Therapeutic Applications of Whey Protein

Whey Protein Review

Keri Marshall, ND, MS

Abstract environment. The components of whey include Whey, a protein complex derived from milk, is beta-lactoglobulin, alpha-lactalbumin, bovine se- being touted as a functional food with a number rum albumin, lactoferrin, immunoglobulins, lac- of health benefits. The biological components toperoxidase enzymes, glycomacropeptides, lac- of whey, including lactoferrin, beta- tose, and minerals.2 In additional, whey derived lactoglobulin, alpha-lactalbumin, from buttermilk versus cheese contains the lipid glycomacropeptide, and immunoglobulins, sphingomyelin. demonstrate a range of immune-enhancing Several cultures consider fermented foods properties. In addition, whey has the ability to part of a healthful diet. Historically, whey was act as an antioxidant, antihypertensive, considered a cure-all used to heal ailments rang- antitumor, hypolipidemic, antiviral, ing from gastrointestinal complaints to joint and antibacterial, and chelating agent. The primary ligament problems. Nanna Rognvaldardottir, an mechanism by which whey is thought to exert Icelandic food expert, describes how whey, called its effects is by intracellular conversion of the syra by the Icelandic people, is fermented and amino acid cysteine to glutathione, a potent stored in barrels. Syra is diluted with water and intracellular antioxidant. A number of clinical ingested or used as a marinade or preservative for trials have successfully been performed using meat and other food. Syra was the most common whey in the treatment of cancer, HIV, hepatitis beverage of Icelandic people and is thought to have B, cardiovascular disease, osteoporosis, and replaced ale, due to lack of grains in the region.3 as an antimicrobial agent. Whey protein has Today, whey is a popular dietary protein also exhibited benefit in the arena of exercise supplement purported to provide antimicrobial performance and enhancement. activity, immune modulation, improved muscle (Altern Med Rev 2004;9(2):136-156) strength and body composition, and to prevent cardiovascular disease and osteoporosis. Advances Introduction in processing technology, including ultrafiltration, In recent years, milk constituents have microfiltration, reverse osmosis, and ion-ex- become recognized as functional foods, suggest- change, have resulted in development of several ing their use has a direct and measurable effect on different finished whey products. Whey protein health outcomes.1 Whey, a by-product of cheese concentrates (ranging from 80-95 percent protein), and curd manufacturing, was once considered a reduced lactose whey, whey protein isolate, de- waste product. The discovery of whey as a func- mineralized whey, and hydrolyzed whey are now tional food with nutritional applications elevated whey to a co-product in the manufacturing of Keri Marshall, MS, ND – 1996 Master of Science Social 2 and Preventive Medicine, State University of New York at cheese. Milk contains two primary sources of Buffalo; 2001 graduate, National College of Naturopathic protein, the caseins and whey. After processing Medicine. Private practice, Sandpoint, Idaho. Correspondence address: 515 Pine Street, Suite H, occurs, the caseins are the proteins responsible for Sandpoint, ID 83864 making curds, while whey remains in an aqueous Email: [email protected]

Table 1. Types of Commercially Available Whey Proteins

Product Description Protein Concentration Fat, Lactose, and Mineral Content

Whey Protein Isolate 90-95% Little if any

Whey Protein Concentrate Ranges from 25-89% Some fat, lactose, and minerals Most commonly available as 80% As protein concentration increases, fat, lactose, and mineral content decreases.

Hydrolyzed Whey Protein Variable Varies with protein concentration Hydrolysis used to cleave peptide bonds Larger proteins become smaller peptide fractions Reduces allergic potential compared with non-hydrolyzed

Undenatured Whey Concentrate Variable Some fat, lactose, and minerals Usually ranges from 25-89% As protein concentration increases, fat, lactose, and mineral content decreases. Processed to preserve native protein structures; typically have higher amounts of immunoglobulins and lactoferrin

available commercially. Each whey product var- and incubated for 30 minutes. Rennet extract is ies in the amount of protein, carbohydrates, im- added and the mixture is stirred, resulting in co- munoglobulins, lactose, minerals, and fat in the agulation of curd. finished product. These variables are important Rennet is derived from the abomasum factors in the selection of whey fractions for spe- (fourth stomach) of newly born calves. Chymosin, cific nutritional applications. Table 1 describes the the active enzyme ingredient of rennet, aids in the various whey protein products available. coagulation of milk by separating it into curds and whey. In a newly born calf, chymosin aides in the Whey Protein Manufacturing digestion and absorption of milk. Adult cows do not have this enzyme. Protein from bovine whole milk consists The liquid whey is drained through a stain- of approximately 20-percent whey protein. When less steel screen and the remaining curd is cut and casein is removed from whole milk, liquid whey cooked at 30 degrees C. Whey liquid is then fil- remains, having a protein concentration of about tered at 45 degrees C and brought to a pH of 3 by 65 percent. The following is a summary of the adding citric acid. The liquid is filtered to one- Ohio State University method of manufacturing fifth its original volume, resulting in whey con- whey protein powder. Milk is high-temperature, centrate that is approximately 80-percent protein. short-time pasteurized (163 degrees F for 30 sec- This can be additionally micro-filtered to increase onds) and held overnight at 40 degrees C. The protein concentration to as high as 95 percent. following morning the mixture is cooled to 30 degrees C, inoculated with a lactic acid culture,

Table 2. Amino Acid Residues in Bovine versus Human fat and lactose. In addition, some manufacturers hydro- Lactoferrin lyze (cleaving peptide bonds via enzymes or heat) the whey Bovine Lactoferrin Human Lactoferrin to provide more peptides and free amino acids in the final Alanine 67 63 product.4 Proline 30 35 The commercial success of whey protein has led Arginine 39 43 to the development of high Lysine 54 46 quality whey protein supple- Asparagine 29 33 ments manufactured as primary products and not as a by- Valine 47 48 product of cheese manufactur- Tr yptophan 13 10 ing. Manufacturers take spe- cial care to preserve the bio- Cysteine 34 32 logical activity, native protein Threonine 36 31 structure, and protein-bound- fats in the finished product. Isoleucine 15 16 Proteins are processed under Serine 45 50 low temperatures and not ex- posed to fluctuating pH Glutamine 29 27 changes to avoid denaturing Glutamic acid 40 42 the native structures. In addition, the source of milk and the Phenylalanine 27 30 health of the milking cows is Methionine 4 5 thought to contribute to immune-enhancing activity of Leucine 65 58 whey products.5,6 Glycine 48 54 Tyrosine 22 21 Biological Components Aspartic Acid 36 38 Amino Acid Content Histidine 9 9 Collectively, whey Total number of residues 689 691 proteins have all the essential amino acids and in higher con- From: Pierce A, Colavizza D, Benaissa M, et al. Molecular cloning and centrations compared to vari- sequence analysis of bovine lactoferrin. Eur J Biochem 1991;196:177-184. ous vegetable protein sources such as soy, corn, and wheat gluten.2 In addition to having a full spectrum of amino ac- The final whey protein concentrate is ids, the amino acids found in whey are efficiently warmed and spray-dried to achieve whey protein absorbed and utilized, relative to free amino acid powder. Whey protein concentrates can then be solutions.7 put through an ion-exchange process to remove

Relative to other protein sources, whey breast-feeding, and serves as a child’s first line of has a high concentration of branched-chain amino immune defense – referred to as “passive acids (BCAAs) Ð leucine, isoleucine, and valine. immunity.” IgA is secreted in breast milk and BCAAs, particularly leucine, are important fac- ultimately transferred to the digestive tract in the tors in tissue growth and repair. Leucine has been newborn infant, providing better immunity than a identified as a key amino acid in protein metabo- bottle-fed child.12 Colostrums contain significantly lism during the translation-initiation pathway of greater concentrations of immunoglobulins than protein synthesis.8 mature milk. Immunoglobulins reach maximum Whey proteins are also rich in the sulfur- concentration the first 24-48 hours post-parturition containing amino acids cysteine and methionine. and decline in a time-dependent manner following With a high concentration of these amino acids, peak concentration.13 immune function is enhanced through intracellu- Similarly, the whey fraction of milk ap- lar conversion to glutathione. pears to contain a significant amount of immunoglobulins, approximately 10-15 percent of total Lactoferrin whey proteins. An in vitro study demonstrated bo- Lactoferrin, an iron-binding glycoprotein, vine milk-derived IgG suppresses human lympho- is a non-enzymatic antioxidant found in the whey cyte proliferative response to T cells at levels as fraction of milk as well as in colostrums. The low as 0.3 mg/mL of IgG. The authors further con- lactoferrin component of whey consists of approxi- clude bovine milk IgG typically ranges between mately 689 amino acid residues, while human 0.6-0.9 mg/mL and is therefore likely to confer 14 lactoferrin consists of 691 residues.9 Whey immunity that could be carried to humans. Stud- lactoferrin is composed of a single polypeptide ies show raw milk from non-immunized cows con- chain with two binding sites for ferric ions. Be- tain specific antibodies to human rotavirus, as well fore processing, bovine lactoferrin is only 15-20 as antibodies to bacteria such as E. coli, Salmo- percent saturated with iron. Iron-depleted nella enteriditis, S. typhimurium, and Shigella 15,16 lactoferrin, defined as containing less than five- flexneri. percent iron, is referred to as apolactoferrin. Hu- man breast milk contains apolactoferrin.10 The beta-Lactoglobulin concentration of lactoferrin in human milk and beta-Lactoglobulin represents approxi- colostrums is approximately 2 mg/mL and 7 mg/ mately half of the total protein in bovine whey, mL, respectively, while in bovine milk and colos- while human milk contains no beta-lactoglobu- trums it is approximately 0.2 mg/mL and 1.5 mg/ lin. Besides being a source of essential and mL, respectively.11 Lactoferrin is a dominant com- branched chain amino acids, a retinol-binding pro- ponent of whey protein in human breast milk; tein has been identified within the beta-lactoglo- however, the concentration in most commercial bulin structure. This protein, a carrier of small whey protein powders is only 0.35-2.0 percent of hydrophobic molecules including retinoic acid, has total proteins. Table 2 illustrates the difference in the potential to modulate lymphatic responses.17 amino acid profiles between bovine and human lactoferrin. alpha-Lactalbumin alpha-Lactalbumin is one of the main Immunoglobulins proteins found in human and bovine milk. It An immunoglobulin (Ig) is an antibody comprises approximately 20-25 percent of whey or gamma-globulin. There are five classes of proteins and contains a wide variety of amino antibodies Ð IgA, IgD, IgE, IgG, and IgM. IgG acids, including a readily available supply of constitutes approximately 75 percent of the essential and branched chain amino acids. Purified antibodies in an adult. IgG is transferred from alpha-lactalbumin is most readily used in infant mother to child in utero via cord blood and by

Table 3. Components Found in Whey Protein

Whey Components % of Whey Protein Benefits

beta-Lactoglobulin 50-55% Source of essential and branched chain amino acids

alpha-Lactalbumin 20-25% Primary protein found in human breast milk Source of essential and branched chain amino acids

Immunoglobulins 10-15% Primary protein found in colostrum Immune modulating benefits

Lactoferrin 1-2% Antioxidant Antibacterial, antiviral , and antifungal Promotes growth of beneficial bacteria Naturally occurs in breast milk, tears, saliva, bile, blood, and mucus

Lactoperoxidase 0.50% Inhibits growth of bacteria

Bovine Serum Albumin 5-10% Source of essential amino acids Large protein

Glycomacropeptide 10-15% Source of branched chain amino acids Lacks the aromatic amino acids phenylalanine, tryptophan, and tyrosine

formula manufacturing, as it has the most Lactoperoxidase structurally similar protein profile compared to Whey contains many types of enzymes, breast milk. However, due to cost effective including hydrolases, transferases, lyases, pro- measures, most dairy-based infant formulas teases, and lipases. Lactoperoxidase, an important contain ingredients such as demineralized whey enzyme in the whey fraction of milk, is the most with higher levels of beta-lactoglobulin, making abundant enzyme and the majority of it ends up in them less similar to human milk. whey following the curding process. Lactoperoxi- In a murine study, alpha-lactalbumin, in dase accounts for 0.25-0.5 percent of total protein both the native and hydrolyzed state, enhanced found in whey. It has the ability to catalyze cer- antibody response to systematic antigen stimula- 18 tain molecules, including the reduction of hydro- tion. The same group proved alpha-lactalbumin gen peroxide.20 This enzyme system catalyzes has a direct effect on B-lymphocyte function, as peroxidation of thiocyanate and some halides well as suppressing T cell-dependent and -independent 19 (such as iodine and bromium), which ultimately responses. generates products that inhibit and/or kill a range of bacterial species.21 During the pasteurization process, lactoperoxidase is not inactivated, suggesting its stability as a preservative.

Glycomacropeptide form. Riboflavin, niacinamide, and glutathione re- Glycomacropeptide (GMP) is also re- ductase are essential cofactors in the reduction of 23 ferred to as casein macropeptide. GMP is a pro- glutathione. As a result of the glutathione/anti- tein present in whey at 10-15 percent, due to the oxidant component of whey, it is being investi- 24 action of chymosin on casein during the cheese- gated as an anti-aging agent. making process. GMP is only present when As a detoxifying agent, glutathione per- chymosin is used during processing; therefore, oxidase (GSHPx), which is derived from selenium cheeses such as cottage cheese not made with and cysteine, is an endogenous antioxidant enzyme chymosin do not produce GMP in the curding pro- with the ability to convert lipid peroxides into less cess.22 GMP is high in branched chain amino ac- harmful hydroxy acids. The peroxidases interact ids and lacks the aromatic amino acids including with hydrogen peroxide to reduce it to water, ne- phenylalanine, tryptophan, and tyrosine. It is one gating its oxidative potential. Both glutathione of the few naturally occurring proteins that lacks peroxidase activity and selenium concentrations phenylalanine, making it safe for individuals with have been shown to decrease as lactation contin- phenylketonuria (PKU). ues, peaking at approximately one month after

Bovine Serum Albumin Figure 1. Synthesis of Glutathione from Cysteine, Glutamate, Bovine serum and Glycine albumin (BSA) is a large protein that makes up approximately 10-15 Step 1Glutamic acid Step 2 g-Glutamylcysteine percent of total whey + + protein. BSA is a source Cysteine Glycine of essential amino + + acids, but there is very ATP ATP little available information regarding its poten- g-Glutamylcysteine Glutathione tial therapeutic activity. synthetase synthetase Table 3 summarizes the components found in whey. g-Glutamylcysteine Glutathione + + ADP ADP Mechanism of + + Action P P Whey has potent antioxidant activity, likely by contribut- ing cysteine-rich proteins that aid in the synthesis of glutathione (GSH), initiation. Practitioners use whey protein products as a source of cysteine to increase intracellular a potent intracellular antioxidant.2 GSH is com- glutathione levels25,26 and it has been reported that prised of glycine, glutamate, and cysteine (Figure 1). Cysteine contains a thiol (sulfhydryl) group GSHPx activity in cow’s milk, and presumably whey, is the same as in human milk.27 that serves as an active reducing agent in prevent- ing oxidation and tissue damage. As an antioxidant, glutathione is most effective in its reduced

Studies on lactoferrin have demonstrated increasing hydrolysis and slowing entrance to the its ability to activate natural killer (NK) cells and small intestine. Whey proteins, on the other hand, neutrophils, induce colony-stimulating factor ac- do not coagulate under acidic conditions. They are tivity, and enhance macrophage cytotoxicity.28-31 considered to be “fast proteins,” as they reach the Lactoferrin also appears to have antivi- jejunum quickly after entering the gastrointesti- ral, antifungal, and antibacterial properties. The nal tract.40 After reaching the small intestine, the antimicrobial effect is likely more potent in or- hydrolysis of whey is slower than that of casein, ganisms that require iron to replicate, as lactoferrin allowing for greater absorption over the length of has the unique ability to chelate iron in a way that the small intestine. deprives microorganisms of this essential nutri- A study by Arnal et al demonstrates that ent for growth.32 In additional, lactoferrin has the whey’s rapid absorption patterns are superior for ability to release the outer membrane of gram- postprandial protein utilization and overall nitro- negative bacteria, the lipopolysaccharide compo- gen balance in elderly women.41 Most recently, a nent, thus acting as an antibiotic.33 randomized, single-blind study examining protein Lactoferrin demonstrates anti-inflamma- satiety found whey protein to exhibit a higher post- tory properties. A mouse study revealed lactoferrin prandial level of plasma amino acids compared to had the ability to regulate levels of tumor necro- casein.42 sis factor (TNF) and interleukin 6 (IL-6), thus A study by Troost et al examined the ef- decreasing inflammation and, ultimately, mortal- fects of an orally administered recombinant hu- ity.34 man lactoferrin (rhLF) in the digestive tract. The In addition to the above-mentioned prop- study population consisted of eight female ileo- erties, alpha-lactalbumin can chelate heavy met- stomy patients who consumed 5 g rhLF and col- als.35 It reduces oxidative stress because of its iron- lected full ileostomy output for 24 hours. In a 24- chelating properties.36 hour period, only 4 µg of lactoferrin were excreted. Whey has been recently touted as a health- The study concluded dietary rhLF does not reach ful dietary supplement to reduce blood pressure. the colon because it is digested in the stomach and Antihypertensive peptides have been isolated in small intestine.43 A similar study by the same group the primary sequence of bovine beta-lactoglobu- was conducted on 12 healthy volunteers. On three lin.37 These peptides give whey significant angio- separate days, volunteers were instructed to con- tensin I converting enzyme (ACE) inhibitory ac- sume 4.5 g lactoferrin. After conclusion of the tivity, which blocks the conversion of angiotensin study, results revealed bovine lactoferrin survived I to angiotensin II, a highly potent vasoconstric- passage through the stomach, demonstrating the tor molecule.38 beta-Lactoglobulin has been de- ability of this protein to survive in an acidic envi- scribed by Nagaoka et al as a cholesterol-lower- ronment.44 ing agent. In animal studies, beta-lactoglobulin inhibited cholesterol absorption by changing mi- Clinical Indications for Whey 39 cellar cholesterol solubility in the intestine. Cancer Absorption and Digestion of Milk Whey protein concentrates have been re- searched extensively in the prevention and treat- Proteins: Casein versus Whey and ment of cancer. Glutathione stimulation is thought Lactoferrin to be the primary immune-modulating mechanism. The physical manifestations of milk in the In a review of whey protein concentrates in the gut vary, based on the type of milk protein. Whey treatment of cancer, Bounous discusses the anti- comprises approximately 20 percent of milk pro- tumor and anticarcinogenic potential. The amino tein, while 80 percent of protein in milk is casein.32 acid precursors to glutathione available in whey These two proteins possess quite different prop- might: (1) increase glutathione concentration in erties. Casein proteins form curds in the stomach, relevant tissues, (2) stimulate immunity, and (3)

detoxify potential carcinogens.45 Other authors breast, one patient with pancreatic cancer, and one conclude the iron-binding capacity of whey may with liver cancer.59 Patients were given 30 g whey also contribute to anticancer potential, as iron may protein concentrate (Immunocal) for six months. act as a mutagenic agent causing oxidative dam- In six patients, blood lymphocyte GSH was el- age to tissues.46 evated initially, suggesting high tumor GSH lev- Many animal studies have examined the els. After completion of the study, two of the pa- effects of whey and its immune-enhancing com- tients showed signs of tumor regression and a re- ponents, including lactoferrin and beta-lactoglo- turn of lymphocyte GSH levels to normal, while bulin.47-54 In animal studies where colon cancer was two patients showed signs of tumor stabilization induced, whey demonstrated significantly lower without normalization of glutathione levels. Three incidence of tumors, as well as fewer aberrant patients had progression of disease and lympho- crypts.47,49,52,54 When whey-based protein powders cyte GSH levels increased from initial measure- were compared to soy-based protein powders, ments. The conflicting results and small size of similar effects were also observed.48,52 Yoo et al this study indicate the need for a larger clinical demonstrated lactoferrin has the ability to inhibit trial to investigate the potential of a whey protein metastasis of primary tumors in mice with can- concentrate singly and as an adjunct to chemo- cer.50 Bovine serum albumin (10-15 percent of total therapy. whey protein) has demonstrated inhibition of In a recent clinical trial, 20 patients with growth in human breast cancer cells in vitro.55 A stage IV malignancies (one bladder, five breast, hamster study demonstrated fractionated whey has two prostate, one neuroblastoma, one ovarian, one the ability to prevent and treat 5-fluorouracil che- gastric, three colon, one mesothelioma, two lym- motherapy-induced oral mucositis.56 This protec- phoma, two non-small cell lung, and one osteosar- tion is thought to occur via induction of tumor coma) received a combination of 40 g/day non- growth factor-beta (TGF-β), which reduces basal denatured whey protein concentrate, 50-100 g/day epithelial cell proliferation. intravenous Transfer Factor Plus¨ (a supplement A recent in vitro study by Kent et al dem- containing several immunoactive components), 1- onstrated that an isolate of whey protein, when 2 g/day oral ascorbic acid, Agaricus blazei tea, a compared to a casein-based protein, increased glu- multiple vitamin/mineral complex, 500 mg tathione synthesis and protected human prostate Andrographis paniculata twice daily, and a soy cells against oxidant-induced cell death.57 An in extract for six months.60 After six months there vitro study on a human hepatoma cell line was were 16 survivors, all of whom had significantly conducted using a high lactoferrin whey concen- higher NK function and higher mean hemoglobin trate (Immunocal¨), a baicalein medium, or a com- and hematocrit levels. All patients noted having bination of the two.58 Baicalein, a potential anti- an improved quality of life during the course of cancer drug, is a flavonoid extracted from the study. No comparison was made between this Scutellaria revularis that is thought to have anti- combination therapy and whey protein alone. tumor activity. Immunocal alone did not have a significant impact on the hepatoma cell line. How- Hepatitis ever, when Immunocal was combined with Whey protein supplementation demon- baicelein, cytotoxicity was enhanced by inducing strates variable effects in patients infected with a higher rate of apoptosis than in the group treated Hepatitis B or C. Initially it was found that bo- with baicalein alone. vine lactoferrin prevented hepatitis C virus (HCV) To date, few clinical trials on whey and infection in vitro in a human hepatocyte line.61 cancer have been conducted. It has been proposed These results prompted further clinical trials. that GSH concentrations are high in tumor cells, A pilot study was conducted on 11 patients giving them resistance to chemotherapeutic with chronic HCV. Each patient received either 59 agents. In 1995, a small trial was conducted on 1.8 or 3.6 g bovine lactoferrin daily for eight five patients with metastatic carcinoma of the

weeks. In patients with low pretreatment viral loads of HCV, de- Table 4. Comparison of Whey Products Immunocal creases in HCV RNA and serum and Protectamin alanine transaminase were observed. In patients with higher pretreatment HCV viral loads, Amino Acid Immunocal (g/100 g) Protectamin (g/100 g) levels did not change signifi- 62 Aspartic 9.80 10.50 cantly. A dose-response trial of Glutamic 16.37 17.48 45 individuals with HCV was Serine 3.56 5.58 conducted at doses of 1.8, 3.6, Glycine 1.64 1.84 and 7.2 g lactoferrin daily for eight weeks.63 A virological re- Histidine 2.04 1.73 sponse was observed in only four Arginine 2.30 2.24 patients, although HCV RNA Threonine 4.63 6.97 was still detectable. Eight patients had a virological response Alanine 4.80 4.87 Ð a 50 percent or greater decrease Proline 3.76 6.01 in HCV RNA Ð eight weeks af- Tyrosine 3.76 1.97 ter the treatment ended. There were no significant variations in Valine 4.53 6.06 dose-dependant responses. This Methionine 2.13 2.27 trial left many unanswered ques- Isoleucine 6.41 6.32 tions for future studies regarding whey supplementation and HCV, Leucine 12.56 9.96 including optimum dose, dura- Phenylalanine 4.00 3.15 tion, and the potential effects of Lysine 10.68 9.19 combining supplementation with conventional treatments such as Tr yptophan 2.86 1.53 interferon therapy. Cysteine/Cystine 4.17 2.28 In an open study on 25 patients diagnosed with either Hepatitis B or C, patients were Minerals given 12 g whey (Immunocal) Sodium 0.3 0.1 twice daily for 12 weeks.64 Prior Potassium 0.5 0.5 to the start of treatment with whey, patients were given 12 g Calcium 0.4 0.3 casein protein daily for two Chloride 0.4 0.1 weeks. Patients were also given Phosphate 0.3 0.3 casein following Immunocal for a four-week period. In the 17 pa- Magnesium 0.3 0.1 tients with HCV, no significant From: Micke P, Beeh KM, Buhl R. Effects of long-term supplementation with changes were noted. In the group whey proteins on plasma glutathione levels of HIV-infected patients. Eur J Nutr with hepatitis B virus (HBV), 2002;41:12-18. serum lipid peroxidase levels decreased, while IL-2 and NK activity increased. In six of eight HBV patients serum alanine

transferase levels were reduced, while plasma glu- Antimicrobial tathione levels increased in five of the same eight. Plasma levels of lactoferrin have been This trial shows promise for the use of whey pro- found to be elevated due to release from neutro- tein in the treatment of HBV. phils during infection, inflammation, tumor development, and iron overload.11 Several studies Human Immunodeficiency Virus have revealed lactoferrin plays a direct role in the (HIV) body’s defense against pathogens, including find- Glutathione deficiency is a common prob- ings that individuals more susceptible to infection 69,70 lem for individuals infected with HIV. In an effort have lower levels of neutrophil lactoferrin. to increase cysteine, and ultimately glutathione, Several studies have examined the effects of whey several studies have been conducted on the use of protein concentrates on the eradication of 71-73 whey proteins in HIV-positive individuals.65,66 In Helicobacter pylori. H. pylori is widely ac- a study by Micke et al, 30 subjects with HIV were cepted as the causative agent in over 90 percent 74 randomized to receive a daily dose of 45 g whey of duodenal ulcer cases. To date, the most effec- protein from one of two sources Ð Protectamin¨ tive form of eradication of H. pylori is one week or Immunocal. The two products have different of a triple antibiotic therapy. Due to the expense amino acid profiles and Immunocal is produced of treatment and concern for antibiotic resistance, at a lower isolation temperature (<72 degrees C). new treatments are being sought. After two weeks of oral supplementation, the In an open, randomized, single-center Protectamin-supplemented group demonstrated study of 150 individuals with diagnosed H. pylori significantly elevated glutathione levels, while the infection, patients were given antibiotics at vary- Immunocal group had statistically non-significant ing doses and durations (range 7-10 days) in con- 73 elevations. Table 4 compares the amino acid pro- junction with 200 mg encapsulated lactoferrin. files of the two products. Analysis of the study revealed 100-percent eradi- The same researchers conducted a subse- cation of H. pylori in the group using the seven- quent six-month study, using the same dose and day antibiotic course with the addition of products.65 Similarly, in the Protectamin group glu- lactoferrin. On the other hand, the “classic” seven- tathione levels increased within a two-week pe- day triple antibiotic group had a success rate of riod, while levels in the Immunocal group did not. 76.9 percent, while the 10-day treatment group All participants were then crossed over to receive demonstrated a 70.8-percent success rate. Protectamin. After six months, all patients had a In a small study, 12 children suffering significantly increased glutathione level when from chronic pharyngitis were administered a compared to baseline numbers. combination of 500 mg erythromycin three times Two small studies have been conducted daily and 100 mg bovine lactoferrin in a gargle. on HIV-positive women examining exercise, whey All children tested positive for Group A Strepto- supplementation, and body composition.67,68 In a cocci. After 15 days of treatment, fewer intracel- 2000 study the author notes an increase in body lular Group A Streptococci were found compared 75 mass composition with whey supplementation, to a group treated with antibiotics alone. whereas the 2001 study notes no effect. Both stud- In a concise review, Shah discusses the ies note an improvement in quality of life with bacteriostatic and bacteriocidal activity lactoferrin increased exercise and whey protein intake. Treat- exhibits against a number of organisms, includ- ment groups received 1.0 g/kg daily of ing Escherichia coli, Salmonella typhimurium, undenatured bovine-derived whey protein powder. Shigella dysenteriae, Listeria monocytogenes, Bacillus stearothermophilus, Bacillus subtilis, and Micrococcus luteus.32,76-79 Shah further discusses that lactoferrin, when in combination with lysozyme, is a more potent bacteriostatic agent

against Pseudomonas aeruginosa, Listeria signaling molecule for initiation of protein syn- monocytogenes, and E. coli.32,80 In addition to the thesis.8,89 It has been speculated that the quality of above-mentioned bacteria, lactoferrin has also a particular protein for enhancing muscle hyper- demonstrated antifungal activity toward Candida trophy and strength is related to its leucine con- albicans.81 tent.36 The amino acid profile in dietary proteins influences their nitrogen utilization, and poor qual- Cardiovascular Disease ity dietary proteins have been shown to increase 90,91 In recent years, studies have linked a high- nitrogen losses and limit protein synthesis. fat diet to an increased risk of cardiovascular dis- Human studies documenting the benefi- ease (CVD). Because CVD is linked to a number cial effect of whey protein supplementation on of other factors, including increased age, genet- muscle size and strength are limited. Burke et al ics, obesity, sedentary lifestyle, and alcohol intake, demonstrated that men engaged in resistance train- quality of dietary fat must be taken into consider- ing programs while supplementing with whey pro- ation. Milk is composed of more than 12 different tein concentrates showed greater improvements types of fat, including sphingolipids, free sterols, in strength than men using resistance training 92 cholesterol, and oleic acid.82 Several studies have alone. Forty-two men, ages 18-31, familiar with found milk intake and milk products lower blood weight training, followed the same high volume, pressure and reduce the risk of hypertension.83-85 heavy load, and free-weight resistance-training A study was conducted on a group of 20 program for 12 weeks. The participants were di- healthy adult males to investigate whether a fer- vided into three groups in double-blind fashion: mented milk supplement with an added whey pro- whey protein (1.2 g/kg body wt/day), a multi-in- tein concentrate would affect serum lipids and gredient whey protein sports supplement (1.3 g/ blood pressure.86 The fermented milk contained kg body wt/day), or placebo (1.2 g/kg body wt/ both Lactobacillus casei and Streptococcus day of maltodextrin ). At baseline there were no thermophilus. During the course of eight weeks, significant differences among the groups in lean volunteers consumed 200 mL of fermented milk tissue mass or strength as determined by four with whey protein concentrate or a placebo in the measurements, including bench press, squat, morning and evening. The placebo consisted of a isokinetic knee extension, and isokinetic knee flex- non-fermented milk product without the addition ion. After 12 weeks men who received whey pro- of whey protein concentrate. After eight weeks, tein or the multi-ingredient whey protein sports the fermented-milk group showed significantly supplement in combination with resistance train- higher HDLs and lower triglycerides and systolic ing showed greater improvements in one of four blood pressure than did the placebo group. While muscle strength measurements. In addition, whey total cholesterol and LDL levels were lower in the supplemented groups showed greater improve- fermented-milk group, the difference was not sta- ments in lean tissue mass over the placebo group. tistically significant. While this study does not demonstrate whey protein’s superiority over other protein sources, it Exercise does show improvement in resistance training with the addition of protein supplementation. Whey protein supplements, including pu- Lands et al showed the effect of three rified alpha-lactalbumin sports drinks, have been months whey protein supplementation (10 g twice readily utilized in the consumer market because daily) versus the same amount of casein as placebo of the high protein quality score and high percent- on muscular performance in 18 men.93 At baseline age of BCAAs.87 Whey proteins can contain up to 2,88 there were no significant differences in age, height, 26-percent branched chain amino acids, which weight, percent ideal body weight, whole leg are efficient substrates for synthesizing new pro- isokinetic cycle testing, peak power, or 30-second teins. For example, the BCAA leucine acts as a work capacity. Peak power and 30-second work

capacity improved significantly in the treatment Obesity group with no change in the placebo group. Body Obesity has reached epidemic proportions weight remained unchanged in both groups, but in the United States. Low-fat, high-carbohydrate the supplemented group had a decrease in percent dietary trends are being eschewed in favor of body fat. higher-protein, lower-carbohydrate diets. For in- While moderate exercise enhances immu- dividuals eating high-protein diets, whey is an at- 94 nity, intense athletic training has been shown to tractive source of dietary protein. Whey protein 95-97 stress the immune system. Free radical produc- isolates can be as high as 95-percent protein, after tion and increased inflammatory activity are the removal of fat and lactose, and contain valu- thought to contribute to impaired immune activ- able minerals and vitamins. Whey has made a sig- ity in over-trained athletes. In highly trained indi- nificant commercial impact in the weight-loss in- viduals muscular performance and recovery can dustry for its protein content alone. The essential 98-100 be hindered by oxidative stress. The availabil- and non-essential amino acids in whey act as sub- ity of glutathione has been shown to reduce oxi- strates for protein synthesis and may improve body 101 dative stress. mass index in individuals participating in exer- Acting as a cysteine donor, whey has been cise programs.92 102 shown to raise intracellular GSH levels in vitro. Calcium is thought to influence energy Lands et al proposed enhanced biosynthesis of metabolism because intracellular calcium regu- intracellular glutathione, shown by an increase of lates adipocyte lipid metabolism and triglyceride lymphocyte GSH levels in test subjects, is the storage.108 Zemel et al demonstrated a greater ef- mechanism responsible for the improved muscu- fect of dairy versus nondairy sources of calcium 93 lar performance observed in their study. for improving body composition. Calcium-forti- Whey’s amino acid profile makes it ideal fied cereal was compared to calcium-fortified ce- for body composition and to support protein syn- real plus nonfat dried milk for accelerating weight thesis and muscle growth. Other bioactive com- and fat loss in mice. The addition of nonfat dried ponents found in whey might benefit additional milk resulted in substantial amplification of these aspects of health in active people and trained ath- effects. The mechanism responsible for increased letes by improving immune function and gas- fat loss found with dairy-based calcium versus trointestinal health and exhibiting anti-inflamma- nondairy calcium has not yet been elucidated. The tory activity. Whey components, such as IgA, researchers speculate, “this additional activity re- glutamine, and lactoferrin, can beneficially impact sides in the whey fraction of milk.”108 The common complaints among athletes, including bioactive components in whey are thought to act repeated infections and gastrointestinal distur- synergistically with calcium to attenuate lipogen- bances. esis, accelerate lipolysis, and effect nutrient parti- 103,104 Lower levels of sIgA and glutamine tioning between adipose tissue and skeletal have been found after intensive exercise and in muscle.108 over-trained individuals, and have been correlated While there is increasing evidence dairy 105 with increased frequency of infection. In addi- intake and whey protein may play a role in the tion, glutamine deficiency has been proposed to prevention and/or attenuation of the rising epi- contribute to gastrointestinal complaints experi- demic of obesity, there is minimal data to suggest 106 enced by highly trained individuals. Free radi- dairy, calcium from dairy, or whey can impact in- cal damage is thought to delay muscle recovery dividuals with established obesity. and impair performance.107 Whey prevents free radical damage through supporting intracellular glutathione levels and supplying lactoferrin for additional antioxidant activity.

Infant Formula and Infantile Colic Osteoporosis Creating a substitute for mother’s milk has Milk has been proposed as a nutritional proved to be challenging. It is estimated a nursing food that aids in the prevention of osteoporosis infant ingests about 3 g lactoferrin daily during the due to its bioavailable calcium content.116 Re- first week of life,109 whereas a calf drinking two li- searchers have begun to examine the different ters of milk a day ingests about 2 g lactoferrin daily. components of milk to determine if a particular It is well accepted that nursing infants have a much isolate is responsible for the bone-protective ef- richer gut flora than do bottle-fed infants, particu- fects. Initially, in vitro and animal studies deter- larly with Bifidobacteria and Lactobacilli.2 Such flora mined milk basic protein (MBP), a component of is normally associated with an increased resistance whey, has the ability to stimulate proliferation and to colonization of the digestive tract with pathogenic differentiation of osteoblastic cells as well as sup- bacteria.110 In a study by Roberts et al it was deter- press bone resorption.117-119 MBP is prepared from mined that the addition of lactoferrin to a feeding fractionated whey through cation exchange resin. formula increased levels of Bifidobacteria in bottle- The total protein concentration of MBP is 98 per- fed babies. The levels of Bifidobacteria in formula- cent, containing lactoferrin, lactoperoxidase, and fed babies that were supplemented with lactoferrin other minor components. Several in vivo studies were not as high as those found in breast-fed babies. on rats determined that both whey protein and frac- In addition, Bifidobacteria in formula-fed babies took tionated whey protein had the ability to increase up to three months to develop, while Bifidobacteria femoral bone strength in young ovariectomized developed more rapidly in nursing infants.111 rats.120-122 In a double-blind, German study, 102 To date three clinical trials have been con- healthy infants less than two-weeks old were ran- ducted to determine the effects of MBP in bone domized to receive either a standard cow’s milk for- metabolism.123-125 In a trial by Toba et al 30 healthy mula or an infant formula containing partially hy- adult men were given a beverage containing 300 drolyzed whey protein.112 Results of this study indi- mg MBP.123 Subjects were instructed to drink one cate the whey-protein fed infants had significantly beverage daily for 16 days while maintaining a more Bifidobacteria in their stools, ultimately afford- normal diet. After 16 days, serum calcium and ing improved gastrointestinal immunity. It has been urinary calcium excretion were unchanged among observed in previous studies that higher levels of all subjects. Both serum osteocalcin and Bifidobacteria in the digestive system decrease the procollagen I carboxy-terminal propeptide (PICP) potential for developing atopic disease for at-risk levels increased after 16 days, indicating increased infants with family history.113 bone formation. Osteocalcin and PICP are bio- A randomized, double-blind, placebo-con- chemical markers released from osteoblasts to trolled study of 43 infants with diagnosed infantile assess bone formation. colic was conducted to determine whether a A similar study was conducted on a group hypoallergenic, hydrolyzed whey formula was su- of 33 healthy adult women to assess bone metabo- perior to a standard cow’s milk formula.114 Infantile lism.124 Women in the study were randomized to colic is described as at least three hours per day of receive either a placebo or a 40 mg MBP bever- crying for at least three days a week for a minimum age daily. Baseline left calcaneus bone-mineral of three weeks.115 Parents kept a 24-hour diary for density tests were performed on all women, and two weeks during the study. After a one-week quali- were repeated again at the completion of the study. fication period, infants in the study were random- Results indicated bone mineral density was sig- ized to receive either whey or cow’s milk formula nificantly increased in the MBP group compared for one week. A clinically relevant result was ob- to placebo. Biochemical indices also revealed the served in the whey formula group, with crying time MBP group exhibited a significant decrease in reduced to less that one hour per day Ð a one-hour urinary cross-linked N-teleopeptides, indicating an greater reduction than found in the cow’s milk inhibition of osteoclast-mediated bone resorption. formula group.

The authors state 400-800 mL of milk is equiva- Whey provides protein necessary for wound heal- lent to the 40 mg dose of MBP. More recently, ing. Surgery stresses the body, altering natural Yamura et al conducted a similar double-blind defenses, leading to various post-surgical compli- study examining 30 women over a six-month pe- cations. Zimecki et al demonstrated lactoferrin riod. Results indicate a daily dose of 40 mg per regulated the immune response and provided pro- day of MBP significantly increases radial bone tective measures in post-surgical patients.132 density.125 Human studies demonstrated whey protein improved cognitive function and coping abil- Gastrointestinal Support ity in highly stressed individuals.133,134 A rise in A small, randomized, double-blind, cross- serotonin is thought to improve adaptation to 135 over study was conducted on 10 children with stress, and the authors proposed the tryptophan short bowel syndrome to examine the effect of a available in whey provides a substrate to increase hydrolyzed versus a non-hydrolyzed whey protein brain serotonin levels. The treatment groups for on growth and development.126 Energy intake, ni- both studies received alpha-lactalbumin-enriched trogen balance, intestinal permeability, and weight whey protein because it has the highest tryptophan gain were similar among all children, indicating concentration of whey protein fractions. the particular form of whey was not an essential Table 5 summarizes the clinical trials us- component. In children experiencing bowel resec- ing whey. tion, food introduction and promotion of normal growth and development is of utmost importance. Conclusion The increased transit time of whey proteins in the Milk is one of the oldest functional foods small intestine makes it an ideal protein source available to mammals. From birth, mammals rely for this small subset of children. on mother’s milk for nutrition and immune pro- Whey protein has demonstrated a protec- tection. Scientists are beginning to develop an tive effect on the gastric mucosa. This effect is understanding of the various components of milk, thought to be related to the sulfhydryl component, including whey, and how they may impact health particularly cysteine and its link with glutamic acid and disease. Considerable positive research re- in the production of glutathione.127,128 Several ani- garding whey and its biological components con- mal studies have demonstrated this protective ef- tinues to be published. It is noteworthy some of fect.129,130 In a study by Rosaneli et al, rats fed a the research has been funded by commercial dairy whey protein concentrate showed a 41-percent organizations, although a significant amount of reduction in ulcerative lesions caused by ethanol clinical data from Asia was not supported by dairy- ingestion, while a 73-percent reduction rate was aligned interest groups. observed following repeat doses of whey.129 A To date, no severe adverse reactions have study by Matsumoto et al demonstrated an iso- been noted following administration of whey pro- lated alpha-lactalbumin formula had a four-fold tein products, although some patients note minor effect on reducing ulcerative lesions compared to gastrointestinal disturbances. For individuals with a whey protein formula containing only 25-per- frank milk allergies, whey products may not be cent alpha-lactalbumin.130 suitable, although many individuals sensitive to dairy find that casein is the culprit and they can Other Uses of Whey Protein tolerate whey. Other dairy-sensitive individuals are The wide range of essential and non-es- lactose-intolerant. Most whey proteins are pro- sential amino acids, minerals, fats, and biologi- cessed to remove lactose and finished whey prod- cally active proteins in whey provide extensive ucts only contain trace amounts. De-lactosed application in clinical nutrition. Adequate protein whey, produced from crystallizing a majority of intake is essential for post-surgical wound heal- the lactose out and recovering the remaining whey, ing and protein depletion delays healing time.131 is appropriate for lactose-intolerant individuals.

Table 5. Summary of Clinical Trials Using Whey Protein

Condition Dose of Whey Protein Study Duration Results

30 g daily 6 months 2 of 5 patients had tumor regression Results suggested an increase in glutathione levels in healthy cells and decreased Cancer glutathione levels in cancer cells

40 g daily 6 months 16/20 survivors at 6 months Stage 4 malignancies Increased NK cell function Used other natural Increased glutathione therapies. Increased hemoglobin and hematocrit Improved quality of life

Hepatitis B 12 g daily 12 weeks Decreased serum lipid peroxidase levels Increased IL-2 and NK activity Decreased serum alanine transferase activity Increased plasma glutathione levels

HIV 45 g daily 2 weeks and Increased glutathione levels in both 6 months trials

Cardiovascular Risk 200 mL of fermented milk 8 weeks Increased HDL Factors combined with liquid Decreased triglycerides whey daily Decreased systolic BP Decreased total cholesterol

1.2 g/kg 12 weeks Improved lean tissue mass body weight daily Improvement in one of four muscle Exercise strength measurements

10 g twice daily 3 months Significant improvements in peak power Significant increase of 30-second work capacity Increased lymphocyte glutathione levels

Infant Formula Standard Cow Formula 12 weeks Increased Bifidobacteria proportion vs Increased gastrointestinal immunity Partially Hydrolyzed Decreased potential for developing Whey Formula atopic diseases (doses varied) 1 week Decreased incidence of infantile colic

A challenge test with a small portion of a particu- foods will develop. In addition to the isolated lar whey supplement for an individual with dairy colostrum, lactoferrin, and alpha-lactalbumin sensitivities would be indicated before beginning products currently available, glycomacropeptide, therapeutic amounts. lactoperoxidase, whey immunoglobulin, and As researchers further investigate whey bovine serum albumin isolated products may be components it can be expected that new functional investigated.

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