. Therefore, the analyses of Pearson correlations between FSR measured for organs and tissues and the plasma cortisol concentrations measured at 30 min were conducted. There were negative correlations (P < 0.05) between FSR measured in plasma (r = –0.91; P = 0.002), cardiac muscle (r = –0.77; P = 0.026) and skeletal muscles (biceps femoris, r = –0.83; P = 0.011; longissimus dorsi, r = –0.73; P = 0.041) and plasma cortisol concentrations measured at 30 min post-tracer administration, respectively.


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 2  Changes in plasma cortisol concentrations with time after an i.p. or i.v. administration of a flooding dose of Phe L-[ring-2H5]Phe in fed weanling piglets. Values are means ± SE, n = 5 (i.p.) or 4 (i.v.). The effect of time was significant in both groups. *Difference between the i.p. group and the i.v. group at 30 min of the post-tracer administration, P < 0.05. For the i.p. group, Y = 278.35 – 115.06e(–0.251x), R2 = 0.92, P < 0.05, n = 30; and for the i.v. group, Y = 197.57 – 42.87e(–0.877x), R2 = 0.97, P < 0.05, n = 24.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The objectives of the study were to systematically compare differences in tracer incorporation patterns and FSR in target organs and tissues measured by the flooding dose technique via the i.p. route compared with the i.v. route and to further reveal factors responsible for potential differences in FSR measurements associated with these 2 routes of tracer delivery. We compared the 2 routes of tracer delivery within 30 min of the post-tracer administration for the 2 following reasons. First, a short duration within 15–30 min of tracer injection is most frequently used by researchers for the flooding dose technique due to time constraint for most research activities. Second, FSR in the cortisol-sensitive tissues or organs such as muscles decreased dramatically with time from 15 to 90 min of the post-tracer injection, with the decreases most profound during the initial time slot of 15–30 min. Several measures were taken to minimize systemic variations in the pigs receiving the different routes of the tracer delivery, including: 1) both the i.p. and the i.v. groups of piglets were pairs of littermate gilts to minimize genetic differences in protein synthesis activity; 2) all the pigs had catheters surgically placed in the carotid artery and jugular vein to minimize differences in FSR attributable to the surgery, although the jugular vein catheter was used in the i.v.-administered pigs for only the delivery of the flooding dose; and 3) sham i.p. injection or sham i.v. infusion was performed on pigs receiving the i.v. or the i.p. administration, respectively, to minimize potential systemic differences in handling procedures.

When the tracer and tracee Phe was injected via the i.p. route, the plasma free-pool tracer isotopic enrichment increased logarithmically over time, consistent with the pattern reported in our previous studies of the i.p. route for flooding dose in piglets (15). However, the plasma free-pool tracer isotopic enrichment decreased linearly over time when the tracer and tracee Phe were infused via the i.v. route consistent with the pattern of several previous studies (2,3,12). Once injected into the cavity via the i.p. route, the tracer had to redistribute from the peritoneal cavity into plasma in the i.p.-injected piglets, thus explaining the lag time needed and the logarithmic increase in the plasma free-pool tracer isotopic enrichment in agreement with the previous observations by Morán et al. (29). In contrast, distribution of the tracer from the circulating blood into intra- and extracellular pools explains the linear decrease in the plasma free-pool tracer isotopic enrichment after the i.v. route of the tracer infusion. Therefore, there are distinctive plasma tracer free pool kinetic patterns in association with the flooding dose technique between the i.p. and the i.v. routes of tracer delivery.

Results from our previous study (15) showed that the plasma free-pool tracer isotopic enrichment was sustained for at least 75 min post-tracer administration, suggesting that the plasma free-pool isotopic enrichment in the i.p.- and the i.v.-administered pigs would be maintained until 30 min. In accordance with our previous findings (15), the pigs used in this study were killed and organ and tissue samples harvested at 30 min after the start of the tracer administration. The significantly higher free-pool tracer isotopic enrichments observed in pancreas, cecum, and colon than in the plasma for the i.p. group of this study were in agreement with our previous study of an i.p. flooding dose (15), suggesting that some tracer Phe might have been transported directly from the i.p.-injected cavity pool into the intracellular space of organs. However, physiological mechanisms of this tracer Phe uptake process are unclear. Despite significant differences in the free-pool tracer isotopic enrichments observed in some target organs and tissues, the differences were small in magnitude and were within the ranges of the values measured for the plasma free pools (25–27 mol%). Therefore, it can be concluded that the tracer flooding was effectively reached in both the i.p. and i.v. groups of pigs and the FSR measurements obtained by the flooding dose technique is valid for both of the compared groups.

Whereas both the i.p. and the i.v. routes of tracer delivery have been widely used for measurements of FSR by the flooding dose technique, this is a comprehensive investigation of how the routes of the tracer delivery affect the measurements of FSR by the flooding dose technique. Our results suggest that FSR measurements in plasma, cardiac muscle, skeletal muscle, spleen, and stomach by the flooding dose technique via the i.p. route of tracer injection significantly underestimate FSR in these tissues or organs. For plasma, cardiac muscle, and skeletal muscle, this was due to the adverse effect of the cortisol surge being more dramatically induced by the i.p. route than the i.v. route for the delivery of the flooding dose (Table 3; Fig. 2). The negative effect of glucocorticoids on protein synthesis is shown to be mediated through affecting some of the key protein synthetic initiation factors as was well demonstrated in skeletal muscles (30). Recent studies suggest that the glucocorticoids inhibit protein synthesis by depressing the mammalian target of rapamycin-signaling pathway through stimulating the regulated in development and DNA damage responses protein 1 but not the regulated in development and DNA damage responses protein 2 (31,32). Data from this (Fig. 1) and our previous study (15) showed that the free-pool tracer enrichment started to reach its plateau in the plasma and in all the organs and tissues at ~11–12 min after the flooding dose of the i.p. tracer injection. Meanwhile, the cortisol concentration surge did not reach its peak and its significant difference from the i.v. group until 30 min after the flooding dose of the i.p. tracer and tracee Phe injection from this study. Thus, the completion of sampling of these cortisol-sensitive tissues and organs within 12 to 20 min after the i.p. tracer injection would be conceivable to minimize the potential negative impacts associated with the i.p. injection-based flooding dose approach for measuring FSR in plasma, cardiac muscle, and skeletal muscles.

An interesting observation of this study was that the handling of piglets during tracer administration and blood sampling caused a transient logarithmical pattern of rise in the plasma cortisol concentration in both the i.p. and the i.v. groups. This observation was consistent with earlier reports that acute human-pig interactions, e.g. handling piglets during blood sampling, caused transient plasma surges of glucocorticoids and glucose concentrations in responses to stress (33,34). The much more dramatic increase in the plasma cortisol concentration in the i.p. group than in the i.v. group is likely due to the fact that the i.p. injection procedure, including the injection of the flooding dose of saline into the peritoneal cavity within a very short period of time, was more stressful and stimulating to causing the cortisol concentration surge in the piglets compared with the i.v. group of piglets. However, it should be pointed out that the cortisol concentration surge patterns due to the routes of tracer administration from this study cannot be simply extrapolated to other experimental conditions. Cortisol concentration surge patterns as well as a potential effect of cortisol on target organ or tissue protein synthesis rates may also be different under different physiological conditions or health status such as prolonged fasting, burn, infection, etc., which need to be investigated in future studies.

In conclusion, the administration of a flooding dose of an amino acid tracer-tracee mixture via both the i.p. and the i.v. routes has effectively flooded the whole body plasma, organ, and tissue tracer free pools with distinctive patterns of extracellular free pool tracer kinetics that are valid for the measurement of FSR. Measurements of FSR in plasma, cardiac muscle, and skeletal muscles may be underestimated by the flooding dose via the i.p. route of tracer delivery due to the effect of plasma cortisol concentration surge. A fast regimen of tissue sampling to be completed within 12 to 20 min after an i.p. route of tracer and trace injection may be helpful to minimize the potential negative impact on the measurement of FSR in plasma and the muscles by the flooding dose technique.


   FOOTNOTES
 
1 Supported by research grants from the Natural Sciences and Engineering Research Council of Canada Discovery Program and the Ontario Ministry of Agriculture, Food and Rural Affairs-University of Guelph Animal Research Program (to M.Z.F.). Back

2 Author disclosures: K. Bregendahl, X. Yang, L. Liu, J. T. Yen, T. C. Rideout, Y. Shen, G. Werchola, and M. Z. Fan, no conflicts of interest. Back

5 Present address: Department of Animal Science, 201 Kildee Hall, Iowa State University, Ames, IA 50011-3150. Back

6 Deceased. Back

7 Abbreviations used: BW, body weight; FSR, fractional protein synthesis rates; mol%, percent molar enrichment. Back

Manuscript received 28 January 2008. Initial review completed 1 April 2008. Revision accepted 24 July 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Henshaw EC, Hirsch CA, Morton BE, Hiatt HH. Control of protein synthesis in mammalian tissues through changes in ribosome activity. J Biol Chem. 1971;246:436–46.[Abstract/Free Full Text]

2. McNurlan MA, Tomkins AM, Garlick PJ. The effect of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem J. 1979;178:373–9.[Medline]

3. Garlick PJ, McNurlan MA, Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J. 1980;192:719–23.[Medline]

4. Garlick PJ, Fern M, Preedy VR. The effect of insulin infusion and food intake on muscle protein synthesis in postabsorptive rats. Biochem J. 1983;210:669–76.[Medline]

5. Davis TA, Reeds PJ. Of flux and flooding: the advantages and problems of different isotopic methods for quantifying protein turnover in vivo. II. Methods based on the incorporation of a tracer. Curr Opin Clin Nutr Metab Care. 2001;4:51–6.[Medline]

6. Fan MZ, Chiba LI, Matzat PD, Yang X, Yin YL, Mine YH, Stein H. Measuring synthesis rates of nitrogen-containing polymers by using stable isotope tracers. J Anim Sci. 2006;84 Suppl:E79–93.[Abstract/Free Full Text]

7. Dunlop DS, van Elden W, Lajtha A. A method for measuring brain protein synthesis rates in young and adult rats. J Neurochem. 1975;24:337–44.[CrossRef][Medline]

8. Jepson MM, Pell JM, Bates PC, Millward DJ. The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem J. 1986;235:329–36.[Medline]

9. Maeda Y, Hayashi K, Hashiguchi T, Okamoto S. Genetic studies of the muscle protein turnover rate of coturniz quail. Biochem Genet. 1986;24:207–16.[Medline]

10. Bernier JF, Calvert CC. Effect of a major gene for growth on protein synthesis in mice. J Anim Sci. 1987;65:982–95.[Abstract/Free Full Text]

11. Martinez JA. Validation of a fast, simple and reliable method to assess protein synthesis in individual tissues by intraperitoneal injection of a flooding dose of [3H]phenylalanine. J Biochem Biophys Methods. 1987;14:349–54.[CrossRef][Medline]

12. Czerwinski SM, Cate JM, Francis G, Tomas F, Brocht DM, McMurtry JP. The effect of insulin-like growth factor-I (IGF-I) on protein turnover in the meat-type chicken (Gallus domesticus). Comp Biochem Physiol. 1998;119:75–80.

13. Yoshida S, Yoshizumi T, Kamei H, Ozaki K, Yahara T, Tajiri T, Yamasaki K, Shirouzu K. Effect of intracerebroventricular injection of tumor necrosis factor-{alpha} on gut mucosal protein turnover in mice fed enterally. J Surg Res. 1999;87:73–6.[Medline]

14. Danicke S, Bottcher W, Simon O, Jeroch H. The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS. Isotopes Environ Health Stud. 2001;37:213–25.[Medline]

15. Bregendahl K, Liu L, Fan MZ, Cant JP, Bayley HS, McBride BW, Milligan LP, Yen JT. Fractional protein synthesis rates are measured by an intraperitoneal injection of a flooding dose of L-[ring-2H5]phenylalanine in pigs. J Nutr. 2004;134:2722–8.[Abstract/Free Full Text]

16. Southorn BG, Kelly JM, McBride BW. Phenylalanine flooding dose procedure is effective in measuring intestinal and liver protein synthesis in sheep. J Nutr. 1992;122:2398–407.[Abstract/Free Full Text]

17. Burrin DG, Wester TJ, Davis TA, Fiorotto ML, Chang X. Dexamethasone inhibits small intestinal growth via increased protein catabolism in neonatal pigs. Am J Physiol. 1999;276:E269–77.[Medline]

18. Nyachoti CM, de Lange CFM, McBride BW, Leeson S, Gabert VM. Endogenous gut nitrogen losses in growing pigs are not caused by increased protein synthesis rates in the small intestine. J Nutr. 2000;130:566–72.[Abstract/Free Full Text]

19. Davis TA, Fiorotto ML, Burrin DG, Reeds PJ, Nguyen HV, Beckett PR, Vann RC, O'Connor PMJ. Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs. Am J Physiol Endocrinol Metab. 2002;282:E880–90.[Abstract/Free Full Text]

20. Faure M, Moënnoz D, Montigon F, Fay LB, Breuillé D, Finot PA, Ballèvre O, Boza J. Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats. Anal Biochem. 2002;307:244–51.[CrossRef][Medline]

21. Hunter RJ, Patel VB, Baker AJ, Preedy VR. Liver dysfunction induced by bile duct ligation and galactosamine injection alters cardiac protein synthesis. Metabolism. 2004;53:964–8.[Medline]

22. NRC. Nutrient requirements of swine. 10th ed. Washington, DC: National Academy Press; 1998.

23. Yen JT, Killefer J. A method for chronically quantifying net absorption of nutrients and gut metabolism into hepatic portal vein in conscious swine. J Anim Sci. 1987;64:923–34.[Abstract/Free Full Text]

24. Canadian Council on Animal Care. Guide to the care and use of experimental animals. In: Olfert ED, Cross BM, McWilliam AA, editors Vol. 1, 2nd ed. Ottawa: CCAC; 1993.

25. MacKenzie SL. Gas chromatographic analysis of amino acids as the n-heptafluorobutyryl isobutyl esters. J Assoc Off Anal Chem. 1987;70:151–60.[Medline]

26. Culea M, Hachey D. Determination of multiply labeled serine and glycine isotopomers in human plasma by isotope dilution negative-ion chemical ionization mass spectrometry. Rapid Commun Mass Spectrom. 1995;9:655–9.[CrossRef][Medline]

27. Wolfe RR. Radioactive and stable isotope tracers in biomedicine. Principles and practice of kinetic analysis. New York:Wiley-Liss; 1992.

28. Byrkit DR. Statistics today: a comprehensive introduction. Menlo Park (CA): Benjamin/Cummings Publishing; 1987.

29. Morán JM, Mahedero G, Salas J, Ribeiro JM, Mariñõ E, Vinagre LM. A pharmacokinetic study of peritoneal absorption of glucose and alanine in rats: nutritional implications. JPEN J Parenter Enteral Nutr. 1998;22:72–6.[Abstract/Free Full Text]

30. Liu Z, Li G, Kimball SR, Jahn LA, Barrett EJ. Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle. Am J Physiol Endocrinol Metab. 2004;287:E275–81.[Abstract/Free Full Text]

31. Wang H, Kubica N, Ellisen LW, Jefferson LS, Kimball SR. Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. J Biol Chem. 2006;281:39128–34.[Abstract/Free Full Text]

32. Yang X, Yang C, Farberman A, Rideout TC, de Lange CFM, France J, Fan MZ. The mammalian target of rapamycin-signaling pathway in regulating metabolism and growth. J Anim Sci. 2008;86 Suppl:E36–50.[Abstract/Free Full Text]

33. Dubreuil P, Farmer C, Couture Y, Petitclerc D. Hematological and biochemical changes following an acute stress in control and somatostatin-immunized pigs. Can J Anim Sci. 1993;73:241–52.

34. Hemsworth PH, Barnett JL. Human-animal interactions and animal stress. In: Moberg GP, Mench JA, editors. The biology of animal stress: basic principles and implications for animals. Oxon (UK): CAB International; 2000. p. 309–35.





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bregendahl, K.
Right arrow Articles by Fan, M. Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bregendahl, K.
Right arrow Articles by Fan, M. Z.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Copyright © 2008 by American Society for Nutrition