Physiological Properties of Whey Protein
...have shown different physiological functions. This review is focus on the main physiological functions such as: antioxidant activity, anticancer activity, immunoenhacing properties anti-aging activity, and antihypertensive activity. The capacity of a cell to recover from an oxidative insult is represented by its ability to regenerate intracellular stores of glutathione (Noelle et al., 1981). GSH, a tripeptide thiol found in all cells, is the major free radical oxygen scavenger (Richie, J. 1992). 2.I Antioxidant Activity The primary components of the physiological antioxidative defense system are the superoxide dismutases, catalase and GSH system (Powers et al., 1999). During exercise oxidative stress could be induced. Oxidative stress is the state where free radicals that are highly reactive atoms (active oxygen), are in larger quantities than the body antioxidant capacity. Most of the oxygen consumed is utilized in the mitochondria for substrate metabolism and the reduction of adenosin-5-triphosphate (ATP) production to H2O. However, a small fraction of oxygen may be converted univalently to several intermediates (O2, OH-), which are classified as reactive oxygen species (ROS) that contribute to the development of muscular fatigue (Gohill et al., 1988). An important consideration when attempting to increase the reactive oxygen species is that GSH is only synthesized inside the cell as is shown in Figure 2. Figure 2. Synthesis of glutathione: the cell¡¦s own antioxidant (adapted from, Bounus et al., 1999). Recent studies show that exogenous GSH increased endurance to physical exercise in mice, this study employed brief bouts of swimming as the exercise challenge (Cazzulani et al., 1991). However, at a dose of 0.25g/kg, GSH did not affect endurance when injected once but did significantly increase endurance when injected once a day for seven days. Furthermore, in a vivo study of whey-based cysteine donor a whey protein preparation, the performance of healthy young adults was enhanced. Subjects ingested 20g/day of Immunocal, resulting in a 35.5% increase in circulating lymphocyte GSH concentrations. At the same time, subjects were able to generate more power to perform more work during a 30 s. maximal effort (Landis et al., 1999). While has been proved that injected GSH per se was poorly available to tissues and it did not influence exercise-induced oxidative stress or endurance to long-duration exhaustive trade-mill run (Chandan et al., 1994). The importance of GSH status was demonstrated in a GSH deficiency model used by Chandan K. (1994) which revealed that the endogenous GSH pool tissues is critically important in the circumvention of exercise induced oxidative stress and maintenance of physical performance. The importance of protein in the diet for the immune response has been the aim of several studies, especially it¡¦s role in the inflammatory aspects of immune functions. Pro-inflammatory cytokines Interleukin 1 (IL1), Interleukin 6 (IL6) and tumor necrosis factor-a (TNF) and reactive species (ROS), play major roles in inflammatory aspects of the immune function. They are closely linked with pathology in a wide range of diseases and condition, which have an inflammatory basis (Klasing, 1988). Evidence of oxidative damage has been observed in sepsis, HIV and hepatitis infection, cancer diabetes mellitus and cystic fibrosis (Staal et al., 1992). Pro inflammatory cytokines and oxidants have the capacity to stimulate production of each other. The stimulation of cytokine production by oxidants is due to activation of nuclear factor kappa B (NFkB) by oxidants. NFkB is a multi component protein, present in the cytoplasm of a wide range of cells, including macrophages, lymphocytes and hepatocytes (Klasing, 1998). While cytokines bring about major changes in protein and amino acid metabolism, whereby amino acids are released from peripheral tissues for nutrition of cells of the immune system and the synthesis of acute phase proteins and glutathione by the liver, the amount increased may not always match demands (Grimble, 1998). It has been estimated that, during major infections in man, the amount of protein required to produce and maintain an increase in circulating white blood cells and acute phase proteins is approximately 45g/d (Moldawer, 1992). Many substances with high content of glycine, serine and sulphur amino acids, methionine and cysteine, are produced in enhanced amounts in response to cytokines. The substances comprise glutathione and metallothionein (Grimble, 1998). 2.2 Anticancer Activity Human cancer cells have shown higher cellular GSH levels than the adjacent normal cells (Russo et al., 1986). This discovery was apparently related to their proliferative activity. In fact, cancer is the only condition in which an elevation of a tightly regulated system such as GSH has been reported (Kennedy et al., 1995). Previous investigation had reported that once the lymphocyte number had dropped in cancer patients, the recovery of the lymphocyte number to a normal level might not be possible. The increase of the lymphocyte number is important because of its potential for improving the granulocytes/lymphocytes (G/L) ratio (Ietomi K., 1990). Whey protein concentrate have established in vitro studies a direct inhibitory effect on cancer cell replication (Barta et al., 1991). In other human cancer cell studies the inhibitory effect was related to the serum albumin component of whey, where albumin was found to present a substantial antimutagenic effect in an in-vitro assay using hamster cells (Bosselaers et al., 1994). Also feeding lactoferrin to mice inhibited the growth of solid tumors and reduced lung colonization by melanomas (Bezault et al., 1994). A study in vivo with a daily dose of 30g of Immunocal TM (Figure 3.), a bovine whey protein concentrate, prepared by Immunotech Research Corporation, found ƒÒ-lactoglobulin has 56.3%, ƒÑ-lactalbumin has 22.8%, serum albumin has 11.1%, of lactoferrin has 0.7% and 9.2% of immunoglobulin. This study showed that all patients (7 patients) gained a sense of well-being. It was found that whey proteins might be a likely supplement in the nutritional management of cancer patients about to undergo chemotheraphy (Kennedy et al., 1995). For example, in an in vivo study made by Dr. Bounus (1988) patient ¡§F¡¨ had solid tumors in her abdomen that could be identified by physical examination and she had received chemotherapy. She had difficulty in consuming the undenatured WPC and was about to withdraw from treatment. However it was established that while she was taking the undenatured WPC, her lymphocyte number increased, and the increasing number of tumor markers even showed signs of decreasing. Serum albumin may also diminish cell proliferation in the human breast cancer cell line in vitro (Inga et al., 1990). With all these studies is proved that whey protein has an important anti cancer activity. This can be attributed to its high content of cysteine, which plays an important role in the synthesis of GSH. It has been found that GSH levels are higher in human cancer cells than in the adjacent normal cells. 2.3 Immune-enhancing Properties The humural immune response and antibody production require rapid protein synthesis. High protein diets should therefore significantly increase the immune response. Evidence was obtained by Bounus (1988) that when male mice were fed with different diets, the main difference was the protein type (lactalbumin, casein, soy protein, wheat protein, corn protein, egg albumin, fish protein, ƒÑ-lactalbumin, ƒÒ-lactoglobulin and Spirulina maxima protein). The immune response to sheep red blood cells was measured by, the number of plaque forming cells per spleen. Table 4. Mouse immune response to different type of diet to sheep red blood cells (Bonous et al., 1988). Diet No. of plaque-forming cells Lactalbumin 150 PFC/spleen 10-3 Casein 35 PFC/spleen 10-3 Soy 20 PFC/spleen 10-3 Wheat 18 PFC/spleen 10-3 Egg albumin 75 PFC/spleen 10-3 Fish protein 90 PFC/spleen 10-3 ƒÑ-lactalbumin 58 PFC/spleen 10-3 ƒÒ-lactoglobulin 90 PFC/spleen 10-3 Hence the efficiency of dietary cysteine (Table 4.) in inducing immune response by glutathione levels seem to be greater when it is delivered in the whey protein than by other sources of protein (Bounus et al., 1988). The favorable effects of undenatured WPC in animal experiments were associated with a sustained elevation of tissue glutathione levels. However, nowhere was this effect was more evident than in the spleen cells during the antigen driven clonal expansion of the lymphocyte pool. These experiments plainly point out that the immune-enhancing activity, i.e. increased lymphocyte proliferation of dietary undenatured WPC, is associated to the detected greater production of GSH in the lymphocytes in comparison to an actual GSH decrease noted in casein-fed controls during a similar antigenic challenge (Bounus et al., 1989). More specifically, the GSH content of the lymphocytes appear to be correlated with the ability of oxidants and thiols to modulate their proliferation (Noelle et al., 1981). In this sense modulation of intracellular GSH may affect immune responsiveness (Fidelus et al., 1986). Another study also made by Bounus et al. (1989) investigated the resistance of mice to pneumococcal infection. He found that mice with lactalbumin diets showed higher resistance to this infection, than mice with other types. In this study the mice were fed 20g/100g of different types of proteins, such as lactalbumin, casein, soy, serum albumin, ƒÒ-lactoglobulin and ƒÑ-lactalbumin. He concluded that the immune-enhancing effect of the whey protein is dependent upon the overall amino acid pattern resulting from the contribution of all its protein components. 2.4 Anti-aging activity The by-products of the normal physiological metabolic processes of life are considered to cause aging. A study where mice were fed a diet of 20g/100 g of either whey protein concentrate or casein, showed that the mean survival time, at the time at which 55% of mice were dead, was increased by about 30% in mice commenced on the whey protein diet, rather than with casein. Also with the whey protein diet, the tissue glutathione levels were higher than with the casein diet. Comparing these results with the theory of aging, which hypothesizes that the degenerative changes related with aging could result from toxic effects of free radicals produced during cellular metabolism, may explain the effect of whey protein on survival (Bounus et a., 1989). Birt (1982) proved a delayed outcome of fatal, age-related diseases in hamsters with glutathione enhancing whey protein diet. Another important role of glutathione related to anti-aging effect are the maintenance of normal tissue hydration in ocular tissue and is the source of the cysteine utilized in the biosynthesis of mercapturic acid conjugates of N-acetylcysteine, end products of a process which serves to detoxify a variety of harmful compounds and xenobiotics (Megaw, 1984). Glutathione binds transitional metals and is an important factor in their elimination. It appears that, the tissue glutathione levels are more consistently reported to decline with old age. 2.5 Antihypertensive activity Milk proteins are the main source of different biologically-active peptides, although other animal and vegetable proteins are recognized as peptides with bioactive sequences. Such peptides are inactive within the sequence of the precursor proteins and can be released by enzymatic proteolysis, for example during gastrointestinal digestion or during food processing. Once they are liberated in the body, bioactive peptides may act as regulatory compounds with hormone-like activity. Peptides from food proteins should be taken into consideration as potential modulators of various regulatory processes in the body. The possible regulatory effects concern nutrient uptake (phosphopeptides, casomorphins), postprandial hormone secretion (casomorphins), blood pressure reduction (casokinins), immune defense (immunopeptides, casokinins, casomorphins) ...