QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moehn, S. Articles by Ball, R. O. Search for Related Content PubMed PubMed Citation Articles by Moehn, S. Articles by Ball, R. O.

---

Nutritional Methodology

Development of the Indicator Amino Acid Oxidation Technique to Determine the Availability of Amino Acids from Dietary Protein in Pigs^1,2

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5; ^* Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 and the Departments of Paediatrics and Nutritional Sciences, University of Toronto, Toronto, ON, Canada

⁴To whom correspondence should be addressed. E-mail: Ron.Ball{at}ualberta.ca.

ABSTRACT

Standardized ileal ("true") digestibility is currently the best estimate of amino acid digestibility, but it does not measure bioavailability. Growth assays to determine amino acid bioavailability are expensive and laborious; thus, a rapid method is needed. Applying the principle of slope-ratio assay to the indicator amino acid oxidation (IAAO) method, we hypothesized that the reduction in indicator oxidation per gram of lysine in feedstuffs relative to that per gram of free lysine represented the bioavailability of lysine, here termed "metabolic availability." Indicator oxidation in pigs was linear over increasing lysine intakes (r = 0.90, P = 0.001) when the dietary lysine contents were 2 SD below the mean lysine requirement of the pigs. Peas were treated (raw, heated to reduce lysine availability, or heated with added lysine) to test the responsiveness of the IAAO to differing lysine availability. Free lysine reduced indicator oxidation by 3.16% of dose oxidized per gram added lysine, whereas the addition of protein lysine as raw (–2.81%) and heated peas (–1.73%) reduced oxidation to a lesser degree. Adding free lysine to heated peas decreased indicator oxidation, evidence that heating had worsened the utilization of pea protein for protein synthesis by reducing the bioavailability of lysine alone. Pea diets differed only in the availability of lysine; therefore IAAO detected differences in lysine bioavailability. Because the IAAO technique responds to lysine available at the sites of protein synthesis, the metabolic availability covers all losses during digestion, absorption, and utilization of lysine. This method can determine the metabolic availability of amino acids of a feedstuff within 2 wk.

KEY WORDS: • pigs • lysine • bioavailability • indicator amino acid oxidation

In pig nutrition, the amino acid content of diets is commonly expressed as standardized ileal digestible amino acids (1). The determination of apparent digestibility involves measuring the disappearance of amino acids at the terminal ileum before microbial metabolism of amino acids in the colon (2). However, some of the amino acids at the ileum are of endogenous origin; therefore this approach underestimates true digestibility of amino acids. In swine nutrition, these apparent digestibility data are standardized by employing an estimate of ileal losses of endogenous amino acids (1,3). However, a single estimate of gut endogenous amino acid losses is inappropriate because these losses differ among feedstuffs depending on protein content, antinutritional factors, and type and quantity of fiber. Additionally, not all of the absorbed amino acids are available for protein synthesis; some lysine can be digested and absorbed but it is in a chemically complexed form that cannot be used by animals for body protein synthesis (i.e., Maillard products) (4). The importance of this discrepancy was shown in slope-ratio growth trials in which pigs were fed diets formulated to various true ileal digestible lysine levels (5). At similar intakes of true ileal digestible lysine, the diets using various protein sources did not result in protein deposition or feed conversion similar to the synthetic lysine diets, even though these diets were formulated on the basis of data supposedly corrected for digestibility differences. Although such growth and slaughter trials serve as the absolute standard for feed evaluation, their laboriousness and expense preclude their use on a routine basis (6). Therefore, there is a need to develop a more rapid, less expensive method for determining metabolic amino acid availability in foods.

We chose to evaluate the indicator amino acid oxidation (IAAO) method to determine the availability of amino acids from dietary proteins for protein synthesis. When one amino acid is limiting protein synthesis, then all other amino acids are in excess and must be catabolized. By monitoring this catabolism using a ¹⁴C-labeled amino acid, one of these excess amino acids will indicate the rate of whole-body protein synthesis as driven by the limiting amino acid. Because the appearance of ¹⁴CO₂ at plateau reflects whole-body protein synthesis, any changes in oxidation rate will reflect the whole-body bioavailability of the limiting amino acid, and thus account for all losses of dietary amino acids during digestion and cellular metabolism. In other words, the higher the oxidation of the indicator, the lower the metabolic availability of the test amino acid for protein synthesis and vice versa. We hypothesized that at a given amino acid intake, differences in indicator amino acid oxidation rate would be proportional to the bioavailability of an amino acid. We propose the term "metabolic availability" for this definition of bioavailability.

To determine the metabolic availability, key conditions must be fulfilled. First, the test amino acid must be first limiting to drive indicator oxidation rates. Second, the response of oxidation rate to increments of the test amino acid must be predictable to allow calculation of availability according to the principle of the standard curve assay (7). Third, the indicator oxidation rate must show good repeatability to allow the accurate determination of metabolic availability. The objective of the proposed research was to demonstrate that the IAAO method is suitable to determine the metabolic availability of amino acids in feedstuffs for pigs, using lysine as an example.

MATERIALS AND METHODS

    Animals and diets. All procedures were approved by the Faculty Animal Policy and Welfare Committee of the University of Alberta. Yorkshire/Landrace barrows [body weight (BW) 15–18 kg] from a specific pathogen–free herd (University of Alberta Swine Unit, Edmonton, AB, Canada) were adapted to individual metabolic cages (1 x 2 m) and the experimental base diet over 3 d before being surgically fitted with catheters in each femoral vein (8). After 3 d of postsurgical recovery, pigs were fed test diets and oxidation regimens were started at 7 d postsurgery. Oxidation studies were performed after 2 d of adaptation to each test diet (9); for duplicate oxidations on a given test diet, studies were performed on consecutive days. At the end of the experiments, the pigs were killed by an injection of sodium pentobarbital.

Pigs were fed twice daily, except for the infusion days, when they received half the daily ration divided into 8 hourly meals during i.v. isotope administration or in 16 half-hourly meals during oral isotope administration; the remaining daily ration was fed in the evening. The effect of feeding frequency on amino acid catabolism was previously evaluated (10). Feed intake was restricted to 90 g/(kg^–0.75 BW · d); water was available at all times. The isoenergetic, isonitrogenous diets were based on barley and soybean meal (Table 1). These diets were formulated to be first limiting in lysine at the highest lysine concentration. In the diets containing the test feedstuffs, peas were added in an amount required to achieve a lysine concentration equivalent to 80% of that recommended by the NRC (3) (i.e., high lysine diet in Table 1); in these pea diets, corn oil was substituted for cornstarch to achieve the desired concentrations of metabolizable energy. Peas were added either raw, or after heating according to the protocol by van Barneveld et al. (11) to render the lysine in peas unavailable via Maillard products (peas were heated to 165°C over 7.5 h and held for 15 min). For the third treatment, free lysine-HCl was added to the diet of heated peas in an amount equal to that in raw peas. Phenylalanine and tyrosine concentrations were kept constant in all test diets.

    Isotope infusion and calculations. All pigs were subjected to repeated primed, constant 4-h infusions. The growing pigs received a priming dose of 1.75 times the constant infusion rate of 473 ±14 kBq/h of L-[1-¹⁴C]phenylalanine (American Radiolabeled Chemicals) mixed in sterile saline. For i.v. infusions, the isotope solution was infused continuously using a syringe pump into one of the femoral catheters. For oral infusions, one-eighth of the total 4-h dose was mixed into one-sixteenth of the study period feed ration and the mash was placed in the feed trough every 0.5 h. All feed aliquots were consumed by all pigs during the 0.5 h before the next isotope aliquot.

With repeated isotopic tracer infusions, there is an accumulation of L-[1-¹⁴C]phenylalanine in the pig’s body protein resulting in a radioactive background in expired ¹⁴CO₂. The steady state radioactive background was determined in 4 growing pigs of the same genetic background and size; the pigs were subjected to similar feeding and isotope infusion regimens (9). The radioactive background estimate was expressed as a percentage of the cumulative dose infused:

The ¹⁴CO₂ plateau values were corrected for radioactive background according to this equation, and oxidation rates were calculated from these corrected plateau values.

    Linearity of response to lysine intake. The linearity of indicator oxidation response was demonstrated in a preliminary experiment and included data from a total of 9 pigs for which the individual lysine requirement had been determined (8). Each of these 9 pigs was studied at lysine intakes below and above the requirement breakpoint. Data below the breakpoint for each pig were used to describe linearity in the oxidation response to increments of lysine intake.

    Metabolic availability of lysine in test ingredients. In 4 pigs, the linearity of oxidation was determined at lysine intakes corresponding to 50, 60, 70, and 80% of the requirement according to NRC (3). This approach used single measurements on alternating days to adapt to each new diet. For the same pigs, peas, heated peas, or heated peas plus lysine were included in the base diet at the expense of cornstarch to achieve a lysine content of 80% of the lysine requirement according to the NRC (3). Phenylalanine oxidation was measured twice for each of the diets. In 2 pigs, determination of linearity and lysine availability in the pea diets was repeated using oral isotope delivery.

    Statistical analyses. Lysine intake was expressed as the intake above that provided by the base diet (50% lysine). The effect of lysine intake on phenylalanine oxidation was estimated using the procedure "mixed" (12) with "oxidation day" as repeated measurement (where applicable) and "pig" as random variable. Nesting lysine intake within type of lysine addition (e.g., free lysine or lysine in peas) gave the change (slope) in phenylalanine oxidation per gram of lysine for each type of lysine addition. The metabolic availability of lysine in feedstuffs was calculated by dividing the slope for lysine from a feedstuff by the slope for free lysine. Covariates and their interactions with main effects were tested but were notsignificant (P > 0.05). The repeatability of oxidation measurements was assessed using mean CV of measurements within animals and treatments. Results were expressed as least square means ± SE. P < 0.05 was regarded as significant.

RESULTS

    Linearity of response to increasing lysine intake. Pigs consumed 89.1 ± 0.9 g/kg^0.75 feed/d. At dietary lysine intakes below the individual pig’s requirement, determined using IAAO (8), phenylalanine oxidation decreased linearly with increasing dietary lysine intake. Within pigs, phenylalanine oxidation decreased by 2.48–5.21% of dose/g added lysine (r = 0.70–0.95). The mean decrease in phenylalanine oxidation was 3.27 ± 0.33%/g added lysine (r = 0.90, P = 0.001, Fig. 1) and did not differ among all pigs except for one that exhibited a greater decline in oxidation rate. The mean rate, over all dietary treatments, of phenylalanine oxidation differed among pigs (P = 0.009). Given the differences among pigs in basal oxidation rates and in oxidation response to added lysine, these parameters must be determined in each pig to determine the availability of lysine in feedstuffs. To avoid lysine intake in excess of any pig’s individual requirement, the upper limit of lysine intake should be set at 80% of the requirement (3), or 2 SD (8) below the requirement according to the NRC (3).

View larger version (20K): [in this window] [in a new window]   FIGURE 1 Linearity of L-[1-14C]phenylalanine oxidation (percentage of dose) during i.v. or oral isotope delivery in pigs fed graded levels of lysine-HCl. Values are means ± SD (percentage of dose with vertical error bars and intake with horizontal error bars); error bars on some means were too small to be displayed. The equation represents linear regression on data. Daily lysine intake from the base diet was 3.9 g/d.

    Repeatability of measurements. The mean CV of repeated oxidation measurements with i.v. isotope delivery for a given diet was 10.3 ± 2.7%. The variability of measurements did not differ for peas and heated peas, but was greater (P = 0.02) for heated peas plus lysine than for peas. For oral isotope delivery (n = 2 pigs), the mean CV was 11.8 ± 5.5%.

DISCUSSION

A rapid method to measure amino acid bioavailability was developed by adapting the IAAO method and using the principle of the standard-curve assay. We hypothesized that the ratio of the oxidation change caused by incremental lysine in feeds to that caused by incremental free lysine represented the "metabolic availability" of lysine in feedstuffs. Our approach takes into account digestion of the dietary protein, the absorption and metabolism of the amino acids from the dietary protein, and utilization for protein synthesis.

The CO₂ output measured in IAAO studies represents an end product of amino acid metabolism, which is reflective of all losses during amino acid digestion, absorption, and metabolism. These constitute all of the components affecting bioavailability (6). In contrast, measuring amino acid digestibility covers the losses occurring only during digestion. Therefore, the metabolic availability can be expected to be more representative of the bioavailability of amino acids than digestibility values. Other methods, such as the homoarginine method (13), are applicable to lysine only, or lack suitability for routine determination of amino acid availability, such as the use of intrinsically labeled foods (14).

The shorter adaptation period (2 d) (9) for the IAAO than for balance or direct oxidation methods (15) allows the determination of the linear response to free amino acids within 8 d, followed by 3 d experimental time to determine the response to amino acids in one feedstuff. This short adaptation time is due to the constancy of indicator amino acid flux (16).

The IAAO technique responds to the partitioning of the indicator amino acid to either protein synthesis or oxidation in a 2-phase manner: decreasing oxidation at increasing intakes of the limiting amino acid when below requirement, and intake followed by a plateau in oxidation when the test amino acid is in excess of the requirement (17). Therefore, to achieve the condition of linearity of response for the application of slope-ratio or standard-curve assays (7,18), dietary test amino acid contents must be kept below the requirement of individual pigs. Testing this critical condition using diets with <80% of the predicted lysine requirement for each experimental pig accommodates differences in oxidation response among pigs and allows the identification of test amino acid dietary concentrations where the condition of linearity is fulfilled (7).

Because the indicator amino acid content must be constant in all diets and in excess of the requirement, any changes in its dietary intake will change the indicator oxidation rate irrespective of the content of the test amino acid. In the absence of "metabolic availability" data for phenylalanine, true ileal digestibility data (3) were used to formulate the diets so that phenylalanine did not differ significantly among the diets. This initial assumption was considered reasonable because phenylalanine does not participate in Maillard reactions and its ileal digestibility was not affected by the heating regimen (11). Although the requirement for phenylalanine must be exceeded to ensure that the test amino acid is first limiting, the inclusion level must not be too great, either. High phenylalanine intake was shown to preclude linear increase in phenylalanine oxidation, probably via overloading of phenylalanine hydroxylase, the rate-limiting enzyme in phenylalanine oxidation (19). Consequently, the diets were formulated to provide phenylalanine at a rate close to the recommendation of House et al. (19) who suggested supplying phenylalanine for IAAO studies at 2 SD above the requirement.

    Indicator oxidation is a measure of amino acid bioavailability. Peas were chosen as a test ingredient because of the exhaustive work by van Barneveld et al. (11,20,21) to characterize lysine availability in this feedstuff using several methods. Peas are normally not heat processed, unlike soybean, canola, or meat meals; this is a major cause of decreased metabolic availability of lysine because of the formation of Maillard products, which are absorbed, but unavailable for protein synthesis (4,5). By using peas, we were able to test the effects of raw vs. heated feedstuff vs. heated feedstuff plus free lysine to demonstrate that this new method detects differences in the metabolic availability of lysine.

Oxidation responded linearly to added free lysine, which was shown to be 100% digestible and available in pigs (22). When protein-bound lysine was added as peas, phenylalanine oxidation was higher than with equivalent amounts of free lysine, indicating that protein synthesis was lower; thus, less of the lysine was metabolically available (88.8%). When peas were heated, a large proportion of the pea lysine was rendered unavailable; protein synthesis was therefore lowered further, and phenylalanine oxidation was highest among treatments, indicating a 54.5% overall metabolic availability of lysine. Van Barneveld et al. (20,21) obtained similar results using the slope-ratio growth assay and similar dietary treatments (85 and 48% availability for lysine in raw and heated peas, respectively). Supplementing free lysine to the heated peas diet reversed the effects of heating, increasing protein synthesis and lowering phenylalanine oxidation to a rate lowest among all treatments. This last diet demonstrated that lysine availability alone was responsible for the lower protein synthesis and higher indicator amino acid oxidation after heating peas. Van Barneveld et al. (20,21) also showed that adding lysine to heated peas reversed the growth depression caused by heating the peas. The differences in oxidation response among free lysine in peas, heated peas, and heated peas plus lysine indicate that phenylalanine oxidation directly reflected the bioavailability of lysine and that the changes in oxidation were due entirely to changes in metabolically available dietary lysine. In our experiment, we added excess free lysine to the heated pea diet, an amount equivalent to the total lysine present in raw peas (i.e., assuming all lysine would be lost via heating). Because 55% of the lysine was still available after heating, the total available lysine in this supplemented diet exceeded each pig’s requirement. Consequently, the phenylalanine oxidation resulting from these diets was lower than that from the raw pea diet, further suggested that lysine alone recovered the effects of heating.

In some of the original studies of IAAO in pigs, oral delivery of isotope was performed as a single or double bolus dose, which did not allow phenylalanine oxidation to reach a steady state (23). Continuous i.v. isotope infusion circumvented this problem but created problems with catheter patency over a prolonged period of time (>2 wk). Because increasing the feeding frequency was shown to reduce the fluctuation of oxidation measurements (10), we offered feed dosed with isotope solution in 16 half-hourly meals. This regimen proved successful in that the plateaus in oxidation were reached and maintained for a similar length of time, repeated oxidation measurements showed a similar variability, and the oxidation response to the different test diets was analogous to the i.v. infusion regimens. The greater oxidation rate with oral isotope delivery (17.9 vs. 13.7% of dose) is probably due to the utilization of the tracer amino acid by the gastrointestinal tract, which is bypassed during i.v. infusion (24). The present data indicate a 30% higher oxidation of the tracer when given orally. However, given orally, dietary amino acids still undergo first-pass metabolism, leading to similar estimates for metabolic availability for both routes of tracer application. This is analogous to amino acid requirement estimates, which were not different when comparing oral isotope to i.v. delivery in pigs (25) and humans (26). Oral isotope delivery is a suitable tool with which to improve the practicality of the proposed method to determine amino acid availability. In particular, oral dosing simplifies the technique by eliminating surgical facilities, specialized housing conditions for catheterized animals, and risk of catheter-related sepsis.

Preliminary work from our group to develop this method further showed that adaptation of the IAAO method to determine amino acid (methionine) bioavailability for humans is promising (27). This indicates that the metabolic availability method is suitable for amino acids other than lysine and for monogastric species other than pigs.

In conclusion, using lysine, the IAAO method was able to determine amino acid bioavailability for protein synthesis in feedstuffs. This new method is based on the principle of standard curve growth assays, and the inverse effect of the limiting amino acid on protein synthesis vs. the utilization of other amino acids. This method allowed us to determine the lysine usable by pigs for protein synthesis by simple amino acid analysis of feedstuffs and a short in vivo experiment (<3 wk). Because the added lysine was the only difference in the diet containing heated peas, the response in phenylalanine oxidation was shown to be due entirely to the changes in the metabolic availability of lysine. Furthermore, the calculated availability in peas was similar to that determined by van Barneveld et al. (20) using the slope-ratio growth assay (85%) and to published values for the true ileal digestibility of lysine in peas (88%) (3). This agreement shows that the proposed method is suitable for detecting differences in the metabolic availability of lysine from feedstuffs. Furthermore, this "metabolic availability" includes all losses during digestion, absorption, and utilization of the test amino acid without the need to make assumptions about endogenous amino acid losses or utilization of absorbed amino acids. Therefore, this new method eliminates the reliance on assumed values to correct ileal digestibility of lysine for endogenous losses and unknown corrections for the chemical availability of absorbed lysine. The "metabolic availability" measured by this technique is therefore equivalent to the true bioavailability of lysine for protein synthesis. This method should be applicable to all essential amino acids, monogastric species, and foodstuffs.

FOOTNOTES

¹ Portions of this study were published previously in conference proceedings: Ball RO, Möhn S, Bertolo RFP, Korver D. Rapid new methods for measuring amino acid requirements and "true" amino acid availability in feeds for swine and poultry. Proc. 23th Western Nutrition Conference; 2002; Edmonton, AB, Canada. p. 151–61 and Möhn S, Bertolo RFP, Pencharz PB, Ball RO. A rapid method to determine "true metabolic availability" of amino acids in feedstuffs for pigs. 9th International Symposium on Digestive Physiology in Pigs; 2003, Banff, AB, Canada. Volume 2, p. 1–3, University of Alberta, Edmonton, AB, Canada.

² Supported by the Alberta Agricultural Research Institute, Alberta Pork and the Ontario Pork Producers Marketing Board. The amino acids were generously donated by Degussa AG, Germany.

³ Present address: Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9.

Manuscript received 16 May 2005. Initial review completed 29 June 2005. Revision accepted 31 August 2005.

LITERATURE CITED

1. Moughan PJ. Amino acid digestibility and availability in feedstuffs. Ball RO eds. Amino acid digestibility and availability in feedstuffs. Proceedings of the 9th International Symposium of the Digestive Physiology in the Pig. :199-221 University of Alberta Edmonton, Canada.

2. Sauer WC, Ozimek L. Digestibility of amino acids in swine: results and their practical application—a review. Livest Prod Sci. 1986;15:367-388.

3. National Research Council. Digestibility of amino acids in swine: results and their practical application—a review. Nutrient Requirements of Swine. 10th rev. ed. National Academic Press Washington, DC.

4. Carpenter KJ, Booth VH. Damage to lysine in food processing: its measurement and its significance. Nutr Abs Rev. 1973;43:423-440.

5. Batterham ES. Availability and utilization of amino acids for growing pigs. Nutr Res Rev. 1992;5:1-18.

6. Lewis AJ, Bayley HS. Amino acid availability. Ammerman CB Baker DH Lewis AJ eds. Amino acid availability. Bioavailability of nutrients for animals—amino acids, minerals, and vitamins. :35-65 Academic Press San Diego, CA.

7. Littell RC, Lewis AJ, Henry PR. Statistical evaluation of bioavailability assays. Ammerman CB Baker DH Lewis AJ eds. Statistical evaluation of bioavailability assays. Bioavailability of nutrients for animals—amino acids, minerals, and vitamins. :5-33 Academic Press San Diego, CA.

8. Bertolo RFP, Moehn S, Pencharz PB, Ball RO. Estimate of the variability of the lysine requirement of growing pigs using the indicator amino acid oxidation technique. J Anim Sci. 2005; In press.

9. Moehn S, Bertolo RFP, Pencharz PB, Ball RO. Indicator amino acid oxidation responds rapidly to changes in lysine or protein intake: studies in growing and adult pigs. J Nutr. 2004;134:836-841.[Abstract/Free Full Text]

10. Möhn S, Fuller MF, Ball RO, de Lange CFM. Feeding frequency and tracer do not affect direct estimates of lysine oxidation in the growing pig. J Nutr. 2003;133:3504-3508.[Abstract/Free Full Text]

11. van Barneveld RJ, Batterham ES, Norton BW. The effect of heat on amino acids for pigs. 1. A comparison of ileal and faecal digestibilities of amino acids in raw and heat-treated field peas (Pisum sativum cultivar Dundale). Br J Nutr. 1994;72:221-241.[Medline]

12. SAS Institute Inc. The effect of heat on amino acids for pigs. 1. A comparison of ileal and faecal digestibilities of amino acids in raw and heat-treated field peas (Pisum sativum cultivar Dundale). SAS/STAT User’s Guide, Release 8.2. SAS Institute Cary, NC.

13. Moughan PJ, Rutherfurd SM. A new method for determining digestible reactive lysine in food. J Agric Food Chem. 1996;44:2202-2209.

14. Bos C, Metges CC, Gaudichon C, Petzke KJ, Pueyo ME, Morens C, Everwand J, Benamouzig R, Tome D. Postprandial kinetics of dietary amino acids are the main determinant of their metabolism after soy or milk protein ingestion in humans. J Nutr. 2003;133:1308-1315.[Abstract/Free Full Text]

15. Motil KJ, Opekun AR, Montadon CM, Berthold HK, Davis TA, Klein PD, Reeds PJ. 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. 1994;124:41-51.[Abstract/Free Full Text]

16. House JD, Pencharz PB, Ball RO. Lysine requirement of neonatal piglets receiving total parenteral nutrition as determined by oxidation of the indicator amino acid L-[1–14C]phenylalanine. Am J Clin Nutr. 1998;67:67-73.[Abstract]

17. Pencharz PB, Ball RO. Different approaches to define individual amino acid requirements. Annu Rev Nutr. 2003;23:101-116.[Medline]

18. Finney DJ. Different approaches to define individual amino acid requirements. Statistical methods in biological assay. Charles Griffin & Company Ltd. London, UK.

19. House JD, Pencharz PB, Ball RO. Phenylalanine requirements determined by using L-[1–14C]phenylalanine in neonatal piglets receiving total parenteral nutrition supplemented with tyrosine. Am J Clin Nutr. 1997;65:984-993.[Abstract/Free Full Text]

20. van Barneveld RJ, Batterham ES, Norton BW. The effect of heat on amino acids for pigs. 2. Utilization of ileal-digestible lysine from heat-treated field peas (Pisum sativum cultivar Dundale). Br J Nutr. 1994;72:242-256.

21. van Barneveld RJ, Batterham ES, Norton BW. The effect of heat on amino acids for pigs. 3. The availability of lysine from heat-treated field peas (Pisum sativum cultivar Dundale) determined using the slope-ratio assay. Br J Nutr. 1994;72:257-275.[Medline]

22. Susenbeth A, Agunbiade A, Dohms J. Bioavailability of lysine of several protein sources determined by the slope-ratio assay in pigs. Février C Aumaitre A Habe F Vares T Zjalic M eds. Bioavailability of lysine of several protein sources determined by the slope-ratio assay in pigs. Protein Feed for Animal Production. EAAP Technical Series. :133-137.

23. Ball RO, Bayley HS. Time course of the total and radioactive carbon dioxide production by piglets receiving dietary [14C]phenylalanine. Can. J. Physiol. Pharmacol. 1985;63:1170-1174.[Medline]

24. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 1997;128:606-614.

25. Cvitkovic S, Bertolo RF, Brunton JA, Pencharz PB, Ball RO. Enteral tryptophan requirement determined by oxidation of gastrically or intravenously infused phenylalanine is not different from the parenteral requirement in neonatal piglets. Pediatr Res. 2004;55:630-636.[Medline]

26. Kriengsinyos W, Wykes LJ, Ball RO, Pencharz PB. Oral and intravenous tracer protocols of the indicator amino acid oxidation method provide the same estimate of the lysine requirement in healthy men. J Nutr. 2002;132:2251-2257.[Abstract/Free Full Text]

27. Humayun AM, Moehn S, Bertolo RF, Ball RO, Pencharz PB. Determination of true metabolic availability of essential amino acids from dairy versus soy proteins (an assessment of protein quality) [abstract]. FASEB J. 2004;18:A540.

This article has been cited by other articles:

				S. B. Myrie, R. F. Bertolo, W. C. Sauer, and R. O. Ball Effect of common antinutritive factors and fibrous feedstuffs in pig diets on amino acid digestibilities with special emphasis on threonine J Anim Sci, March 1, 2008; 86(3): 609 - 619. [Abstract] [Full Text] [PDF]

				R. Elango, R. O. Ball, and P. B. Pencharz Indicator Amino Acid Oxidation: Concept and Application J. Nutr., February 1, 2008; 138(2): 243 - 246. [Abstract] [Full Text] [PDF]

				S. Moehn, A. K. Shoveller, M. Rademacher, and R. O. Ball An estimate of the methionine requirement and its variability in growing pigs using the indicator amino acid oxidation technique J Anim Sci, February 1, 2008; 86(2): 364 - 369. [Abstract] [Full Text] [PDF]

				M. A. Humayun, R. Elango, S. Moehn, R. O. Ball, and P. B. Pencharz Application of the Indicator Amino Acid Oxidation Technique for the Determination of Metabolic Availability of Sulfur Amino Acids from Casein versus Soy Protein Isolate in Adult Men J. Nutr., August 1, 2007; 137(8): 1874 - 1879. [Abstract] [Full Text] [PDF]

				H. H. Stein, B. Seve, M. F. Fuller, P. J. Moughan, and C. F. M. de Lange Invited review: Amino acid bioavailability and digestibility in pig feed ingredients: Terminology and application J Anim Sci, January 1, 2007; 85(1): 172 - 180. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moehn, S. Articles by Ball, R. O. Search for Related Content PubMed PubMed Citation Articles by Moehn, S. Articles by Ball, R. O.