Protein and the Bariatric Patient

Protein and the Bariatric Patient July 2008

by Laura Frank, PhD, MPH, RD, CD Dr. Frank is with the St. Francis Center for Weight Management, Franciscan Health System, Federal Way, Washington.

Part 1 of a 3-part series.

INTRODUCTION The importance of protein in nutrition cannot be overstated. The word protein is derived from the Greek word proteos, meaning primary, or “taking first place.”1 Protein is made up of building blocks of amino acids that provide nitrogen to the body. Nitrogen-based compounds are constantly being degraded and resynthesized, which is a process called protein turnover. When protein turnover results in a net synthesis of body protein, the body is in a state of positive nitrogen balance.

Positive nitrogen balance is associated with increased protein tissue synthesis (e.g., building of lean body mass) and growth. In contrast, net degradation of protein is termed negative nitrogen balance and occurs in conditions such as malnutrition.

Protein is an important macronutrient for bariatric patients because it provides energy (4 calories per gram) and promotes wound healing and muscle protein synthesis. Furthermore, calories provided by protein may be more thermogenic and may help the bariatric surgery patient achieve weight loss goals more efficiently than the equal amount of calories provided from either carbohydrates or fats.2 However, due to restriction of dietary intake and/or malabsorption of dietary protein, bariatric surgery can put patients at risk for protein malnutrition.3 It is important for clinicians to have a thorough understanding of protein and its metabolism, including digestion and absorption pre- and post-surgery. It is also important for clinicians to monitor protein status either by serum protein biomarkers or body composition measures. Close monitoring of bariatric patients can decrease the patient’s risk of protein malnutrition.

Dietitians can determine patients’ protein needs and help patients identify high-quality food and supplemental sources of protein in order to meet these needs. However, best practice for nutrition guidelines pre- and post-surgery has not been established for any type of bariatric surgery. Continued multidisciplinary efforts to establish best practice for protein intakes are warranted in order to promote optimal health for our patients.

Overview of Protein The building blocks of protein are referred to as amino acids.1 Amino acids are molecules that contain a nitrogen group attached to two hydrogens (-NH2), referred to as an amine group. There are essentially 20 amino acids.4 Nine of these 20 amino acids need to be obtained by the diet as the body cannot synthesize them; these are called the indispensable amino acids (IAAs). Approximately six amino acids are termed the conditionally indispensible (essential) amino acids (CIAAs), which become indispensable when the body is put under additional stress as in wound healing4 or after bariatric surgery. The Institutes of Medicine (IOM) have established a recommended dietary allowance (RDA) for the IAAs that should be used as a reference value when assessing dietary intake and protein supplements, shown in Table 1.5 Roles of Protein in the Body Protein plays functional roles in the body, providing constituent amino acids for the synthesis of enzymes, hormones, and immunoproteins.1 Enzymes are catalysts used in chemical reactions. Hormones are signaling proteins that regulate many of our body’s metabolism and function. Immunoproteins are proteins that help protect our bodies from infection and illness.6,7 Structural proteins include fibrous proteins that make up our tissues, including collagen (found in our skin, bone, cartilage, and blood vessels), elastin (found in our skin, bone, cartilage, and blood vessels), keratin (found in our hair and nails), and the muscle contractile proteins (actin and myosin) are also considered structural proteins.

Transport proteins are vehicles of transport for several substances, including biomarkers of nitrogen balance, such as albumin, prealbumin (transthyretin), hemoglobin, myoglobin, transferrin, and ceruloplasmin.1,7 Each of these transport proteins is responsible for carrying nutrients and/or oxygen to various places in the body, and are often used as indicators of nutritional status. Lipoproteins are lipid-containing proteins, which transport cholesterol, triglycerides, and phospholipids to and from the liver.1,6 Digestion and Absorption Once swallowed, protein foods enter the stomach from the esophagus. Protein digestion begins in the stomach with the action of hydrochloric acid (HCL). The release of HCL is stimulated by several mechanisms, including the release of gut peptides and hormones (gastrin, gastrin-releasing peptide [GRP], the neurotransmitter, acetylcholine [Ach], and the amine [nitrogen compound] histamine).1 HCL denatures or modifies the protein by causing the protein to unfold

or uncoil. HCL also activates the conversion of the proteolytic enzyme pepsinogen (inactive pre-enzyme form or zymogen) to pepsin (active form). Pepsin hydrolyzes peptide bonds at a pH of less than 3.5 and creates large polypeptides, oligopeptides, and/or free amino acids (especially leucine, methionine, phenylalanine, tyrosine, tryptophan, glutamate, and aspartate).8 The end products of stomach protein digestion mixed with an acid-chyme are emptied through the pyloric sphincter into the duodenum for continued digestion.1 In the duodenum, the acid-chyme product stimulates gut peptides, such as secretin and cholecystokinin (CCK), released from the mucosal endocrine cells.1 Both secretin and CCK stimulate the pancreas to release pancreatic juices and alkaline digestive zymogens, which need to be converted by catalysts into active proteolytic enzymes (Table 2). Enteropeptidase is secreted from the intestinal brush border in response to secretin and CCK. Once trypsin is formed, it too can activate trypsinogen and chymotrypsinogen to yield active proteolytic enzymes.

The cavity of the small intestines, called the lumen, is a fertile ground for the initial holdings of free amino acids as well as oligopeptides. Proteolytic peptidases, located in the brush border of the intestine, hydrolyze and allow further breakdown of oligopeptides into tripeptides and dipeptides.

Although protein absorption occurs throughout the small intestine, the jejunum and ileum are particularly important in this role.3 Digestibility and absorption rates of several foods have been established, specifically for milk, peas, casein, whey, and free amino acids from enteral protein products. However, it is less clear about the specific absorption rates of meat, chicken, fish, and legumes.9 Absorption rates from the gut can vary from 1.4g/h for raw egg white to 8 to 10g/h for whey protein isolate; however, there have been no studies that have evaluated the upper limit of amino acid intake,10 especially in the bariatric community.

Liver Processing of Protein and Elimination The urea cycle, which is found in the liver, is important for the removal of ammonia (NH4+) from the body. Ammonia is toxic because of its effects on the pH and can lead to brain malfunction and coma.1 The liver’s capacity to deaminate proteins and produce urea for excretion of excess nitrogen through the kidneys is one of the rate-limiting factors of protein digestion and is termed the maximum rate of urea excretion (MRUE).9,12 Rudman et al12 studied 10 healthy males and the MRUE associated with various levels of protein intakes (measured in grams (g) protein nitrogen (N)/kg of body weight (BW)/day). This study showed that with a protein dose of 0.53g protein N/kg/day, a MRUE was reached, averaging 55mg urea N/h/kg, persisting for 16 hours. Higher doses of protein (~1.33g protein N/kg/day) did not further accelerate urea excretion, but prolonged the duration of MRUE to 28 hours. These findings indicate that MRUE corresponds to a period of maximal rate of urea synthesis (MRUS). According to Rudman et al, the MRUS in healthy individuals has been reported as an average of 65mg N/h/kg (range of 55–76). Based on the range of MRUS, the recommended daily protein intake for structural use would be 56g for a 70kg individual.

However, the level of dietary protein that can be deaminated and processed through the urea by the liver in a 24-hour period is dependent upon body weight and individual variation in efficiency.

The elimination of protein continues through the colon. However, in general, less than one percent (approximately 10g) of ingested protein is excreted daily in the feces.1 Usually, colonic bacterial proteases have little effect on protein digestion and amino acid absorption. However, the colon over time has been shown to adapt to protein absorption among post-bariatric patients.13 More research is needed within the bariatric population to determine how much the colon contributes to overall amino acid kinetics.

Sources of Protein Protein is found in both animal and plant sources. Good quality protein is an expression of a protein’s ability to provide the nitrogen and amino acids requirement for growth, maintenance, and repair,5 and therefore contains the IAAs. Animal sources of protein, including meat, poultry, fish, eggs, and dairy, are good quality proteins and provide all of the IAAs. Plant sources of protein other than soy, (legumes, vegetables, and grains) tend to have one or more indispensable amino acids lacking, thereby decreasing protein quality.

There are biologic methods used to assess protein quality, including the net protein utilization (NPU), defined as the nitrogen retained in the body/nitrogen consumed; biologic value (BV), defined as the nitrogen retained in the body/nitrogen absorbed from the gut; and the protein efficiency ratio (PER), defined as weight gain/nitrogen consumed.14 Other researchers have utilized the efficiency of nitrogen utilization (ENU) to measure protein quality.

Rand et al15 reported that the ENU for nitrogen retention is approximately 50 percent in healthy adults, with no apparent differences in sex, diet, or climate. Significantly lower ENU was found for older subjects compared to younger subjects (p=0.003). In 1991, the protein digestibility corrected amino acid score (PDCAA) was established as a superior method for the evaluation of protein quality.14 This score indicates the overall quality of a protein because it represents the relative adequacy of its most limiting amino acid.5 The PDCAA indicates the body’s ability to use that product for protein synthesis. The PDCAA is equal to 100 for milk, casein, whey, egg white, and soy.4,5 Protein Requirements in Humans The protein requirement for humans is the sum of the requirement for each of the IAAs plus the non-specific nitrogen requirement.5 The 2005 dietary reference intakes (DRIs)5 suggest that the average protein requirement for adults is 0.66g/kg/day. Nitrogen balance data (corrected for nitrogen intake and dermal and miscellaneous nitrogen losses) are valuable in assessing protein requirements. A meta-analysis from 235 subjects were gathered from 19 studies

examining the estimation of basal or maintenance requirements of nitrogen intakes in healthy adults.15 The authors reported that the median estimated average requirement (EAR) of nitrogen from these data was 105mg N/kg/day and the 97.5th percentile (defined as the recommended dietary allowance [RDA]) was estimated as 132mg N/kg/day or 0.65 and 0.83g good quality protein/kg/day. Therefore, the RDA for protein is set at 0.8g/kg/day or approximately 50g per day for normal adults.5 The Institute of Medicine (IOM) has also published RDAs for each of the IAAs (Table 1).5 The protein requirement for an individual is significantly increased when an individual is not meeting his or her overall energy/caloric needs compared to an individual who is in positive nitrogen balance.16 Pellett and Young17 showed that nitrogen balance was severely compromised when dietary energy intakes were less than 35kcal/kg. Researchers have concluded that adding 100g of carbohydrate per day decreases nitrogen loss by 40 percent in modified protein fasts.7, 18 Because muscle protein accretion or anabolism is indicative of positive nitrogen balance, measurements of protein synthesis are important. Indispensable amino acids, including branched chain amino acids (BCAA), are primarily responsible for acute amino acid-stimulated muscle anabolism.19 Studies on resting human muscle indicate that administration of BCAAs, particularly leucine, has an anabolic effect on protein metabolism either by increasing the rate of protein synthesis, decreasing the rate of protein degradation, or both.20, 21 Furthermore, IAAs, including the BCAAs, have been shown to activate key enzymes in protein synthesis after physical exercise.22, 23 Therefore, postprandial leucine balance can be used as an index of protein deposition9, 24 and can help in the clinical assessment of nitrogen balance. However, practically speaking, assuming adequate total energy intakes, a diet providing all of the IAAs, including the BCAAs, should result in positive nitrogen balance and protein accretion.

Upper Limits of Protein Ingestion Less is known about the maximum amount of nitrogen that a body can digest and utilize. To date, there have been no studies that have established a tolerable upper limit (TUL) of amino acid uptake.10 According to the IOM, there is insufficient data in humans to establish a TUL for either total protein or any of the amino acids.5 Lack of scientific data about TULs, however, does not diminish the fact that consuming large amounts of amino acids, especially disproportionate amounts of specific amino acids, may cause adverse physiological effects.10 For example, large quantities of methionine, cysteine, and histidine have been found to cause tissue damage with chronic administration.25 The maximum safe protein intakes for humans have been estimated at approximately 285g/d for an 80kg male.9 However, Bilsborough and Mann9 suggest the maximum protein intake should be based on bodily needs, weight control evidence, and avoidance of protein toxicity. These authors recommend that maximum protein intakes should be approximately 25 percent of total energy intakes at approximately 2 to 2.5g/kg/d, corresponding to 176g protein per day for an 80kg individual on a 2,800-calorie diet.

There is conflicting data regarding the metabolic disadvantages of high-protein diets. Bernstein et al26 concluded that long-term consumption of high-protein diets by persons with normal kidney function may cause renal injury; however, other researchers have shown discordant results.27-29 Matrin et al29 concluded that high-protein diets promote normal adaptive responses that are well within the function of a normal healthy kidney. More studies are needed to determine if high-protein diets are deleterious to the kidneys. Also, promoting a high-protein diet from high-saturated fat food sources (animal sources of protein) versus monounsaturated or polyunsaturated food sources (plant sources of protein) would not be prudent dietary practice due to the increased risk of heart disease.

Conclusion Clinicians should have a broad understanding of protein nutrition, including food sources, digestion and absorption, and the various roles that protein plays in the body. Protein’s role of providing energy and nitrogen to the body underscores its importance in human nutrition. Continued research regarding protein intake in the pre- and postbariatric patient is warranted in order to establish best practice nutritional guidelines.

References 1. Groff JL, Gropper SS. Advanced Nutrition and Human Metabolism, 3rd ed. Belmont, CA: Wadsworth; 2000.

2. Halton TL, Hu FB. The effects of high protein diets on thermogenesis, satiety and weight loss: a critical review. J Am College Nut. 2004;23(5):373–385.

3. Dobratz JR, Earthman CP. Review of protein status and body composition after gastric bypass surgery and very low-calorie diet therapy. Bariatric Nurse Surg Pt Care. 2006;1(3):195–204.

4. Castellanos VH, Litchford MD, Campbell WW. Modular protein supplements and their application to long-term care.

Nutr Clin Pract. 2006;21:485–504.

5. Institute of Medicine of the National Academies. Protein and amino acids. In: Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids. Food and Nutrition Board: National Academy of Sciences. Washington, DC: National Academy Press; 2005.

6. Mahan LK, Escott-Stump S, eds. Krause’s Food, Nutrition, and Diet Therapy, 9th ed. Philadelphia: WB Saunders Company;1996.

7. Shils, ME, Olson, J, Shike M, eds. Modern Nutrition in Health and Disease, 8th ed. Philadelphia: Lea and Febriger; 1994.

8. Kilberg MS, Stevens BR, Novak DA. Recent advances in mammalian amino acid transport. Ann Rev Nutr.

1993;13:137–65.

9. Bilsborough S, Mann N. A review of issues of dietary protein intake in humans. Int J Sport Nutr and Exer Metab.

2006; 16: 129-152.

10. Bier DM. Amino acid pharmacokinetics and safety assessment. J Nutr. 2003: 133: 2034S-2039S. 11. Wolfe RR, Miller SL. Amino acid availability controls muscle protein metabolism. Diabetes Nutr Metab.

1999;12(5):322-8.

12. Rudman D, Galambos JT, Smith RB 3rd, et al. Maximal rates of excretion and synthesis of urea in normal and cirrhotic subjects. J Clin Invest. 1973;52:2241–2249.

13. Elliott K. Nutritional considerations after bariatric surgery. Crit Care Nurs Q. 2003; 26(2):133–138.

14. FAO/WHO Expert Consultation. Protein Quality Evaluation: Food and Agriculture Organization of the United Nations. Rome: Food and Agriculture Organization; 1991. FAO Food and Nutrition Paper, No. 51.

15. Rand WM, Pellett PL, Young VR. Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults. Am J Clin Nutr. 2003;77:109–127.

16. Hoffner LJ. Protein and energy provision in critical illness. Am J Clin Nutr. 2003;87:906–911.

17. Pellett PL, Young VR. The effects of different levels of energy intake on protein metabolism and of different levels of protein intake energy metabolism: a statistical evaluation of the published literature. In: Scrimshaw NS, et al, eds.

Protein-Energy Interactions. Switzerland: International Dietary Energy Consultative Group; 1992:81–121.

18. Contaldo F, Di Biase G, Scalfi L, et al. Protein-sparing modified fast in the treatment of severe obesity: weight loss and nitrogen balance data. Int J Obes. 1980;4(3):189–196.

19. Volpi E, Kobayashi H, Sheffield-Moore M, et al. Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am J Clin Nutr. 2003 Aug;78(2):250–258.

20. Louard RJ, Barrett EJ, Gelfand RA. Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man. Clin Sci (Lond). 1990; 79(5):457–466.

21. Nair KS, Schwartz RG, Welle S. Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. Am J Physiol. 1992 ;263(5 Pt 1):E928–E934.

22. Blomstrand E, Eliasson J, Karlsson HK, Köhnke R. Branched-chain amino acids activate key enzymes in protein synthesis after physical exercise. J Nutr. 2006;136(1 Suppl):269S–273S. 23. Tipton KD, Elliott TA, Cree MG, et al. Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise. Med Sci Sports Exerc. 2004;36(12):2073–2081.

24. Dangin M, Boirie Y, Guillet C, Beaufrère B. Influence of the protein digestion rate on protein turnover in young and elderly subjects. J Nutr. 2002; 132(10):3228S–3233S. 25. Garlick, PJ. The nature of human hazards association with excessive intakes of amino acids. J Nutr. 2004: 134(6 suppl): 1633S–1639S. 26. Bernstein AM, Tryzon L, Li Z. Are high-protein, vegetable-based diets safe for kidney function? A review of the literature. J Am Diet Assoc. 2007;107:644–650.

27. Manninen AH. Are high-protein diets safe for kidney function? J Am Diet Assoc. 2007;8(20):1722.

28. Pecoits-Filho R. Dietary protein intake and kidney disease in Western diet. Contrib Nephrol. 2007;155:102–112.

29. Matrin WF, Armstrong LE, Rodriques NR. Dietary protein intake and renal function. Nutr Metab. 2005;2:25.

Protein and the Bariatric Patient October 2008

by Laura Frank, PhD, MPH, RD, CD Part 2 of 3 INTRODUCTION All macronutrients (proteins, carbohydrates, and fat) have interrelationships in terms of bioenergetics.

Amino acids, primarily alanine and glutamine, interact with glucose metabolism both as carbon substrates and by recycling glucose carbon via transamination/deamination reactions. Therefore, protein metabolism provides the body with essential building blocks from amino acids to provide carbon substrates for energy metabolism and/or nitrogen compounds needed for nitrogen balance.1 When nutrition is scarce, or when humans experience anorexia, prolonged vomiting, diarrhea, food intolerance, depression, alcohol/drug abuse, or other reasons for food deprivation, protein-energy malnutrition (PEM) will most likely ensue.2 When inadequate protein intake occurs (especially inadequate intake of indispensable amino acids), coupled with adequate carbohydrate and energy intake, this condition is termed protein malnutrition (PM).

Hormonal state is an important covalent modulator for metabolic processes in the body.1 In PEM, the body is generally in a hypoinsulinemic and hyper-gluconemic state.2,3 Low levels of insulin allow for a shift in substrate utilization to stored body fuels, such as muscle and liver glycogen and stored fat. Initially, weight loss occurs as a result of water loss due to the metabolism of liver and muscle glycogen stores. Muscle protein breakdown also occurs in order to supply the amino acids needed to preserve the visceral protein pool. Carbon liberation from either fatty acid oxidation or from the deamination of amino acids, such as glutamate, glutamine, and alanine (process of gluconeogenesis), help to supply fuel for the body and to maintain vital organs (brain, heart, and kidney).1 The body eventually enters a “protein sparing” state as ketone-bodies (byproducts of fat breakdown in the absence of carbohydrate intakes) increase in abundance. This condition is called ketosis. Weight loss occurs with the breakdown of muscle mass and a reduction of adipose tissue as the body strives to maintain homeostasis.

Continued negative nitrogen balance will result in decreased hepatic proteins, including albumin, with concomitant muscle wasting, asthenia (weakness), and alopecia (hair loss). PEM is typically associated with anemia related to iron, B12, folate, and/or copper deficiency. Deficiencies in zinc, thiamin, and B6 are commonly found with a deficient protein status. In addition, catabolism of lean body mass (LBM) and diuresis cause electrolyte and mineral disturbances with sodium, potassium, magnesium, and phosphorus.2 During PM, the protein deficit occurs in conjunction with excessive intake of carbohydrate calories, resulting in a hormonal state of hyperinsulinemia, inhibiting fat and muscle breakdown.1 When the body is not able to hormonally adapt to spare protein, a decrease in visceral protein synthesis will result along with hypoalbuminemia, anemia, and impaired immunity. If left undiagnosed, this may result in an illness where fat stores are preserved, LBM is decreased, and appropriate weight loss is not seen due to accumulation of extra-cellular water. This edema is associated with PM.2 Preoperative Risk No risk of preoperative PM among pre-gastric bypass and duodenal switch patients has been reported;4, 5 however, a thorough history and physical is needed in the preoperative patient. It is also prudent medical or dietetic practice to complete a thorough screening of nitrogen balance and body composition in the preoperative bariatric patient. Note that increased body mass index (BMI) does not ensure that a patient does not have PM. It is important to emphasize that an obese patient can still have low muscle mass (sarcopenia) and/or PM.6 Routine preoperative screening, including laboratory measures of visceral proteins, can be used to assess the risk of PM. Serum albumin (normal range 3.5–5.0g/dL) can be used as an indicator of long-term protein status (half-life approximately 3 weeks). Serum transferrin and serum transthyretin (or thyroxine-binding prealbumin) are referred to as intermediate- (half-life 8–10 days) and short- (half-life 2 days) term indicators, respectively.7 Retinol binding protein (RBP) may also be useful in diagnosing acute changes in protein status.2 Body composition measures, such as fat free mass (FFM) or lean body mass (LBM) versus fat mass (FM) (or the ratio of FFM:FM or LBM:FM), are helpful to assess functional nitrogen balance among patients.3 Sophisticated models of body composition assessment, such as underwater (hydrostatic) weighing or air displacement plethysmography (e.g., Bod-Pod), are generally more practical in academic settings. Dual-energy absorptiometry (DEXA) and computed tomography (CT) can also be used to measure total body fat and intraabdominal or visceral adiposity, respectively.8 A less expensive option to measure body composition includes bioelectrical impedance (BIA) (e.g., Tanita body-fat analyzer, Arlington Heights, Illinois). Good agreement between BIA measures and DEXA has been reported by some9 but not all10 researchers. In addition, creatinine-height index is reflective of somatic or skeletal muscle protein and can be used as a surrogate biomarker for LBM.7 Clinical signs of PM may also include

excess hair loss, edema, anemia, and impaired wound healing.2 Protein deficiency may be masked by the adipose tissue, edema, and general malaise potentially shown by the patient.11 It would not be appropriate to assume that the obese patient has good nutritional status and appropriate dietary intake. Therefore, a complete dietary assessment is also recommended for the preoperative bariatric patient in order to determine typical protein intakes and determination of patient’s protein food preferences. This information can then be used to recommend an appropriate food plan with adequate healthy proteins prior to the patient’s surgery.

Postoperative Risk Bariatric patients can be at risk for PM due to inadequate protein intakes possibly from food intolerances to good quality protein, such as meat.12 This can occur after restrictive surgeries (e.g., adjustable gastric band [AGB] or gastric sleeve [GS]) and restrictive/malabsorptive surgeries (e.g., Roux-en Y gastric bypass [RYGBP]). Other reasons why bariatric patients are at risk for PM and/or PEM depend upon the type of surgery that they have undergone and include small upper stomach pouch with an approximate 20–30mL capacity, decreased intestinal limb length (and therefore decreased and compromised absorption capacity), and increased protein needs due to wound healing immediately post-surgery.7,13 For post-RYGB patients, the degree of protein malabsorption will depend on the length of the bypassed segment of the small intestine.14 Nutritional deficiencies in patients after RYGB operations have been mainly attributed to dietary restriction.15 Other variables include the patient’s food adversions or food faddisms, failure to comply with or ignorance of post-surgical nutrition guidelines, and/or the inability to recognize or afford high quality protein.

Outcomes of studies investigating the incidence of protein malnutrition after surgery have been equivocal.

Furthermore, several of these studies have been performed during the period of greatest caloric restriction.15 In a recent review of the literature, Shah et al16 stated that protein deficiency, assessed by serum albumin levels, is less common than most other nutrient deficiencies among post-surgical patients. To confirm these findings, other investigators have reported that PEM and PM are rarely found among patients after RYGBP.7,17 Two hundred patients who were morbidly obese were followed for 6 to 8 years (mean: 6.7 years) after RYGBP surgery. Meat intolerance was observed in 51 percent, 60.3 percent, 59.5 percent, and 55.1 percent of the patients during zero to 12 months, 13 to 24 months, 25 to 72 months, and 73 to 96 months after surgery, respectively. However, these patients had no caloric malnutrition or protein deficiency. Moize et al12 studied 93 patients retrospectively over 12 months post-RYGBP. Significant differences in total calorie intakes occurred comparing three-month (849±329 calories) to 12-month intakes (1,101±400 calories) (p=.009) and significant differences in protein intakes (g/day) were also observed over the 12-month period (45.6±14.2 g/day at three months versus 58.5±17.1 g/day at 12 months).

Although protein intakes met the minimum recommendation of 0.8g/kg/day, these intakes were significantly different than the recommended 1.5g/kg/day (p=0.01). At 12 months, protein intakes were significantly lower among patients with protein intolerance (p=0.02). However, despite low energy and protein intakes, normal protein status (as measured by serum albumin) was reported throughout the 12-month follow-up.

As stated, the degree of protein malabsorption will depend to some extent on the length of the bypassed segment of the small intestine.14 In a prospective, randomized, clinical study, Brolin and LaMarca et al14 reported that 13 percent of patients status-post-two-years distal (long-limb) RYGB had hypoalbuminemia (albumin <3.5mg/dL) compared to normal albumin levels reported among patients with short Roux limbs (<150cm). In agreement with this study, Nelson et al18 followed 257 super-obese patients after receiving a long Roux limb of 400 to 500cm and a 100cm common channel (for digestion and absorption). Eighty-two percent of patients returning the survey an average of 48 months postoperatively (range 12–148 months) lost greater than 50 percent of excess body weight. Nine patients (4%) who developed or were developing impending protein/calorie malnutrition required proximal relocation of the enteroenterostomy with symptom resolution. In contrast, protein deficiency was not found in long-limb super-obese (BMI>50kg/m2) patients who were a mean 43 months out from surgery.19 Among patients with malabsorptive surgeries (e.g., biliopancreatic diversion [BPD] and duodenal switch [DS]), the degree of malabsorption will determine the patient’s risk status for PM. Secondary PM caused by malabsorption of protein has been reported to be more common after BPD/DS procedures compared to RYGBP procedures, especially during the first or second year postoperative.7, 17 Overall, the incidence of PM after BPD has been reported to be approximately 15 percent.20 Scopinaro et al21 reported that intestinal albumin and nitrogen absorption was 73 percent and 57 percent, respectively, among BPD patients (N=15). It was concluded that loss of endogenous nitrogen (approximately 5-fold the normal value) plays a significant role in the development of PM after BPD, especially during the early postoperative period when restricted food intake may cause a negative balance of both calories and protein. In a prospective study of 65 RYGBP versus 65 BPD patients (BMI range 35–50kg/m2), Scroubis et al22 showed negligible risk of protein deficiency (as measured by albumin) after two-year follow-up; hypoalbuminemia occurred in one (1.5%) RYGBP patient and in 6six (9.2%) BPD patients. In a 15-year follow-up using questionnaires on BPD patients (N=858), there were 32 re-hospitalizations for severe protein deficiency.23 Revisions were performed in 54 (6.3%) patients mainly due to recurrent protein malnutrition. Totté et al24 followed 180 BPD patients (using Scopinaro’s method with a 50cm common channel) and found that protein deficiency, unrelated to the surgery, developed in only two patients at 16 and 24 months postoperative, requiring parenteral nutrition, conversion of the alimentary tract, and psychiatric counseling for correction. They concluded that metabolic complications after BPD were the result of patient non-compliance with dietary recommendations (70–80g protein per

day) during preoperative counseling administered by a registered dietitian. Other researchers have reported sporadic cases of recurrent late PM requiring 2 to 3 weeks of parenteral feeding for correction20 and/or elongation of the common limb.24 Scopinaro et al21 has reported that by varying the length of the intestinal limb and the common channel, varying degrees of protein malabsorption can be increased or decreased.

In DS patients, the common channel is 75 to 100cm or 125cm in length for absorption of nutrients.25 Protein malnutrition occurs in 3 to 5 percent of this cohort. Rabkin et al5 followed a cohort of 589 sequential DS patients (with a gastric sleeve and common channel of 100cm) and found annual laboratory measures of serum markers for protein metabolism to slightly decrease at one year postoperative but then stabilize at two and three years postoperative well within normal limits.

The relationships between body composition and dietary protein intakes have been studied by few researchers.

Several researchers have reported that the weight loss associated with bariatric surgery results in appropriate proportions of lean body mass (LBM) versus fat mass (FM).26-28 In contrast, in a prospective study, Carey et al29 measured body composition and metabolic changes for one year following bariatric surgery without concomitant dietary intake data in 19 bariatric patients (14 female, 5 male). Data collection occurred within one week preoperatively and one, three, six, and 12 months postoperatively. Two female subjects were lost to the study between six months and one year, resulting in 17 subjects (12 female, 5 male) completing the entire 12-month followup. Significant losses in LBM were observed in all time periods except 6 to 12 months, where no change in LBM (60.6 vs. 61.1kg) was observed. The equivocal results of these studies emphasize the need to establish an optimal evidence-based level of protein intakes among bariatric patients for maximal LBM preservation.

Based on the outcomes of most of these studies as indicated by serum protein markers, with and without dietary intake data, it does appear that bariatric surgery poses a relatively low risk of developing obvious signs of protein malnutrition. However, evidenced by substantial LBM loss and below-standard creatinine-height index values, clinicians should also use body composition and somatic protein assessments in order to detect early signs of low protein nutriture.7 It also appears that the BPD procedure has the greatest impact on protein nutrition. However, prospective, longitudinal, randomized, clinical studies are needed to determine the effects of all types of weight loss surgeries on the risk of protein deficiency, negative nitrogen balance, PM, and/or PEM. Frequent continued monitoring of signs of PM or PEM are necessary in this population.

Protein Prescription for the Bariatric Patient Usual protein recommendations for the post-bariatric surgery patient are anywhere from 1 to 2g protein per kg of adjusted body weightâ&#x20AC;&#x201D;calculated as current body weight (CBW) minus ideal body weight (IBW) multiplied by 25 percent plus IBW [CBW-IBW x 25% + IBW]).25 A minimum of 60 to 70g of protein per day should be ingested.13 Many programs recommend a range of 60 to 80 grams total protein intake per day or 1.0 to 1.5g/kg IBW, although exact needs have yet to be defined. The use of 1.5g/kg IBW/day beyond the early post-surgical phase is probably above metabolic requirements for non-complicated patients and may prevent the consumption of other macronutrients in the context of volume restrictions. An analysis of the RYGB patientâ&#x20AC;&#x2122;s typical nutrient intake at one year postoperative found no significant changes in albumin with daily protein consumption at 1.1g/kg IBW.12 Following BPD/DS procedures, the amount of protein should be increased by approximately 30 percent to accommodate for malabsorption, making the average protein requirement for these patients approximately 90g/day.30 Treatment for Protein Malnutrition in the Bariatric Patient Unfortunately, loss of muscle mass is an inevitable part of the weight loss process after obesity surgery or any very low calorie diet (VLCD).31 Patients, especially those undergoing BPD/DS, should be encouraged to focus on proteinrich foods of good quality protein (providing a complete protein of all IAAs). Patients need to eat slowly and to masticate their food well to compensate for the reduced grinding capacity of the stomach.25 Foods that are moist, either by preparation methods or by saliva, can also facilitate food to pass from the esophagus to the pouch.

When a deficiency occurs and there is no mechanical explanation for vomiting or food intolerance, patients can often be successfully treated with a high-protein liquid diet and slow progression to a regular diet.11 As the protein deficiency is corrected and edema is decreased around the anastomosis, food tolerance and vomiting may resolve. If protein deficiency cannot be corrected with dietary protein intake, the patient may require hospitalization for enteral or parenteral treatment to sufficiently return the total body protein to normal levels. Patients may also require psychological evaluation and/or counseling to determine the degree to which a psychosocial etiology is involved. It is important to rule out or treat all possible underlying mechanical and behavioral causes before considering a surgical revision such as lengthening of the common channel or surgery reversal.

Conclusion Due to the equivocal nature of current research, clinicians need to continually assess the prevalence and incidence of PM pre- and post-bariatric surgery. Clinicians need to screen and monitor pre- and post-bariatric patients for protein malnutrition using tools, such as dietary assessment, serum biomarkers of protein status, and body composition measures. Recommendations of types and amounts of good quality protein from food and supplemental sources can assist in the prevention and treatment of protein malnutrition.

References 1. Groff JL, Gropper SS. Advanced Nutrition and Human Metabolism, Third Edition. Belmont CA, Wadsworth; 2000.

2. Modern Nutrition in Health and Disease, Eighth Edition. In: Shils ME, Olson J, Shike M, eds. Philadelphia: Lea and Febriger;1994.

3. Krause’s Food, Nutrition, and Diet Therapy, Ninth Edition. In: Mahan LK, Escott-Stump S, eds. Philadelphia: WB Saunders Company;1996.

4. Flancbaum L, Belsley S, Drake V, et al. Preoperative nutritional status of patients undergoing Roux-en-y gastric bypass for morbid obesity. J Gastrointest Surg. 2006;10(7):1033–1037.

5. Rabkin RA, Rabkin JM, Metcalf B, et al. Nutritional markers following duodenal switch for morbid obesity. Obes Surg. 2004;14:84–90.

6. Zamboni M, Mazzali G, Fantin F, et al. Sarcopenic obesity: A new category of obesity in the elderly. Nutr Metab Cardiovasc Dis. 2008; Apr 3 [Epub ahead of print].

7. Dobratz JR, Earthman CP. Review of protein status and body composition after gastric bypass surgery and very low-calorie diet therapy. Bariatric Nurse Surg Pt Care. 2006;1(3):195–204.

8. Institute of Medicine of the National Academies. Emerging Technologies for Nutrition Research: Potential for Assessing Military Performance Capability. National Academy of Sciences. Washington, DC: National Academy Press;1997.

9. Thomson R, Brinkworth GD, Buckley JD, et al. Good agreement between bioelectrical impedance and dual-energy X-ray absorptiometry for estimating changes in body composition during weight loss in overweight young women. Clin Nutr. 2007;26(6):771–777.

10. Jebb SA, Cole TJ, Doman D, et al. Evaluation of the novel Tanita body-fat analyser to measure body composition by comparison with a four-compartment model. Br J Nutr. 2000;83(2):115–122.

11. Mason EE. Starvation injury after gastric reduction for obesity. World J Surg. 1998;22:1002–1007.

12. Moize V, Geliebter A, Gluck ME, et al. Obese patients have inadequate protein intake related to protein intolerance up to one year following Roux-en-Y gastric bypass. Obes Surg. 2003;13:23–28.

13. Stocker DJ. Management of the bariatric surgery patient. Endocrinol Metab Clin North Am. 2003;32:437–457.

14. Brolin RE, LaMarca LB, Kenler HA, et al. Malabsorptive gastric bypass in patients with super-obesity. J Gastrointest Surgery. 2002;6:195–203.

15. Avinoah E, Ovnat A, Charuzi I. Nutritional status seven years after Roux-en-Y gastric bypass surgery. Surgery.

1992;111(2):137–42.

16. Shah M, Simha V, Garg A. Review: long-term impact of bariatric surgery on body weight, comorbidities, and nutritional status. J Clin Endocrinol Metab. 2006;91(11):4223–4231.

17. Faintuch J, Matsuda M, Cruz ME, et al. Severe protein-calorie malnutrition after bariatric procedures. Obes Surg.

2004; 14:175–181.

18. Nelson WK, Fatima J, Houghton SG, et al. The malabsorptive very, very long limb Roux-en-Y gastric bypass for super obesity: results in 257 patients. Surgery. 2006;140(4):517–522.

19. Brolin RE, Kenler HA, Gorman JH, et al. Long-limb gastric bypass in the superobese. A prospective randomized study. Ann Surg. 1992;215:387–395.

20. Scopinaro N, Gianetta EM, Friedman D, et al. Evolution of biliopancreatic bypass. Clin Nutr. 1985;5(S):137–146.

21. Scopinaro N, Marinari GM, Pretolesi F, et al. Energy and nitrogen absorption after biliopancreatic diversion. Obes Surg. 2000;10(5):436–441.

22. Skroubis G, Anesidis S, Kehagias I, et al. Roux-en-Y gastric bypass versus a variant of biliopancreatic diversion in a non-superobese population: prospective comparison of the efficacy and the incidence of metabolic deficiencies.

Obes Surg. 2006;16(4):488–495.

23. Marinari GM, Murelli F, Camerini G, et al. A 15-year evaluation of biliopancreatic diversion according to the Bariatric Analysis Reporting Outcome System (BAROS). Obes Surg. 2004;14(3):325–328.

24. Totté E, Hendrickx L, van Hee R. Biliopancreatic diversion for treatment of morbid obesity: experience in 180 consecutive cases. Obes Surg. 1999;9(2):161–165.

25. Elliott K. Nutritional considerations after bariatric surgery. Crit Care Nurs Q. 2003;26(2):133–138.

26. Benedetti G, Mingrone G, Marcoccia S, et al. Body composition and energy expenditure after weight loss following bariatric surgery. J Am Coll Nutr. 2000; 19:270-274.

27. Tacchino RM, Mancini A, Perrelli M, et al. Body composition and energy expenditrue: relationship and changes in obese subjects before and after biliopancreatic diversion. Metabolism. 2003;52:552–558.

28. Strauss B, Marks S, Growcott J, et al. Body composition changes following laparoscopic gastric banding for morbid obesity. Acta Diabetol. 2003;40:S266–S269.

29. Carey DG, Pliego GJ, Raymond RL. Body composition and metabolic changes following bariatric surgery: effects on fat mass, lean mass and basal metabolic rate: six months to one-year follow-up. Obes Surg. 2006;16(12):1602– 1608.

30. Scopinaro N, Gianetta E, Adami GF, et al. Biliopancreatic diversión for obesity at eighteen years. Surgery.

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31. Hoffer LJ, Bistrian BR, Young VR, et al. Metabolic effects of very low calorie weight reduction diets. J Clin Invest.

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32. Avinnoah E, Ovnat A, Charuzi I. Nutritional status seven years after Roux-en-y gastric bypass surgery. Surgery.

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Protein and the Bariatric Patient: Supplemental Protein or Amino Acids November 2008

by Laura Frank, PhD, MPH, RD, CD Part 3 of 3 INTRODUCTION Calories provided by protein may be more thermogenic and may help the bariatric surgery patient achieve weight loss goals more efficiently than the equal amount of calories provided from either carbohydrates or fats.1 Therefore, protein supplements are often recommended to help the bariatric patient achieve weight loss. Also, during the early postoperative period, supplemental protein can provide an important source of calories and indispensable amino acids (IAAs) that help prevent the loss of lean body mass and aide in wound healing. However, there is controversy regarding the safety and efficacy of protein supplements.2,3 Caution should be used with supplements that also contain high calories.4 It has been reported that the amino acid found in the highest concentration may be absorbed, but other amino acids in lesser concentration may not be absorbed.3 Therefore, amino acid supplements may result in impaired or imbalanced amino acid absorption. Furthermore, absorption of peptides (obtained from foods) may be absorbed more rapidly than the absorption of an equivalent mixture of free amino acids from dietary supplements.

Nitrogen assimilation following the ingestion of protein-containing foods has also been shown to be superior to that of the ingestion of free amino acids from dietary supplements. Moreover, the necessity of protein supplementation after bariatric surgery has not been established.5 There are several factors that should determine the type of protein supplement used by an individual, including convenience, taste, texture, and price. However, the productâ&#x20AC;&#x2122;s amino acid profile should be the first priority. A protein supplement that provides all of the indispensable amino acids (IAAs) is of the utmost importance.6 If protein supplements are to be used, the patient should choose complete, good quality proteins such as that provided by milk (whey and casein), egg, and soy. Reputable manufacturers should be able to provide accurate nutrition facts substantiating claims made about the amino acid profiles of their products.

Collagen-based supplements should not be used as a means of complete protein because collagen is missing tryptophan, one of the IAAs.6 In the presence of IAA deficiency, loss of lean body mass can occur despite meeting a daily oral intake protein goal. Collagen-based supplements, however, contain a high level of nitrogen within a small volume. These supplements may therefore be useful for the patient who is able to consume enough good-quality dietary protein to supply the needed IAAs, yet is not consuming enough total nitrogen to meet the nonspecific nitrogen requirement, or the patient who is not achieving nitrogen balance to support muscle protein synthesis.

Digestion Rate Protein digestion rate is unequivocally an independent regulating factor of post-prandial protein retention.7 Whey protein and casein, two milk-based proteins, have very different absorption kinetics.2 Whey protein is soluble, allowing for faster gastric emptying; whereas casein clots in the acid environment of the stomach delay gastric emptying, resulting in a slower release of amino acids. It has been reported that slowly digested protein, by inhibiting proteolysis, induces a higher post-prandial protein gain than rapidly digested protein, which stimulates protein synthesis but also oxidation.7, 8 Post-prandial leucine balance over seven days was higher after slow (casein) versus rapid (whey) protein meals.8 These results were confirmed by others9 concluding that digestion of milk soluble proteins (e.g., whey) is too rapid to sustain the anabolic post-prandial amino acid requirement. However, protein digestibility may also be modified by age. In young adults, slowly digested protein resulted in a better protein gain than rapidly digested protein; however, the opposite was found true among older individuals.10 Whether rapid versus slow uptake of amino acids are better for protein gain among bariatric patients have not been explored.

Whey protein typically comes in the following three forms: isolates, concentrates, and hydrolysates. There is greater processing involved in the isolate form, in which most of the fat and lactose are removed. Therefore, whey protein isolates generally contain greater percentages of protein (90â&#x20AC;&#x201C;98% protein by weight) but are also generally more expensive. Whey protein concentrates naturally contain varying amounts of lactose, minerals, and fat.5 These products are generally 29- to 89-percent protein by weight, but are also generally less expensive and have greater palatability. Hydrolysates are predigested, partially hydrolyzed whey proteins that consequently are more easily absorbed, but their cost is generally higher. However, both whey protein isolates and whey protein concentrates have been characterized as rapidly and easily digestible sources of dietary protein. Boirie et al7 and Dangin et al8 determined that whey protein produced a large but transient rise in postprandial plasma amino acid levels and subsequent increased protein synthesis. Peak amino acid levels were reported in approximately 90 minutes and returned to baseline within five hours.

Numerous types of whey protein products have been produced commercially, all having various percentages of protein, molecular weights, lactose, and mineral content. Manufacturing process, product composition, and protein source can affect their digestibility, and therefore the effectiveness of the product.11 Furthermore, processing

techniques, such as spray-drying, pressurized microfiltration, ultrafiltration, hydrolysis, and ion exchange, are used to produce whey protein products with different physicochemical and functional properties. For example, increasing the degree of hydrolysis concomitantly increases the solubility of the product, thereby making the protein more accessible to proteolytic enzymes and promoting better absorption.12 The source of the whey protein, whether from cheese or milk, as well as the type of milk, can also make a difference in the properties of the final product.13 Animal studies have shown that protein can be denatured or damaged during processing, making these products less soluble and digestible.14 In an in-vitro study of six different milk protein ingredients, Patel and Kilara13 reported that as much as 37 percent of the lysine found in whey protein concentrate products was chemically altered from overprocessing or inadequate storage conditions. It was reported that whey protein from cheese made with whole milk, skim milk, or a skim and buttermilk blend varied considerably in their solubility and other physicochemical properties.

How Much Can We Absorb?

One popular myth is that only 30g of protein can be absorbed and utilized at one time.15 While this is commonly found in both lay and some professional literature, no scientific studies to date have tested this claim, especially in the bariatric community. Arnal et al16 studied the effect of protein-pulse feeding (7% [~5g] dietary protein given at 800, 79% [~55g] given at 1200, and 14% [~10g] given at 2000) versus protein spread-feeding (25% [~17.5g] dietary protein at each meal given at 800, 1200, 1600, and 2000) on protein retention in elderly patients (mean age 68±1 year). Nitrogen balance was more positive with the pulse versus spread diet. Protein turnover rates were also higher with the pulse versus spread diet, due to higher protein synthesis. These authors concluded that a protein pulsefeeding pattern was more efficient than a protein-spread pattern in whole-body nitrogen retention. However, in young women (mean age 26±1 year), these protein feeding patterns did not have significantly different effects on protein retention.17 These data refute the “30g myth.” These authors conclude that protein source, time of consumption, quantity, and composition are factors determining the effect of protein on short-term satiety and food intake in young men. However, more studies are needed to determine how ingested protein load predicts nitrogen balance outcomes, especially among those with varying types of weight loss surgery.

Metabolic Rate Understanding the body’s metabolic state is also important when supporting or refuting the “30g myth.” Protein, whether from food or supplements, provides amino acids. An important concept in protein metabolism is the amino acid pool(s) that contain amino acids of dietary origin (food and supplemental), plus those contributed by the breakdown of body tissue. The amino acids comprising the pool(s) are used for synthesis of new proteins for growth and/or replacement of existing body protein, for production of important non-protein, nitrogen-containing components in the body; for oxidation as a source of energy; and/or for glucose, ketones, or fatty acid synthesis.3 What Supplement Type Is Best?

Another myth commonly shared by bariatric surgeons is the claim that a powdered protein supplement is nutritionally superior or “works better” compared to liquid, ready-to-feed protein supplements. Again, there are no data to support this claim. A comprehensive review of modular protein supplements6 references all liquid, ready-to-feed products; however, there are no studies that compare the nutritional quality of liquid, ready-to-feed versus powdered protein supplements. Patients should be encouraged to utilize ready-to-feed protein supplements if these products improve compliance with ingestion of good-quality protein and can improve protein nutriture.

The Patient’s Role It is the consumer’s responsibility to contact protein supplement manufacturers or raw material suppliers to identify the quality assessment and quality control factors associated with the product. Additional issues not explored within this article series are issues of added ingredients, including artificial sweeteners, vitamins, minerals, and botanicals.

For the lactose-intolerant patient, casein has the lowest lactose content of the dairy proteins; however, due to the manufacturing process of hydrolysis, whey protein isolates are lactose free and therefore may be the product of choice for those who wish to use a supplemental versus food form of good quality protein.5 Continued follow-up with healthcare providers can shed light on whether the patient’s chosen supplement provides the needed nutrition to prevent or treat protein deficiencies.

Conclusion Protein is an important macronutrient that provides energy and aids in wound healing, weight loss, and muscle protein accretion. Further research studies are needed in several areas with regard to protein needs in the bariatric patient in order to establish best-practice guidelines. The dietitian should work closely with the patient to help identify good quality proteins and recommend appropriate dietary protein intakes. A thorough nutrition assessment, including blood work and body composition measures, should be completed at regular follow-up intervals. It is important for the multidisciplinary team to work together to optimize patients’ care and outcomes.

References 1. Halton TL, Hu FB. The effects of high protein diets on thermogenesis, satiety and weight loss: a critical review. J Am College Nut. 2004;23(5):373–385.

2. Bilsborough S, Mann N. A review of issues of dietary protein intake in humans. Int J Sport Nutr and Exer Metab.

2006;16:129–152.

3. Groff JL, Gropper SS. Advanced Nutrition and Human Metabolism, Third Edition. Belmont CA, Wadsworth;2000.

4. Fujioka K. Follow-up of nutritional and metabolic problems after bariatric surgery. Diab Care. 2005;28(2):481– 484.

5. Jacques J. Protein basics. Bariatr Times. 2007;4(4):24–28.

6. Castellanos VH, Litchford MD, Campbell WW. Modular Protein Supplements and their application to long-term care. Nutr Clin Pract. 2006;21:485–504.

7. Boirie Y, Dangin M, Gachon P, et al. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci. 1997;94(26):14930–14935.

8. Dangin M, Boirie Y, Garcia-Rodenas C, et al. The digestion rate of protein is an independent regulating factor of postprandial protein retention. Am J Physiol Endocrinol Metab. 2001;280:E340–E348.

9. Lacroix M, Bos C, Léonil J, et al. Compared with casein or total milk protein, digestion of milk soluble proteins is too rapid to sustain the anabolic post-prandial amino acid requirement. Am J Clin Nutr. 2006;84(5):1070–1079.

10. Dangin M, Boirie Y, Guillet C, Beaufrère B. Influence of the protein digestion rate on protein turnover in young and elderly subjects. J Nutr. 2002;132(10):3228S–3233S. 11. de Wit JN. Nutritional and functional characteristics of whey proteins in food products. J Dairy Sci. 1998;81(3):597–608.

12. Sindayikengera S, Xia WS. Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex. J Zhejiang Univ Sci B. 2006;7(2):90–98.

13. Patel MT, Kilara A. Studies on whey protein concentrates. Foaming and emulsifying properties and their relationships with physicochemical properties. J Dairy Sci. 1990;73:2731–2740.

14. Sarwar G, Peace RW, Botting HG. Differences in protein digestibility and quality of liquid concentrate and powder forms of milk-based infant formulas fed to rats. Am J Clin Nutr. 1989;49:806–813.

15. Venuto T. Available at: www.tomvenuto.com/free_newsletter.shtml. AUT: Need more info; what is this website?

16. Arnal MA, Mosoni L, Boirie Y, et al. Protein pulse feeding improves protein retention in elderly women. Am J Clin Nutr. 1999;69(6):1202–1208.

17. Arnal MA, Mosoni L, Boirie Y, et al. Protein feeding pattern does not affect protein retention in young women. J Nutr. 2000;130(7):1700–1704.