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* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada, T6G 2P5 and
Research Institute, Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8
4To whom correspondence should be addressed. E-mail: Ron.Ball{at}ualberta.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • adaptation • amino acid kinetics • pigs • indicator amino acid oxidation
A traditional method for the determination of amino acid requirements in humans and animals is the N balance method. This method has been criticized (1,2), mainly for systematically overestimating the balance (3). A more recent method, the indicator amino acid oxidation (IAAO) technique avoids these errors (4), but has been criticized because the short period of adaptation to a change in amino acid intake has not been validated (5). A detailed review of the method was published recently (6).
Adaptation is a concept that arose from the peculiarities of the N balance technique. First, the determination of amino acid requirements by the N balance method requires the equilibration of the body’s urea pool and the contents of the gastrointestinal tract after a change of diet. Because of the slow turnover of the urea pool (7) and the long time required to completely replace the contents of the gastrointestinal tract, adaptation periods of at least 5 d are recommended before the start of an experiment. The experimental period should also last for at least 5 d, preferably 10–14 d, to compensate for day-to-day variation in urine and feces excretion (8). The ensuing experimental period of at least 10 d clearly poses problems if a researcher intends to study the amino acid requirement under rapidly changing physiologic conditions, such as for human infants, neonatal pigs, during lactation, or in situations in which a prolonged deficient intake is ethically unacceptable. In spite of the advantages of the N balance technique, such as its low cost and ease of conductance as well as extensive reference data, studies that take place under these changing physiologic conditions require more sensitive, but technically demanding methods such as isotope dilution techniques.
The IAAO is based on the partitioning of amino acids between either protein synthesis or oxidation (4,6). This concept has two important implications for the issue of adaptation: first, measurements made using the IAAO are the net result of amino acid metabolism, which includes all fates of the amino acid under study, including protein synthesized in skin and hair and other uses (5). Therefore, results of the IAAO should be more accurate than results obtained by the N balance method, especially under maintenance conditions. Second, the inability of the body to store amino acids for a prolonged period of time or in substantial amounts (9) means that an animal’s metabolism is forced to adjust to changing amino acid supplies in a very short time period. Indeed, a study in human subjects showed that phenylalanine oxidation had already reached a new equilibrium 3 d after a change in diet (10), with no further change in oxidation when the dietary regimen was maintained for 6 or 9 d. Comparable observations in pigs are not available, and adaptation for IAAO requires study in both humans and animals. A further question to be addressed is the repeatability of measurements made using the IAAO within individuals. Previous applications of this method have relied on a single oxidation measurement per dietary treatment (6). Our hypothesis was that indicator amino acid oxidation responds as rapidly in growing or adult pigs as in humans, i.e., within 3 d or less, and that the repeatability of the measurements compares favorably with those made by N balance.
These open questions led us to investigate the effect of adaptation time on indicator amino acid oxidation in growing pigs and adult pigs at maintenance. The design of the experiment also allowed us to derive an estimate of the repeatability of oxidation measurements within the same pig.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For the growing pig study, Yorkshire-Landrace castrated male pigs (n = 4) weighing 15–18 kg were adapted to an excess lysine diet over a period of 5 d. After the pigs underwent overnight food deprivation, one catheter was inserted into each femoral vein under halothane anesthesia and advanced to the inferior vena cava; catheters were staggered to ensure that blood was not sampled downstream from the isotope infusion. Catheters were then tunneled under the skin from the incision sites to a point of exit between the shoulders on the back. Cotton mesh netting was fitted around the pig’s chest to secure the externalized catheters. Pigs were treated with antibiotics and analgesic and left to recover in separate metabolism pens. After 7 d of recovery while being fed the excess lysine diet, indicator oxidation was measured. Then the pigs were switched to a lysine-deficient diet. Oxidation measurements were performed in each pig at 2, 3, 4, and 6 d after the change in lysine intake. Then diets were switched again to excess lysine, and oxidation was measured again 2, 3, 4, and 6 d after the change in diet.
Fourth-parity adult Yorkshire-Landrace nonpregnant sows (n = 4), with a mean body weight of 242.5 ± 8.3 kg, were adapted for at least 1 wk to a diet providing 200% of the daily maintenance requirement for amino acids (11). After adaptation, sows were surgically fitted with catheters in the cephalic veins for repeated isotope infusions and blood sampling. Catheters were tunneled under the skin from the incision sites to a point of exit between the shoulders on the back. Pigs were treated with antibiotics and analgesic and left to recover for at least 1 wk in individual pens (2.13 m x 1.52 m). During this time, sows were fed the diet providing 200% of the daily maintenance requirement for amino acids. In the evening after the first isotope infusion, the pigs were switched to a diet providing 100% of the daily maintenance requirement for amino acids. For 2 sows, oxidation studies were performed 1, 5, and 9 d after the change of diets; for the other two sows, oxidation was measured 2, 6, and 10 d after the diet change. After the last infusion for that level of amino acid intake, the pigs were switched to a diet providing 50% of the daily maintenance requirement for amino acids and oxidation studies were performed again at the same time points after the diet change.
Diets and feeding.
All pigs were fed twice daily, except for the infusion days, when they received half the daily ration divided into 8 hourly meals; the remaining daily ration was fed in the evening. Feed intake was monitored on a daily basis. Any feed refusals were collected, dried, and weighed.
For the growing pig study, two isonitrogenous and isoenergetic diets based on corn gluten meal, wheat, and cornstarch (Table 1) provided all nutrients, except lysine, at the same level of >120% of requirements according to the NRC (11). The lysine-deficient diet provided lysine at 5.4 g/kg diet, whereas the excess lysine diet was adjusted to 11.8 g/kg by the addition of L-lysine-HCl, at the expense of glutamic acid. Daily feed intake for growing pigs was restricted to 90 g/kg0.75.
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Tracer infusion and sampling procedures.
All pigs were subjected to repeated primed, constant 4-h infusions. In sows, a priming dose of 1.75 times the hourly infusion dose was given within 1 min, followed immediately by the constant infusion at a rate of 567.8 ± 54.9 kBq/h of L-[1-14C]phenylalanine. The growing pigs received a priming dose of 1.75 times the constant infusion rate of 472.6 ± 14.4 kBq/h of L-[1-14C]phenylalanine (ARC).
The equipment consisted of respiration chambers fitted with feeders and drinkers, air flow meters, and a series of gas-washing bottles for 14CO2 collection. The respiration boxes had a volume of 
1.2 m3 for growing pigs and 2 m3 for sows. Air was drawn through these boxes by rotary vane pumps (Gast Model 1023, Gast Manufacturing) via an inlet at the rear and an outlet above the trough at rates of 140 L/min for growing pigs and 240 L/min for sows. After passing through a cold water condenser to remove water from the air, the airflow was divided between a series of gas washing bottles for CO2 collection and a line by-passing the collection. The flow rate through each diversion was measured with two commercial air meters (Canadian Meter). The gas washing bottles were changed at 30-min intervals throughout the studies. The CO2 absorber (ethanolamine:2-methoxyethanol, 1:2, v:v; Caledon) was weighed, sampled, and mixed with a scintillation cocktail (Atomlight, Canberra Packard) for scintillation counting. The samples were counted for 15 min or to an error of 2% in a liquid scintillation counter (Beckman LS3000, Beckman).
Because the pigs were repeatedly dosed with radioactivity, the background radioactivity in breath was expected to increase. Background measurements in growing pigs proved highly variable due to the excited physical activity of pigs during the first 2 daily feedings; this activity increased carbon dioxide excretion and rendered it erratic and unrelated to earlier isotope infusion. Therefore, radioactive background in 4 other growing pigs of the same genetic background and subjected to similar feeding and isotope infusion regimens was determined in a separate experiment using repeated steady-state measurements of 14CO2 excretion beginning after the second feeding, which corresponds to the time of collection for the adaptation experiment. 14CO2 was collected for 6 consecutive 20-min periods at 1, 2, 3, 5, and 9 d after the infusion studies. Using these steady-state estimates of background 14CO2, the decline in expired 14CO2 over this period (i.e., biological decay) was used to predict the radioactive background for each infusion day. Total cumulative dose and time elapsed from previous infusion were used as variables to calculate the steady-state background during infusions.
The sows were calmer during the experimental procedures than growing pigs; therefore, the radioactive background in breath was determined during a 30-min CO2 collection immediately before the next isotope infusion. This collection was started as soon as the CO2 content in the air drawn from the respiration chamber had reached equilibrium as monitored by a CO2 analyzer (Beckman LB 2, Beckman).
Blood samples were collected at 30-min intervals to determine the specific radioactivity (SRA, Bq/mmol) in plasma free phenylalanine and tyrosine, and to measure plasma amino acid concentrations. Blood was not collected in growing pigs because of the failure of one of the two catheters in most pigs.
Analytical procedures.
Feed and urine nitrogen contents were determined by the micro-Kjeldahl technique according to the AOAC (12). Urinary creatinine was determined by a colorimetric method using the appropriate analytical kit (Sigma-Aldrich). Plasma amino acids were determined using precolumn derivatization with phenylisothiocyanate (13) and HPLC (Waters, Millipore). U-14C-leucine was added to the plasma samples as an internal standard to determine the recovery of radioactivity during sample preparation. The fractions containing tyrosine, leucine, and phenylalanine were collected quantitatively for the determination of the specific radioactivity using a fraction collector (Foxy II, Isco). The collected fractions (3 mL) were mixed with 13 mL of a scintillation cocktail (BCS, Amersham) and counted for 30 min or to an error of 2% in a liquid scintillation counter (Beckman LS 3000).
Calculation of results.
Plateaus in breath 14CO2 were defined as those time periods during an infusion study that gave a regression with a slope that was not significantly different from zero. The following variables were calculated for sows only because the loss of one of the two catheters in growing pigs resulted in missing values for more than half of the observations. Phenylalanine oxidation was expressed as a percentage of infusion dose oxidized during plateau periods, where the expiration of 14CO2 was the total radioactivity per collection period divided by the fraction of air flow collected. Phenylalanine flux was calculated as the hourly L-[1-14C]phenylalanine infusion rate divided by plasma free phenylalanine SRA, and extrapolated to 24-h values. Daily phenylalanine oxidation rates (g/d) were calculated as the percentage of oxidation multiplied by phenylalanine flux. Phenylalanine balance (g/d) was calculated as phenylalanine intake minus phenylalanine oxidation. The conversion of phenylalanine to tyrosine was expressed as the percentage of phenylalanine flux and calculated as plasma free tyrosine SRA divided by plasma free phenylalanine SRA.
Statistical analyses.
The effects of time of adaptation were assessed using the procedure MIXED (14), with dietary treatment and time of adaptation nested in dietary treatment as independent variables. "Pig" was included in the model as a random effect. Comparisons between means were performed using the Student-Newman-Keul’s option of the procedure MIXED. Additionally, the effect of time of adaptation on the percentage of the dose oxidized was tested using the general linear model procedure within treatment groups. The repeatability of oxidation measurements was assessed using the mean CV of measurements within pigs and treatments. The data were expressed as least square mean ± SE. Differences were considered significant at P < 0.05 and as tendencies at P < 0.1.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The body weight (19.5 kg ± 0.7) and feed intake (730 g/d ± 32) of the growing pigs increased during the experiment, but did not affect phenylalanine oxidation. The mean oxidation rate obtained in pigs fed the lysine-deficient intake (3.8 g/d) was greater (16.94 vs. 9.64%, pooled SE 0.72; P = 0.001) than that in pigs fed the excess lysine intake (8.8 g/d). When the lysine intake was decreased from the initial excess intake to the deficient intake, phenylalanine oxidation almost doubled within 2 d of the diet change (Fig. 1). There was no change in phenylalanine oxidation when the measurements were repeated 3, 4, or 6 d after the diet change, and the regression equation for the percentage of the dose oxidized vs. day of adaptation had a slope that did not differ from zero. After switching the pigs back to the excess lysine intake, the phenylalanine oxidation dropped within 2 d to a level that did not differ from the initial level of oxidation observed after 2 wk of adaptation. There was no further change in the phenylalanine oxidation when the measurements were repeated for the excess lysine intake at 3, 4, or 6 d after the diet change. Regression analysis showed that the time after a change in lysine intake did not affect the indicator oxidation rate.
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The phenylalanine oxidation rate responded to increasing protein intake in a quadratic manner (r = 0.83), implying that the current estimate for amino acid maintenance requirements for sows may be too low. The minimum oxidation was predicted at 63.1 g/d protein intake, which amounted to 1.5 times the maintenance amino acid intake according to the NRC (11). However, the decrease in oxidation rate does not allow determination of which amino acid(s) were supplied in insufficient amounts in sows fed at 100% of the maintenance requirement.
Repeatability of measurements. The repeatability of the oxidation measurement was derived from the CV of the oxidations within pigs and diet level. The mean CV for phenylalanine oxidation within sows and dietary treatments was 8.80%. The CVs for the plasma concentrations of essential amino acids ranged from 16.3 to 35.8%, whereas the CV for the urinary N to creatinine ratio was 24.8%. In growing pigs, the mean CV of the oxidation measurements within diet and pig was 11.2%.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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45 min for phenylalanine; data not shown) necessitating an immediate response in metabolism to an influx of nutrients. However, this rapid response does not mean that adaptation occurs within a short time frame. Rather, the criterion for adaptation should be a repeatable, steady-state response over a period of time, after a nutritional intervention was performed. Using this criterion, adaptation should be regarded as complete at the first time point of a steady-state plateau in a series of oxidation measurements. Indeed, it was shown in adult humans (10) that phenylalanine oxidation did not differ at 3, 6, or 9 d after a change in phenylalanine intake. Those data indicated that an adaptation had occurred by at most 3 d after a diet change. A more recent study by Motil et al. (8) showed that the adaptation to a change in protein intake was complete within 2 d. Furthermore, Young et al. (18) showed that leucine oxidation returned to its original level within 2 d after a 3-wk leucine-deficient diet was replaced by an adequate diet. However, these direct oxidation studies (8,10,18) involved a change in the pool size of the amino acid being oxidized due to changing the dietary intake of the same amino acid. In contrast, because the IAAO technique employs an unchanging intake and pool size for the oxidized amino acid, it should be expected that the adaptation period required for the IAAO would be at least as short as that for the direct oxidation method, and possibly shorter.
In the present study, we showed that phenylalanine oxidation in growing pigs adapted within 2 d to a change in lysine intake, regardless of the direction of change in dietary lysine intake. It is possible that an adaptation occurred even earlier; however, we did not test the oxidation the day after the change in diet, as we did for sows. For adult nonpregnant sows, a decrease in protein intake caused the phenylalanine oxidation to respond within 18 h, with no further change when the new dietary regimen was maintained for 10 d. It should be noted that the short adaptation time for the IAAO was demonstrated irrespective of the manipulation of a single amino acid or of all dietary amino acids (except for phenylalanine and tyrosine). Furthermore, the short adaptation time was maintained regardless of the rate of body protein turnover, which differs widely between growing and adult pigs or human subjects (7). Therefore, it can be concluded that 2 d are fully adequate to achieve adaptation when using the IAAO. In adult humans, 2 d of adaptation to the protein intake level also is adequate when using the IAAO technique (19). This short adaptation is a major advantage of the technique and allows us to measure the response to several test diets within a relatively short period of time within the same individual. This advantage is important for studies under many conditions.
An important question is how to define adaptation. Young and Marchini (20) reviewed the debate surrounding adaptation vs. accommodation, the latter describing the long-term physiological responses to a habitual dietary regimen. With long-term adaptation to a deficient diet, the subject will "accommodate" to this situation and possibly become more efficient in its metabolism. However, does this accommodation come at the cost of other unmeasured metabolic functions (i.e., work capacity, immune function, stress response)? If such costs are incurred, then it violates Waterlow’s (21) definition of adaptation: the process that permits the organism to respond to dietary change without adverse consequences. Therefore, short-term adaptation will by definition be more reflective of the requirement of the healthy animal. We also must consider the definition of amino acid requirement as proposed by Young and Borgonha (22): the minimal intake level needed to maintain a specific nutritional criterion such as growth, body composition, body amino acid balance, organ, or system function. In spite of this ongoing debate, studies in humans have methodically shown that oxidation methods have the distinct advantage over the nitrogen balance technique of requiring only very short adaptation periods, thus resulting in more time-efficient and cost-effective studies. Indeed, in human studies, the estimates of lysine requirement were very similar whether an adaptation period of hours (23–25), 7 d, or 21 d (26) was employed. This finding is profound in the context of the aforementioned ongoing debate regarding adaptation vs. accommodation.
This experiment showed that the minimum adaptation time required when using the IAAO technique is at most 2 d and probably less. This adaptation time is valid whether the animals are growing or at maintenance and whether the dietary intake of one or all amino acids has been manipulated. The short adaptation time makes the IAAO method suitable to study the effect of several dietary manipulations within individual animals over a short period of time. This distinct advantage of short adaptation over the classical nitrogen balance or growth assays is profound when studying animals in rapidly changing physiologic states such as growth, gestation or lactation, or disease.
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FOOTNOTES |
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2 Supported by grants from the Alberta Agricultural Research Institute, Alberta Pork and Canadian Institutes of Health Research and by Degussa AG, Germany.
3 Present address: Memorial University of Newfoundland, St. John’s, NL, Canada, A1B 3X9.
Manuscript received 31 October 2003. Initial review completed 9 December 2003. Revision accepted 30 December 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Fuller, M. F. & Garlick, P. J. (1994) Human amino acid requirements: can the controversy be resolved?. Annu. Rev. Nutr. 14:217-241.[Medline]
2. Young, V. R. (1987) McCollum award lecture. Kinetics of human amino acid metabolism: nutritional implications and some lessons. Am. J. Clin. Nutr. 46:709-725.
3. Wallace, W. M. (1959) Nitrogen content of the body and its relation to retention and loss of nitrogen. Fed. Proc. 18:1125-1130.[Medline]
4. Brunton, J. A., Ball, R. O. & Pencharz, P. B. (1998) Determination of amino acid requirements by indicator amino acid oxidation: applications in health and disease. Curr. Opin. Clin. Nutr. Metab. Care 1:449-453.[Medline]
5. Bos, C., Gaudichon, C. & Tomé, D. (2002) Isotopic studies of protein and amino acid requirements. Curr. Opin. Clin. Nutr. Metab. Care 5:55-61.[Medline]
6. Pencharz, P. B. & Ball, R. O. (2003) Different approaches to define individual amino acid requirements. Annu. Rev. Nutr. 23:101-116.[Medline]
7. Waterlow, J. C., Garlick, P. J. & Millward, D. J. (1978) Protein Turnover in Mammalian Tissues and in the Whole Body 1978 North-Holland Publishing Oxford, UK .
8. Motil, K. J., Opekun, A. R., Montadon, C. M., Berthold, H. K., Davis, T. A., Klein, P. D. & Reeds, P. J. (1994) Leucine oxidation changes rapidly after dietary protein intake is altered in adult women but lysine flux is unchanged as is lysine incorporation into VLDL-apolipoprotein B-100. J. Nutr. 124:41-51.
9. Holt, L. E., Jr., Halac, E. & Kadji, C. N. (1962) The concept of protein stores and its implications in diet. J. Am. Med. Assoc. 181:699-705.
10. Zello, G. A., Pencharz, P. B. & Ball, R. O. (1990) Phenylalanine flux, oxidation and conversion to tyrosine in humans studied with of L-[1-13C]phenylalanine. Am. J. Physiol. 259:E835-E843.[Medline]
11. NRC National Research Council (1998) Nutrient Requirements of Swine 10th ed. 1998 National Academy Press Washington, DC.
12. Association of Official Analytical Chemists (1990) Official Methods of Analysis 15th ed. 1990 Arlington VA.
13. Bidlingmeyer, B. A., Cohen, S. A. & Tarvin, T. L. (1984) Rapid analysis of amino acids using precolumn derivatisation. J. Chromatogr. 336:93-104.[Medline]
14. SAS Institute Inc. (1999) Version 8.0 1999 SAS Institute Cary, NC.
15. Taveroff, A., Lapin, H. & Hoffer, L. J. (1994) Mechanism governing short-term fed-state adaptation to protein restriction. Metabolism 43:320-327.[Medline]
16. Moehn, S., Fuller, M. F., Ball, R. O. & de Lange, C.F.M. (2003) Feeding frequency and tracer do not affect direct estimates of lysine oxidation in the growing pig. J. Nutr. 133:3504-3508.
17. Garlick, P. J., McNurlan, M. A. & Patlak, C. S. (1999) Adaptation of protein metabolism in relation to limits to high dietary protein intakes. Eur. J. Clin. Nutr. 53(suppl. 1):S34-S43.
18. Young, V. R., Fukagawa, N., Bier, D. M. & Matthews, D. (1986) Some aspects of in vivo human protein and amino acid metabolism, with particular reference to nutritional modulation. Verh. Dtsch. Ges. Inn. Med. 92:640-665.[Medline]
19. Thorpe, J. M., Roberts, S. A., Ball, R. O. & Pencharz, P. B. (1999) Prior protein intake may affect phenylalanine kinetics measured in healthy adult volunteers consuming 1 g protein·kg-1 · d-1. J. Nutr. 129:343-348.
20. Young, V. R. & Marchini, J. S. (1990) Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am. J. Clin. Nutr. 51:270-289.
21. Waterlow, J. C. (1985) What do we mean by adaptation?. Blaxter, K. L. Waterlow, J. C. eds. Nutritional Adaptation in Man 1985:1-10 John Libbey London, UK. .
22. Young, V. R. & Borgonha, S. (2000) Nitrogen and amino acid requirements: the Massachusetts Institute of Technology amino acid requirement pattern. J. Nutr. 130:1841S-1849S.
23. Zello, G. A., Pencharz, P. B. & Ball, R. O. (1993) Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C]phenylalanine. Am. J. Physiol. 264:E677-E685.
24. Duncan, A. M., Ball, R. O. & Pencharz, P. B. (1996) Lysine requirement of adult males is not affected by decreasing dietary protein. Am. J. Clin. Nutr. 64:718-725.
25. Kriengsinyos, W., Wykes, L. J., Ball, R. O. & Pencharz, P. B. (2002) Effect of oral and intravenous tracer on the estimate of lysine requirement in healthy adult males using L-[1-13C]phenylalanine as an indicator amino acid. J. Nutr. 132:2251-2257.
26. Kurpad, A. V., Regan, M. M., Raj, T., el-Khoury, A., Kuriyan, R., Vaz, M., Chandakudlu, D., Venkataswamy, V. G., Borgonha, S. & Young, V. R. (2002) Lysine requirements of healthy adult Indian subjects receiving long-term feeding, measured with a 24-h indicator amino acid oxidation and balance technique. Am. J. Clin. Nutr. 76:404-412.
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