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Nutrient Metabolism

Feeding Frequency and Type of Isotope Tracer Do Not Affect Direct Estimates of Lysine Oxidation in Growing Pigs¹

S. Möhn, M. F. Fuller^*, R. O. Ball and C. F. M. de Lange²

Department of Animal and Poultry Science, University of Guelph, Guelph, Canada N1G 2W1; ^* The Rowett Research Institute, Aberdeen, UK; and Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, Canada

²To whom correspondence should be addressed. E-mail: cdelange{at}uoguelph.ca.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Oxidation contributes to the inefficiency of lysine utilization for protein deposition. The influences of feeding frequency and type of isotope tracer on estimated lysine oxidation were studied in growing pigs fed lysine-limiting diets. Yorkshire gilts (n = 11) weighing 40–45 kg were fitted with venous catheters. They were fed, in 3 or 8 equal meals daily, a purified diet based on casein and cornstarch. Lysine intake limited the pigs’ protein deposition to 70% of their potential. After a 5-d N-balance period, lysine oxidation was estimated by a primed, constant 26-h infusion of [1-¹⁴C]L-lysine and [6-³H]L-lysine. Feeding frequency and type of tracer did not affect lysine oxidation (P > 0.1). Increasing feeding frequency from 3 to 8 times daily reduced the variance and fluctuation of lysine oxidation by 46 and 30%, respectively. The mean lysine oxidation, as a fraction of the true ileal digestible lysine intake, was 9.2% based on the free lysine specific radioactivity (SRA) in plasma, 20.1% based on free lysine SRA in liver and 21.8% calculated from N-balance data. On the basis of liver free lysine SRA, tracer dilution methods and N-balance data give similar quantitative estimates of lysine oxidation (P > 0.10). Isotope tracer studies that cover one or more complete feeding cycles, i.e., feeding-to-feeding periods, can be used to obtain valid daily lysine oxidation values.

KEY WORDS: • pig • lysine oxidation • isotope tracers • feeding frequency • body protein deposition • pigs

In diets for growing pigs, lysine is usually the first-limiting amino acid for body protein deposition (PD)² (1). Oxidation of absorbed available lysine is thought to be one of the main contributors to the inefficiency of lysine utilization for PD (2–6). Minimum or basal oxidation, also referred to as inevitable catabolism, of lysine has been estimated to range from 3% (2) up to 40% of the available lysine intake (3). This wide range in estimates may reflect biological variation or methodological difficulties in measuring amino acid catabolism. Accurate estimates of minimum lysine oxidation are required because it is fundamental to our understanding of amino acid metabolism.

Estimates of lysine oxidation can be obtained indirectly from N-balance or serial slaughter experiments, or directly from isotope tracer studies. Indirect estimates represent a residual value after other uses of lysine have been accounted for and is sometimes deemed less reliable. However, tracer studies also involve uncertainties, mainly concerning identification and sampling of the appropriate precursor pool for oxidation and the quantification of tracer recovery in lysine breakdown products (7–9). In the case of lysine, both [1-¹⁴C]L-lysine and [6-³H]L-lysine may be used as tracers while monitoring the appearance of label in expired CO₂ and body water, respectively (7,8).

Provided that feeding frequency does not affect lysine oxidation, daily lysine oxidation values may be estimated from measurements made during short feeding-to-feeding periods. In both humans (10,11) and rats (12), the oxidation rates of amino acids increased as feeding frequency increased. In contrast, feeding pigs more than once daily improved the utilization of dietary free but not protein-bound lysine for growth (13).

Our objectives were as follows: 1) quantify minimum lysine oxidation in growing pigs, 2) determine the influence of feeding frequency (3 or 8 times daily) and type of isotope tracer ([1-¹⁴C]L-lysine or [6-³H]L-lysine) on estimates of lysine oxidation, and 3) compare estimates of lysine oxidation determined by isotope studies with values calculated from N balance.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and management.

Yorkshire gilts (n = 12) were selected from the University of Guelph herd at 30 kg body weight (BW) and housed individually in adjustable metabolism crates in a temperature-controlled room (14). At 35 kg BW, the pigs were surgically fitted with catheters in the femoral vein for isotope infusion and the jugular vein for blood sampling (15) and assigned to one of two feeding frequencies. At 40 kg BW, their rates of N retention were determined over a period of at least 5 d (16). Immediately thereafter, pigs were placed in an open-circuit respiration chamber for direct measurement of lysine oxidation using a primed, constant 26 h-infusion of [1-¹⁴C]L-lysine and [6-³H]L-lysine. The experimental procedures were approved by the Animal Care Committee of the University of Guelph.

Diets and feeding.

Six pigs were fed 8 times daily (F8) at 3-h intervals and six were fed three times daily (F3) at 0700, 1400 and 2100 h. These feeding regimens were imposed at least 5 d before the N-balance measurements began and were continued to the end of the trial. The diets consisted of a "nutrient mix" and a protein-free "energy mix" (Table 1). The daily allowance of "nutrient mix" was set to achieve the targeted lysine intake of 11 g/d, equivalent to 70% of requirements for maximum PD in this population of pigs (16) to ensure that lysine oxidation was due solely to inevitable catabolism. At each meal, the allowance of nutrient mix was blended with the appropriate amount of "energy mix" to achieve the target energy intake of 2.6 times the maintenance requirement [458 kJ metabolizable energy/(kg BW^0.75 · d)] (17). Combined, the ration provided lysine at 6 g/100 g protein (N x 6.25) or at 0.55 g/MJ metabolizable energy. The ratios of the other essential amino acids to lysine exceeded the recommendations of Wang and Fuller (18) by at least 10%, so that lysine was the first-limiting amino acid. Minerals and vitamins were provided in excess of the recommendations of the NRC (17). Feed was offered as a mash (feed to water = 1:2.5). Additional water was available to the pigs at all times.

The oxidation studies were performed in an open-circuit respiration system. Fresh air was drawn through the system at 70 L/min. Expired CO₂ was absorbed by a mixture of ethanolamine and 2-methoxyethanol (1:2, v/v) (19) in five consecutive gas-washing bottles, followed by a final trap containing 1 mol/L NaOH.

Both [1-¹⁴C]L-lysine (declared purity > 99%) and NaH¹⁴CO₃ were purchased from American Radiolabeled Chemicals (St. Louis, MO). Two batches of [6-³H]L-lysine (declared purity > 99%) and D₂O (98% atom % excess) were purchased from DuPont NEN Research Products (Stevenage, Herts, UK).

Preliminary studies.

To determine the recovery of CO₂ in the respiration system, CO₂ was released from a 0.25 mol/L NaHCO₃ solution (4 L) that contained a known amount of NaH¹⁴CO₃. The solution was placed in the respiration system and CO₂ was released over a 1-h period by infusing HCl into the solution. The recovery of ¹⁴CO₂ was 100.3 ± 0.7% (n = 6).

The recovery of ¹⁴CO₂ infused intravenously was determined in pigs between 40 and 50 kg BW fitted with venous catheters. After pigs were placed in the respiration system, a priming dose of 450 kBq NaH¹⁴CO₃ was given within 1 min, followed by a constant infusion of 300 kBq/h NaH¹⁴CO₃ for 6 h. Once the rate of appearance of ¹⁴CO₂ in expired CO₂ reached a plateau, the mean recovery of ¹⁴CO₂ from the pigs was 84.7 ± 2.3% (n = 12) of the infused dose of NaH¹⁴CO₃.

Infusion protocol and sampling.

For measuring the size of the body water pool (20), 12 mL D₂O was injected 15 min before feeding and the start of lysine infusions. A priming dose of 10 kBq NaH¹⁴CO₃ and 1.5 times the hourly infusion rates of [1-¹⁴C]L-lysine or [6-³H]L-lysine was injected manually via the femoral catheter within a 1-min period. Immediately thereafter, the constant infusion was started at rates of 180 kBq/h [1-¹⁴C]L-lysine and 105 kBq/h [6-³H]L-lysine (F8) or 290 kBq/h for the second batch of [6-³H]L-lysine (F3). The infusion rate of [6-³H]L-lysine was increased because the higher rate facilitated the precise determination of plasma free lysine specific radioactivity (SRA).

Expired CO₂ was collected quantitatively over 0.5-h periods throughout the 26-h infusion period. Blood samples (4 mL) were taken from the jugular catheter at 0.5-h intervals and 15 min after CO₂ was sampled. Additional blood samples (4 mL) were taken before the injection of D₂O and 15, 30, 105 and 345 min thereafter. Blood samples (10 mL) for the determination of ³H₂O were taken at 3-h intervals, starting 105 min after the start of infusions. The blood samples were transferred to heparinized tubes. The plasma was separated by centrifugation for 10 min at 3000 x g, and stored at -20°C until analysis. Urine was collected quantitatively over 3-h periods using bladder catheters, and stored frozen until analysis. At the end of the infusion, the pigs were killed by an injection of pentobarbital. Samples of the right central liver lobe were taken immediately after death, frozen in liquid N₂ and stored at -20°C until further analysis.

Analytical procedure.

The N contents of feed, urine and feces were determined by the Kjeldahl method (21). Feed amino acid contents were determined by ion exchange chromatography (22). Plasma and liver free lysine concentrations were determined by HPLC (Waters, Milford, MA) after precolumn derivatization with phenylisothiocyanate (23). The eluate containing the lysine fraction was collected for subsequent scintillation counting. The recovery of radioactivity during plasma sample preparation was determined using norleucine as an external standard. Urine samples were prepared for HPLC in the same manner as plasma.

To determine the ³H radioactivity and D₂O enrichments in plasma and urinary water, the respective samples were vacuum distilled for subsequent scintillation counting. D₂O enrichment was determined by MS at the Rowett Research Institute as described by Beckett et al. (20).

For scintillation counting, samples of the CO₂-absorber were mixed with Atomlight scintillation cocktail (Canberra Packard, Mississauga, Canada). For all other samples, the BCS cocktail (Amersham, Oakville, Canada) was used.

Calculation of results.

Lysine disappearance (g/d) was calculated from the N-balance data as the true ileal digestible lysine intake minus lysine retained in body protein minus predicted lysine losses via the integument (24) and intestine (25). The true ileal digestibility was previously determined to be 99% (14), whereas retained body protein (N x 6.25) was assumed to contain 7.0% lysine (17).

For the [1-¹⁴C]L-lysine infusion, the oxidation rate was calculated for each 0.5-h period as ¹⁴CO₂ collected divided by the estimated ¹⁴C-lysine SRA at the site of lysine oxidation (Bq/µmol), corrected for the recovery of CO₂ in the pigs. The SRA at the site of lysine oxidation was estimated from SRA in free plasma lysine and from SRA in free plasma lysine multiplied by the ratio of SRA in liver and plasma free lysine at the end of the infusion period. Further calculations were based on the values of individual 0.5-h periods. Within each pig, oxidation patterns were considered stable and used for further analyses when sequential removal of data from initial feeding cycles resulted in the absence of collection period effects on 0.5-h lysine oxidation values. Daily (24 h) values for lysine oxidation were calculated as 48 times the 0.5-h period values.

For the [6-³H]L-lysine infusion, lysine oxidation rates were calculated as the sum of the appearance rate of ³H in body water (kBq/0.5 h) and the rate of ³H excretion with water in urine (kBq/0.5 h) divided by the ³H-lysine SRA (Bq/µmol) at the site of lysine oxidation. Plasma water was assumed to represent the body water pool. The appearance rate of ³H in body water (kBq/0.5 h) was derived from the slope of linear regressions of ³H in body water (MBq/L) on time (0.5 h), multiplied by the size of the pig’s body water pool (L), which was calculated according to Beckett et al. (20). Other calculations were similar to those for [1-¹⁴C]L-lysine.

Statistical analyses were performed using SAS (26). General linear models were used to test the effects of type of tracer, feeding frequency and the interaction between tracer and feeding frequency. Least-square means were compared with the protected t test. Regression analyses were conducted within individual pigs and feeding cycles, and across pigs within treatments, to evaluate time effects on plasma lysine concentrations, plasma lysine SRA and lysine oxidation values. Relationships between the variables of the isotope study and between measurements in the isotope study and the N-balance were assessed by Pearson’s correlation coefficients. Values were expressed as means ± SEM. Differences were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
General observations.

All pigs, except for one in F3, readily consumed the experimental diets. All data from the pig with feed refusals were excluded before statistical analyses. No problems were observed during the isotope tracer study.

N-balance.

The growth performance during the N-balance was not affected by feeding frequency (Table 2). The mean PD of 105.8 ± 2.2 g/d was close to the targeted value, 70% of the previously determined maximum PD in a similar group of pigs (16). Lysine disappearance, based on N-balance, in F3 did not differ (P > 0.1) from that for F8 (Table 2).

The recovery of radioactivity over the entire experimental period in either body water for the ³H label or expired ¹⁴CO₂ for the ¹⁴C label was not affected by feeding frequency. The mean recovery of radioactivity in urine was 2.1 ± 0.4% of the infused tritium label and 1.9 ± 0.1% of the ¹⁴C label. Urinary water contained 0.58 ± 0.11% of the infused ³H; <1% of the total ³H or ¹⁴C radioactivity in urine was found in the fraction containing lysine. These values were not influenced by feeding frequency (P > 0.10).

Plasma free lysine concentrations over the 24-h period were 194 ± 20 µmol/L for F3 and 191 ± 9 µmol/L for F8 and were not affected (P > 0.1) by feeding frequency. The recovery of ¹⁴C from breath and plasma free ¹⁴C lysine SRA increased linearly with time (P < 0.01) during the 24-h infusion. Calculated lysine flux decreased (P = 0.001) with time. Consequently, lysine oxidation (g/0.5 h) did not change (P > 0.1) during the infusion period.

Daily lysine oxidation was not influenced by tracer or feeding frequency (P > 0.10). Across feeding frequencies and tracer types, lysine oxidation was 9.17 ± 0.85% of the true digestible lysine intake, when based on plasma lysine SRA. Because the ratio of liver free lysine SRA to plasma free lysine SRA was not affected by tracer (P > 0.1) and feeding frequency (P = 0.11, Table 3), the value of 0.456 ± 0.041% was used to calculate the lysine oxidation rates on the basis of liver free lysine SRA. Using this approach, lysine oxidation was estimated as 20.1 ± 1.9% of the true ileal digestible lysine intake. These values were not influenced by feeding frequency or type of isotope tracer (P > 0.10; Table 3). For all variables, the interaction between type of tracer and feeding frequency was nonsignificant (P > 0.1).

Within feeding cycles, i.e., feeding-to-feeding periods, lysine oxidation and plasma free lysine concentration followed distinct patterns (P < 0.01). Overlaying feeding cycles clarified the influence of time after feeding on lysine oxidation and plasma lysine concentration (Fig. 1). The effect of time after feeding on the amplitude of lysine oxidation was more pronounced in F3 (P = 0.001) than in F8 (P = 0.088). In F3, oxidation increased for 2 h after feeding, with a subsequent slow linear decline back to its initial values. In F8, oxidation began at a higher level, reached a peak 1 h after feeding, and declined to its initial value by 2 h. The amplitude of the residual lysine oxidation in F8 was 30% of the amplitude in F3. The mean SD of residual lysine oxidation at all time points after feeding in F8 was 46% of that in F3.

View larger version (22K): [in this window] [in a new window]   FIGURE 1 Lysine oxidation and plasma free lysine concentrations as affected by time after feeding in pigs fed 3 or 8 times daily. Values are residuals of regressions on pig and feeding cycle and represent means ± SEM for each time point after feeding.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

From N-balance observations (Table 2), we calculated a lysine disappearance of 22% of lysine intake. Marginal utilization of lysine in serial slaughter experiments of 86% (4), 74% (5) and 75% (27) imply oxidative losses of lysine of 14, 26 and 25% of lysine intake, respectively.

Lysine oxidation, based on isotope tracers and plasma lysine SRA, was 9.2% of the true digestible lysine intake. On the basis of liver free lysine SRA, the lysine oxidation rate in the present experiment was 20.1%, similar to the lysine disappearance calculated from the N-balance (21.8%). Mnilk et al. (8), who measured liver free lysine SRA to estimate SRA at the site of lysine oxidation, reported oxidation rates of 12.7% of the true digestible lysine intake.

Retention of CO₂ by the pig influences the quantification of lysine oxidation when using [1-¹⁴C]L-lysine as a tracer. Our estimate of 15.3% for CO₂ retention is comparable to values compiled for growing pigs (28) and humans (7). However, it may underestimate the true CO₂ retention because CO₂ released intracellularly during lysine oxidation may be used for carboxylation reactions, contrary to extracellular CO₂, which is transported to a limited degree into cells (29). This bias in estimation of CO₂ recovery would result in an underestimation of lysine oxidation. If the substantial urinary ¹⁴C label excretion reflects excretion of carboxylation reaction products, then urinary ¹⁴C label excretion is accounted for in the adjustment for CO₂ retention by pigs. On the other hand, if urinary ¹⁴C label excretion represents excretion of lysine-containing peptides or small proteins, then this label excretion should not be attributed to lysine oxidation. A more thorough characterization of the urinary ¹⁴C–containing products would elucidate potential implications of urinary ¹⁴C label in urine on estimates of lysine oxidation. The use of [6-³H]L-lysine may underestimate lysine oxidation because one tritium label may be taken up intracellularly by NADP and be sequestered in synthetic reactions (8). In spite of the different assumptions that are required when these two different tracers are used, estimates of lysine oxidation for the ³H tracer were very similar to those of the ¹⁴C tracer.

The absence of an influence of type of tracer on the estimate of amino acid oxidation was also shown for valine (20) and for leucine, phenylalanine and histidine (30). Our finding that lysine oxidation rate was similar for both feeding frequencies is consistent with the only other study in which the effect of feeding frequency on lysine utilization was evaluated in pigs (13). Only when pigs were fed free synthetic lysine, feeding pigs more than once daily improved the utilization of lysine intake (13). In contrast, in rats (12) and adult humans (9,11), a slight increase in leucine oxidation rates was found when the feeding frequency was increased. This difference may be due to differences in metabolism across amino acids, different species or metabolic conditions, i.e., growing pigs are in an anabolic state retaining body protein, whereas adult humans and rats do not generally retain body protein.

Increasing the feeding frequency from 3 to 8 times daily reduced the amplitude of the feeding-induced pattern of lysine oxidation and plasma free lysine concentrations (Fig. 1). A similar attenuation of the pattern of leucine oxidation rates in humans given hourly meals rather than three discreet meals per day was reported by El Khoury et al. (9,11). They also reported a close relationship between leucine oxidation and plasma free leucine concentrations similar to that between lysine oxidation rates and plasma free lysine concentrations in our experiment. This feeding-related pattern of lysine oxidation means that in future studies, complete measurement periods, from feeding to feeding, can be used to accurately extrapolate shorter infusion periods to daily oxidation rates.

Although measurements of lysine oxidation using [1-¹⁴C]L-lysine and [6-³H]L-lysine as tracers require different sets of assumptions, both isotopes gave similar estimates of lysine oxidation. When based on plasma free lysine SRA, lysine oxidation (9.2% of intake) was lower than when based on liver free lysine SRA (20.1% of intake), which was similar to the lysine losses calculated from N-balance data (21.8% of intake). Our data indicate that 20% of the true ileal digestible lysine intake is lost to minimum oxidation. Increasing the feeding frequency from 3 to 8 times daily had no effect on daily lysine oxidation. However, both the amplitude of the feeding-induced pattern in lysine oxidation rates and the variation within feeding cycles were reduced by increased feeding frequency. The reduced amplitude and variability of oxidation measurements indicate a greater reproducibility of values obtained at the higher feeding frequency. Observations made during entire feeding cycles, i.e., periods between feedings, can be used to predict 24-h lysine oxidation values.

	FOOTNOTES

 
¹ Supported by Archer-Daniel Midland, Degussa AG, Eurolysine SpA., Rhone Poulenc Animal Nutrition, Ontario Ministry of Agriculture, Food and Rural Affairs and the Natural Science and Engineering Research Council. S.M. was supported by a stipend from the Deutsche Forschungsgesellschaft.

³ Abbreviations used: BW, body weight; F3/F8, experimental treatments with pigs fed 3/8 times daily; PD, protein deposition; SRA, specific radioactivity.

Manuscript received 28 March 2003. Initial review completed 19 May 2003. Revision accepted 31 July 2003.

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