Challenges in Nutrition Research

Ronald O. Ball

Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, AB, Canada, T6G 2P5

Introduction

This report will not be a comprehensive discussion of all the challenges that exist in nutrition research. There are too many - almost as many as there are nutritionists working in the area! Instead I will focus on the specific challenges which I have taken up. I will describe the research that I am currently pursuing and those that I intend to take on in the next couple years at the University of Alberta.

My research is primarily in the area of amino acid metabolism, however, I am currently working in several other areas of swine nutrition including: the prevalence of ulcers in sows and finished pigs, effects of ulcers on growth and feed conversion (Ayles et al., 1996; Mackin et al., 1997), dietary treatments to prevent or reduce ulcers (Ayles et al., 1996), genetic influences on pork quality (the Ontario pork carcass project - a multi year study involving 3300 pigs with complete data on growth performance, body composition, meat quality and DNA analyses) (Ball et al., 1996b, Canadian Center for Swine Improvement, Ottawa), and use of recycled restaurant and food processing wastes for swine feeding (McNaughton et al., 1997).

My amino acid research includes:

These areas are too many and too broad to be discussed here. Therefore, the following discussion will concentrate on some aspects of my work on amino acid metabolism in the young pig. First a discussion of the general concepts underlying the study of amino acid metabolism will be presented. This will be followed by a brief review of the methods for determining amino acid requirements. Next descriptions will be given of the tools, techniques and methods we apply to answer questions about amino acid metabolism. Finally a review of some recent research and its application to swine nutrition will be presented.

Concepts of Amino Acid Kinetics Body Pool of Amino Acids

An overview of the major concepts in amino acid metabolism is represented by Figure 1. Amino acids enter the body via the diet, either as dietary protein or free amino acids. The metabolic amino acid pool is considered to represent all the amino acid pools in the body. Although there are many sub pools of amino acids (plasma, intracellular, cells within an organ, organelles within cells etc.) there is rapid transport and interconversion between them. When we infuse radioactive amino acid we find that we can achieve uniform labelling in nearly all these pools within one to two hours. This metabolic pool then provides amino acids for protein synthesis and receives amino acids from protein breakdown. Amino acids in excess of the metabolic needs for synthesis of protein or synthesis for non-protein compounds (hormones and neurotransmitters) are oxidized and the carbon excreted as CO2 and the nitrogen excreted as urea and a few other nitrogen compounds. Amino acids are also catabolized at a basal rate often called obligatory oxidation.

These paradigms of amino acid metabolism allow us to use radioactive amino acids to trace these various events that define amino acid requirements, rates of protein synthesis and breakdown, and the influences of diet and other factors on these important events.

Amino acid requirements for protein synthesis can be measured under a variety of conditions by applying these concepts. The two most sensitive methods of measuring amino acid requirement are Direct and Indicator Amino Acid Oxidation.

Direct Oxidation. Direct amino acid oxidation refers to the measurement of oxidation of the limiting amino acid when graded dietary concentrations of that amino acid are provided. A typical response is illustrated in Figure 2. The technique is based upon the concept that amino acids in excess of the amounts needed for protein synthesis are oxidized preferentially. Therefore, when an amino acid is present at deficient levels, most of the amino acid will be efficiently used for protein synthesis and oxidation will occur at a low basal rate. As dietary supply of the amino acid increases to exceed the animal's requirement for protein synthesis, increased catabolism of the amino acid occurs.

The oxidation is measured by giving an isotopically labelled amino acid either orally or intravenously and quantitating the isotope occurring in CO2. The first application of the method is generally credited to Brookes et al. (1972). They demonstrated that the oxidation of lysine, measured after an injection of 14C-lysine, did not increase markedly until the intake of lysine was similar to that shown to maximize average daily gain and feed efficiency, and the estimates of lysine requirement were similar for all response criteria measured. Since then, the method has been applied to estimate the requirement of several different amino acids in growing and adult rats, young pigs (Kim and Bayley 1983; House et al., 1997) and the adult human (e.g., Zello et al., 1990).

The direct amino acid oxidation technique, although it has made major contributions to understanding amino acid kinetics and metabolism, has several drawbacks (see recent review Zello et al., 1995) that restrict its use or affect its accuracy and interpretation. For example, the direct amino acid oxidation technique requires the use of a suitably labelled isotopic tracer (i.e., 1-13C or 1-14C label position) in order to specifically quantify the end-products (1d3CO2 or 14CO2) of amino acid metabolism. Suitable tracers are not available for every amino acid of interest (e.g., methionine, threonine).

Indicator Amino Acid Oxidation. The oxidation of an indicator amino acid as a means of estimating the requirement for another amino acid was first used by Kim et al. (1983a, b) and Ball and Bayley (1984). These authors suggested that the limitation by one amino acid of protein synthesis might effectively be represented by the oxidation of a different amino acid. The method is based upon the concept that the partition of all essential amino acids between protein synthesis and oxidation is sensitive to the level of the most limiting amino acid. When one amino acid is limiting for protein synthesis (i.e., its dietary concentration is less than in the 'ideal' amino acid profile) then all other amino acids must be present in relative excess and, therefore, are oxidized. Each addition of the limiting amino acid will increase protein synthesis and thus reduce the oxidation of the other amino acids until the requirement point is reached. Thereafter, further increments in the amino acid will have no effect on protein synthesis or oxidation of the other essential amino acids. Figure 3 shows how indicator oxidation (phenylalanine) changes as tryptophan is added to the diet of the young pig (Ball and Bayley, 1984).

The indicator oxidation method is used under quite different conditions compared to the direct amino acid oxidation technique. In the indicator approach, the same diet, containing all the nutrients exceeding their estimates of requirement, is fed for an adaptation period, and the diets deficient in the test amino acid were offered only on the day of the oxidation study. Zello et al. (1990) used a similar approach in estimating the lysine requirement of adult male humans. This approach of feeding a deficient diet for only a short period of time is ethically more acceptable. Furthermore, the estimates of amino acid requirement derived from the indicator oxidation method uses only the sampling of breath, bypassing the need to calculate absolute oxidation rates by determining the specific radioactivity in tissues or enrichment of the amino acid in the plasma. This is supported by previous studies (Kim et al., 1983a, b) which showed no marked difference in the estimate of amino acid requirements when determined by the oxidation data, expressed as a percentage of the dose oxidized or corrected for the flux of the amino acid following tissue and blood sampling. The indicator technique has been used to estimate the amino acid requirements in baby pigs (Kim and, Bayley 1983; Kim et al., 1983a, b; Ball and Bayley, 1984, 1986; Ball et al., 1986), growing swine (Lin et al.,, 1986,1988), rainbow trout and, most recently, humans (Zello et al., 1993; Duncan et al., 1996).

The indicator amino acid oxidation technique is an improvement on the direct amino acid oxidation method, in terms of adaptation time and end-product (CO2) sampling. In addition, the use of one specific amino acid as an indicator simplifies the techniques and methods used, as methods can be developed for the optimization of the recovery conditions for one specific amino acid, instead of many. Also, the requirement for amino acids that are difficult to study because of the their complex metabolism can be readily determined by indicator oxidation (e.g., threonine and methionine).

Surgical and Oxidation Techniques Used to Study Amino Acid Metabolism in Pigs

A number of new techniques and methods were required to allow us to study amino acid requirements in pigs using these newer and more sensitive technologies. Several years of development, validation and experimentation were required. These techniques are briefly described below.

Animals and Surgery

We have previously described in detail the surgical procedures, animal housing and diet formulations of our oral and intravenously fed piglet models (Murphy et al., 1996; Wykes et al., 1993). Briefly, male Yorkshire piglets are obtained from a minimal disease herd. Upon arrival the pigs are weighed, anaesthetized and prepared for the surgical insertion of catheters, including (Figure 4) external jugular vein, femoral vein and carotid artery, portal vein (via the umbilicus) and stomach (via Stamm gastrostomy) catheters. Following surgery, the piglets are fitted with an adjustable cotton jacket with an anchoring button. The piglets are then placed in individual circular metabolic cages set in banks of four, thus allowing aural and visual contact with littermates. A tether and swivel system, attached to the anchor button on the piglet's jacket, allows the piglet freedom of movement, while providing a means of affixing the diet infusion sets that preclude their tangling. Toys are placed in the cages to enrich the environment.

Diet Regimen

Diets were designed that can be fed either orally, intragastrically, or intravenously. This required the development of a completely purified and sterile diet (Wykes et al., 1993). The reason for developing such a specialized diet was to allow the study of how the gut and liver vs the rest of the body metabolize amino acids when identical amino acid profiles were fed. The goal in designing the oral and intravenous diet solutions was the provision of nutrients at rates sufficient to meet the estimated nutrient requirements of swine weighing 1-5 kg (NRC, 1988). The regimen provides 15 gkg-1d-1 of amino acids and 1.1 MJkg-1d-1 of metabolizable energy, with lipid and glucose each providing 50% of the non-protein energy component. Two approaches were used for the provision of amino acids in the nutrient solutions. Initial nutrient solutions were formulated using a commercially available general purpose amino acid solution (Vamin, Kabi Pharmacia, Stockholm) used in human clinical nutrition. However, use of commercially prepared solutions severely limited experimental treatments. We addressed this issue by using crystalline amino acids to create solutions with amino acid compositions of our own design (House et al., 1994; Wykes et al., 1994).

Study Protocol

As outlined in Figure 5, diet administration is initiated immediately post-operative, at 50% of calculated requirement, with infusion rates adjusted upwards for two days, reaching full rates by the morning of the second day. The animals are weighed daily for a period of eight days post-surgery and diet infusion rates adjusted accordingly. Blood and urine are collected for biochemistry (ions, protein, glucose, etc.) haematological profiles (complete blood cell counts, packed cell volume, etc.), plasma amino acid concentrations and nitrogen excretion.

On the final day of the study, the animals are used in amino acid oxidation studies (Figure 5). An L-114C-amino acid is administered via a primed-continuous infusion into the infusion catheter (femoral) while the piglet is housed in a covered plexiglass box. Amino acid oxidation rates are determined by the complete collection of 14CO2, with adjustments for the retention of label in the bicarbonate pool determined during a constant infusion of 14C-sodium bicarbonate one day prior to the amino acid oxidation study. Blood samples are also withdrawn at 30 min intervals and the plasma collected for the analysis of the specific radioactivity of the infused amino acid (plus metabolites). Animals are then killed and body composition analysed.

Figure 5. Typical study protocol of experiments designed to investigate amino acid metabolism during TPN in neonatal piglet.

Using stochastic modelling techniques (Figure 1), amino acid flux rates are calculated from the plasma specific radioactivity using isotope dilution principles and expressed as a rate per unit of body mass. Oxidation rates are calculated as the product of the flux rate and the percentage of the 14C label transferred from the infusate to the breath. By measuring the rate of amino acid oxidation, an estimate of the net retention of the amino acid (ie the rate of protein synthesis) can be derived by subtracting the oxidation rate from the rate of intake of the amino acid. This approach offers a unique opportunity to compare kinetic estimates of amino acid retention to those derived from traditional measures of nitrogen retention or protein accretion.

Our experience with this model is that piglets tolerate the surgery and the diet regimen well. Complications involving health of the piglets and catheter patency have been minimal. Nutrients from the diet solution are metabolized well: piglets demonstrate normal blood glucose, low triglyceride level, low serum urea and high nitrogen retention. Most biochemical and hematological results are similar between sow-reared and artificially-reared piglets, with the differences being those normally seen with early- weaned piglets.

This piglet model allows for specific investigations requiring detailed and invasive procedures. We have used this model to study amino acid metabolism across the gut, liver and the integration of whole body metabolism and to develop new methods for the estimation of amino acid requirements in pigs.

Application of Amino Acid Kinetic Methods to Swine Nutrition

Early weaning of piglets is often characterized by initial reductions in performance and slower growth (Tokach et al., 1994). A high quality post-weaning diet is critical to mitigate the transition from highly-digestible sow's milk to a usually less-digestible solid feed. The problem is that the gastrointestinal tract of the early weaned piglet is not yet fully developed in terms of digestive and absorptive capabilities to properly handle a typical weaning diet based on cereal grains and vegetable proteins (Pettigrew et al., 1994). As a result, a growth lag phase is common. This problem can be amplified by decreased feed intakes due to environmental stresses. Poor digestibility of diets, along with variable intakes often results in health problems such as diarrhoea (Ball and Aherne, 1987a, b). Current ways to combat this problem involve feeding highly-digestible and very expensive diets containing milk and blood proteins (Pettigrew et al., 1994).

The inclusion of specific ingredients or nutrients in the diet to shorten the adaptation period from the pre- to post-weaning diet would be beneficial to both the animal and the producer. A better developed digestive tract during weaning could reduce health and growth problems for the pig, and the producer could benefit with decreased veterinary bills, and lower overall feed cost, while maintaining or improving growth rates and survival. The period when the high cost diet is required may be shortened by finding dietary ways to stimulate the maturation and development of the gastrointestinal tract.

Research in the area of gut metabolism of amino acids will also have implications for other periods in the pig's life. For example, after any change in the diet there is a period of reduced digestion and absorption while the gut enzymes, hormones and tissues adapt. Malnutrition of the gut also occurs due to stress (ie transport, mixing, etc) and intestinal disease or any condition that reduces feed intake. Inclusion of nutrients or ingredients that specifically feed the gut and either increase or aid in recovery of gut function should improve overall production.

A number of nutrients and dietary compounds have been identified that could increase gastrointestinal development and maturation (see Ball and Ewtushik, 1997 for review). These include several amino acids (glutamine, glutamate, arginine, ornithine, citrulline), polyamines, and nucleotides. Epidermal growth factor (EGF) and insulin-like growth factors (IGFs) and other hormones also have substantial effects on the gut, however, they very unlikely to be of practical use because they are not nutrients and thus dietary supplementation will require they go through the government drug approval process.

Most of the research in amino acid oxidation in pigs has been in the piglet at approximately 3 kg live weight and 10 days of age with a few reports in growing pigs. These have been recently summarized (Bayley, 1993). Most recently, in our piglet studies, we studied glutamine metabolism, determined the requirements for both phenylalanine and tyrosine and the optimum balance between them, and repeated lysine requirement (reviewed by Ball et al., 1996). We have also recently studied the amino acid precursors for proline synthesis and arginine synthesis in the piglet and showed that dietary glutamate can provide only 40% of the proline requirement for protein synthesis, suggesting that proline may be a semi-indispensable amino acid for the young pig (Murphy et al., 1996; Murch et al., 1996).

Currently we are applying amino acid kinetics and oxidation techniques to determine the threonine requirements of piglets, separate the amino acid requirements for gut growth from whole body requirements in the young pig, and quantify the basal level of lysine and threonine oxidation in growing pigs as a function of PDmax (maximum rate of protein deposition). We have recently developed protocols for providing the isotopically labelled phenylalanine in frequent oral meals with oxidation measured by breath collection and urine analysis. This extends the potential application of the technique to mature animals at maintenance or during lactation and simplifies the protocol. In the near future we hope to further adapt the method and build the equipment necessary to carry out amino acid requirement studies in gestating and lactating sows because there is very little quantitative data on amino acid metabolism in sows. We also plan to use these techniques to develop a method for estimating the 'true availability' for protein synthesis of amino acids in feedstuffs (Ball et al., 1995).

Conclusion

The methods for studying amino acids have changed several times over the years, with gradually increasing sophistication and application of technology. Today we are able to use isotopically labelled amino acids to accurately measure amino acid kinetics at the level of the whole body and individual tissues. These data will be useful in increasing the precision of diet formulation and growth modelling. The short term and rapid nature of the technique allows for repeated measurements on individual animals and more sensitive results during periods of rapid change in requirement, such as post natal growth, lactation and gestation.

References

Ayles, H.L., R.M. Friendship and R.O. Ball. (1996) Effect of dietary particle size on gastric ulcers, assessed by endoscopic examination, and relationship between ulcer severity and growth performance of individually fed pigs. Swine Health & Prod. 4(5): 211-216.

Ayles, H.L., R.O. Ball, R.M. Friendship and G.A. Bubenik. (1996) Effect of graded levels of melatonin on growth performance and gastric ulcers in pigs. Can. J. Anim. Sci. 76:607-611.

Ball, R.O. and H.S. Bayley. (1984) Tryptophan requirement of the 2.5 kg piglet determined by the oxidation of an indicator amino acid. J. Nutr. 114:1741-1746.

Ball, R.O. and H.S. Bayley. (1986) Influence of dietary protein concentration on the oxidation of phenylalanine by the young pig. Br. J. Nutr. 55:651-658.

Ball, R.O., J.L. Atkinson and H.S. Bayley. (1986) Proline as an essential amino acid for the young pig. Br. J. Nutr. 55:659-668.

Ball, R.O. and F.X. Aherne. (1987a) Influence of dietary nutrient density, level of feed intake and weaning age on young pigs. I. Performance and body composition. Can. J. Anim. Sci. 67:1093-1103.

Ball, R.O. and F.X. Aherne. (1987b) Influence of dietary nutrient density, level of feed intake and weaning age on young pigs. II. Apparent nutrient digestibility and incidence and severity of diarrhea. Can. J. Anim. Sci. 67:1105-1115.

Ball, R.O., E.S. Batterham and R.J. van Barneveld. (1995) Lysine oxidation by growing pigs receiving diets containing free and protein-bound lysine. J. Anim. Sci. 73(3): 785-792.

Ball, R.O., J.D. House, L.J. Wykes and P.B. Pencharz. (1996a) A piglet model for neonatal amino acid metabolism during total parenteral nutrition. IN: Advances in Swine in Biomedical Research. (Tumbelson, M.E. & Schook, L.B. eds.) Plenum Press. Vol. 2 pp. 713-731.

Ball, R.O., J.P. Gibson, C.A. Aker, K. Nadarajah, B.E. Uttaro and A. Fortin. (1996b) Differences among breeds, breed origins and gender for growth, carcass composition and pork quality. In: Proceedings of the Ontario Pork Carcass Appraisal Project. Eds. J.P. Gibson, C.A. Aker and R.O. Ball. pp 12-20.

Ball, R.O. and A.L. Ewtushik. (1997) Amino acids as substrates for enhancing gut growth and development in the neonate. Proceedings of the 9th Biokyowa International Amino Acid Council. St Louis, MO. Oct 1997.

Bayley, H.S. (1993) Methods of determining the amino acid requirements of pigs. In: Recent Developments in Pig Nutrition 2. Eds. D.J.A. Cole, Nottingham University Press.

Brookes I.M., F.N. Owens and U.S. Garrigus. (1972) Influence of amino acid level in the diet upon amino acid oxidation by the rat. J. Nutr. 102:27-36.

Duncan, A., P.B. Pencharz and R.O. Ball. (1996) Lysine requirement of adult males is not affected by decreasing dietary protein. Amer J. Clin. Nutr. 64: 718-725.

House, J.D., P.B. Pencharz and R.O. Ball. (1994) Glutamine supplementation during total parenteral nutrition promotes fluid retention in the neonatal pig. J. Nutr. 124(4):396-405.

House, J.D., P.B. Pencharz and R.O. Ball. (1997) Phenylalanine requirements determined using L-[114C]-phenylalanine in neonatal piglets receiving total parenteral nutrition supplemented with tyrosine. Amer. J. Clin. Nutr. 65: 984-993.

Kim, K.I. and H.S. Bayley. (1983) Amino acid oxidation by young pigs receiving diets with varying levels of sulphur amino acids Br. J. Nutr. 50:383-390.

Kim, K.I., J.I. Elliot and H.S. Bayley. (1983a) Oxidation of an indicator amino acid by young pigs receiving diets with varying levels of lysine or threonine and an assessment of amino acid requirements Br. J. Nutr. 50:391-399.

Kim, K.I., I. McMillan and H.S. Bayley. (1983b) .Determination of amino acid requirements of young pigs using an indicator amino acid Br. J. Nutr. 50:369-382.

Lin, F.D., T.K. Smith and H.S. Bayley. (1986) Tryptophan requirement of growing swine as determined by the oxidation of an indicator amino acid. J. Anim. Sci. 62:660-664.

Lin, F.D., T.K. Smith and H.S. Bayley. (1988) A role for tryptophan in the regulation of protein synthesis in porcine muscle. J. Nutr 118:445-449.

Mackin, A.J., H.L. Ayles, R.M. Friendship, R.O. Ball and B. Wilcock. (1997) Development and evaluation of an endoscopic technique permitting rapid visualization of the cardiac region of the porcine stomach. Can. J. Vet. Res. 61: 121-127.

McNaughton, E.P., R.O. Ball and R.M. Friendship. (1997) Effects of feeding a chocolate waste product on growth performance and meat quality of finishing swine. Can J. Anim. Sci. 77: 1-8.

Murphy, J.M., S.J. Murch and R.O. Ball. (1996) Proline is synthesized from glutamate during intragastric infusion but not during intravenous infusion in the neonatal pig. J. Nutr. 126(4): 878-886.

Murch, S.J., R.L. Wilson, J.M. Murphy and R.O. Ball. (1996) Proline is synthesized from intravenously infused arginine by piglets consuming low proline diets. Can. J. Anim. Sci.76:435-441.

National Research Council. (1988) Nutrient requirements of swine, Ninth revised edition. National Academy Press, Washington, D.C.

Pettigrew J.E., L.J. Johnston, G.C. Shurson and J.D. Hawton. (1994) Nutrition of the early weaned pig: Feeding nursery pigs. Ann Mtg Amer Assoc Swine Pract.1-36.

Tokach, M.D, R.D. Goodband and J.L. Nelssen. (1994) Recent developments in nutrition for the early weaned pig. Food Anim Comp 4:407-419.

Wykes, L.J., R.O. Ball and P.B. Pencharz. (1993) Development and validation of a total parenteral nutrition model in the neonatal piglet. J. Nutr. 123(7):1248-1259.

Wykes, L.J., J.D. House, R.O. Ball and P.B. Pencharz. (1994) Amino acid profile and aromatic amino acid concentration in TPN: Effect on growth, protein metabolism and aromatic amino acid metabolism in the neonatal piglet. Clin. Sci. 87:75-84.

Zello, G.A., P.B. Pencharz and R.O. Ball. (1990) Phenylalanine flux, oxidation and conversion to tyrosine in humans studied with L-[113C]-phenylalanine. Amer. J. Physiol. 259: (Endocrinol. Metab. 22): E835-E843.

Zello, G.A., R.O. Ball and P.B. Pencharz. (1993) Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C]phenylalanine. Am. J. Physiol. 264 (Endocrinol. Metab. 27): E677-E685.

Zello, G.A., L.J. Wykes, P.B. Pencharz and R.O. Ball. (1995) Recent advances in methods of assessing the dietary amino acid requirements for adult humans. J. Nutr. 125: 2907-2915.