Whey Antiox Bounous

Rivista Italiana di Nutrizione Parenterale ed Enterale / Anno 22 n. 4, pp. 193-200

Wichtig Editore, 2004

Rassegna - Review

The role of whey protein in antioxidant defense S.G. SUKKAR1, G. BOUNOUS2 1 2

Clinical Nutrition Unit, Azienda San Martino University Hospital Genova - Italy Research and Development Department, Immunotec Research Ltd, Vaudreuil-Dorion, Quebec - Canada

ABSTRACT: Whey protein concentrates (WPC) are a heterogeneous group of proteins (β-lactoglobulin, α-lactalbumin, serum albumin and immunoglobulins) obtained in milk after casein (CAS) precipitation. WPC also contains bioactive substances, such as hormones, growth factors (insulin-like growth factors (IGFs), transforming growth factor-β (TGF-β) platelet derived growth factor) and cytokines, which can have an important physiological role. In in vitro studies, WPC demonstrate anticancer actions such as the inhibition of cancer cell growth and antimutagenic effects while, in experimental tumor animal models, tumor burden reduction. Furthermore, WPC present immuno-enhancing properties due to glutathione (GSH) synthesis of lymphocytes, which can represent a possible tool in oxidative stress related diseases. Finally, WPC consumption in adults is associated with a significant increase in protein synthesis, with no change in protein catabolism when compared with CAS, according to their different enteric absorption. The physiological mechanisms by which WPC interact with the antioxidant system, cancer prevention and metabolism, as well as the possible role of WPC in immunonutrition are discussed. In conclusion, WPC could be an interesting integrative protein source in clinical nutrition. Human clinical trials are required to verify the efficacy of WPC in counteracting oxidative stress and in favoring protein anabolism in protein-energy malnutrition syndromes. (RINPE 2004; 22: 193-200) KEY WORDS: Antioxidants, Whey protein, Cancer, Immune system, Immunonutrition, Oxidative stress, ROS PAROLE CHIAVE: Antiossidanti, Proteine del siero di latte, Cancro, Sistema immunitario, Immunonutrizione, Stress ossidativo, Radicali liberi

The current interest in complementary medicine has led to the increasing use and misuse of terms such as “free radicals” and “antioxidants”. A free radical can be defined as any species that contains one or more unpaired electrons, an unpaired electron being one that is alone in an orbital. Most biological molecules contain only paired electrons. A radical either can donate its unpaired electron to another molecule or can acquire an electron from another molecule in order to pair. The characteristic of events involving free radicals is the development of a chain reaction with subsequent damage to adjacent biological structures, which could be involved in the pro-in-

flammatory cascade (Fig. 1). The majority of free radicals originate in the final stage of cell respiration, in which electrons flow from organic substrates to oxygen, yielding energy. When mitochondria are functioning, an electron passing through the respiratory chain can leak directly onto the oxygen molecule resulting in superoxide radical formation: O2 + e- O.-2. Figure 2 demonstrates the sequence of biochemical events following the appearance of O.-2. Overall, antioxidants include endogenous and exogenous elements such as antioxidant enzymes (superoxide dismutase catalase), vitamins (vitamin C and vitamin E), the glutathione (GSH) system and non-vitamin substances such as catechins and β-carotene (1). More▼

INTRODUCTION

© SINPE-GASAPE

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Fig. 1 - Possible role of nutritional intervention as complementary therapy in chronic oxidative stress and chronic inflammation: signal transduction and gene expression can be down-regulated by antioxidant natural systems and their precursor (cysteine, glutamate, whey protein concentrates and antioxidant vitamins), by diet derived antioxidants substances, by diet derived inhibitors of NF-kB or by n-3 fatty acids (from: Sukkar & Rossi Autoimmunity Rev 2004; 3: 199-206) (35). ROS: reactive oxygen species; EGCG: epigallocatechin 3-gallate; SL: sesquiterpene lactones; AP-1: activator protein-1; NF-kB: nuclear factor-kB; AA: arachidonic acid; ECA: eicosapentaenoic acid; COX: cycloxygenase; LOX: lipoxygenase; PG: prostaglandines; TX: tromboxans; LT: leucotriens.

over, the term “antioxidant” was used initially to define the cell’s own protective mechanisms. It is noteworthy that the two major barriers against released free radicals are located close to the mitochondria, in proximity to the oxidant formation source. GSH (L-gamma-glutamyl-L-cysteinylglycine) can spontaneously, or with the help of peroxidase, easily deliver the H necessary for the reduction in the radicals (Fig. 2). The redox system is represented by GSH in the cells and by albumin and cysteine in plasma. In both cases, the active element is the SH (thiol) cysteine group which, when performing its antioxidant activity, is oxidized to cystine or cysteine

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disulfide (Fig. 3). Therefore, the cysteine/cystine ratio defines the redox state that represents the major determinant of optimal cell function. The tripeptide GSH is largely distributed inside the cells where it is a substrate for two classes of enzymes, the selenium-dependent GSH peroxidase and a family of GSH transferases, which are considered to catalyze detoxification by two different reactions: spontaneous or enzyme-associated. The involvement of GSH in cancer protection (in particular of cancer-induced DNA damage) includes a reduction in 9Y GSH peroxidase of hydrogen peroxide, free radicals and reactive oxygen

Sukkar and Bounous

Fig. 2 - Interactions between ROS and GSH.

Fig. 3 - Cellular and plasma redox system.

species (ROS). GSH transferases catalyze the GSH conjugation of electrophilic compounds biotransformed from xenobiotics (mutagens and carcinogens), which are eliminated easily from the body (2-4). Dietary GSH sources are few (1, 5, 6) and excess dietary protein or sulphur amino acids do not enhance the maximum hepatic GSH amount beyond the normal physiological maximum level obtained with adequate dietary components. The liver, the key organ for xenobiotic detoxification and elimination, is the major site of GSH synthesis. This organ has the unique and particular ability to convert the sulphur amino acid methionine to cysteine required for GSH synthesis (2, 7). Almost 95% of GSH synthesized in the liver is released in the blood stream, which supplies the extra-hepatic tissues (2), and bile. The latter is the main GSH source for the intestinal mucosa, where its concentration is relatively high (50-60% of liver content), but its distribution is not uniform. The colorectal mucosa seems to have lower GSH transferase activity in comparison to other parts of the intestinal mucosa, and this could partially explain the susceptibility to cancerogenesis of this anatomic district (8). GSH biosynthesis is strictly dependent on precursor

amino acid concentrations (glutamate, glycine and cysteine) and competes with albumin synthesis for the available cysteine (1, 9). The kinetic characteristics expressed by the km rate for amino acid activating enzymes (the rate limiting enzymes for protein synthesis) is 0.003 mmol/L, whereas that for gamma glutamyl cysteine synthetase (the rate limiting enzymes for GSH synthesis) is 0.35 mmol/L. This means that the biosynthetic pathway for proteins works maximally at a concentration approximately 166-fold lower than for GSH synthesis, whose production is subsequently impaired in greater amounts than that of proteins at low cysteine availability (1). Grimble and Grimble demonstrated that rats nourished with different amounts of sulphur amino acids, undergoing inflammatory stimuli, show differences in net responses to protein and GSH synthesis by the liver (10). Therefore, a redox mechanism that links receptormediated signals to low intracellular GSH levels favors those cells that are engaged in high protein synthesis (11). The albumin concentration reflects a measure of the degree of stress rather than the nutritional status. Several studies have demonstrated that the fractional synthesis rate is increased rather than decreased in both traumatized patients and in cachectic stressed patients

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(12). This effect can be due to the presence of the cysteinil radical in albumin. A possible suggestion claims that albumin enters tissues to repair damage inflicted by the stress conditions, and here it is oxidized. Therefore, in many catabolic clinical conditions the increased turnover and breakdown of albumin could be associated with cysteine consumption. It is noteworthy that nutritional intervention is often ineffective in raising the albumin content of plasma, whereas N-acetylcysteine (NAC), a cysteine analog, can raise plasmatic albumin levels (9). Another possible cysteine source is whey protein, which has been proved to increase the lymphocyte concentration of GSH in animal fed models (13).

Fig. 4 - Reports of studies showing the immunoenhancing role of specially prepared dietary WPCs.

Immune system and antioxidant systems If Figure 3 represents part of the lymphocyte machinery, we can understand why in vitro studies have demonstrated that the oxygen-requiring antigen driven clonal expansion and antibody synthesis in immune cells depends on their capacity to reconstitute GSH to neutralize the increased production of oxygen-derived radicals; therefore, facilitating a sustained immune response (14, 15). This principle was verified by in vivo experiments where animals fed cysteine rich whey protein concentrate (WPC) showed enhanced immune responses to a T-dependent antigen (13, 17) (Fig. 4). This effect was abolished by the administration of buthionine sulfoximine, which inhibited the GSH synthesis; therefore, demonstrating that the role of these proteins on the immune system depends on the GSH system (18). Dietary whey protein concentrates Biological characteristics WPC are a heterogeneous group obtained in milk after casein (CAS) precipitation at pH 4.6 (19). The concentrations in milk vary from approximately 5-7 g/L. The major components in decreasing amounts are β-lactoglobulin (2-4 g/L), α-lactalbumin (0.6-1.7 g/L), bovine serum albumin (0.2-0.4 g/L) and immunoglobulins (0.5-1.8 g/L). Commercial WPC (approximately 75% protein) is prepared in the diary industry as a byproduct of cheese and CAS manufacture. WPC contains carbohydrate (4% lactose) and 5% lipids (approximately 25% fatty acids and 25% phospholipids) (19), it also contains bioactive substances such as hormones, growth factors (insulin-like growth factors, transforming growth factor-β (TGF-β) and platelet growth factor) and cytokines, which can have an important physiological role. For example, TGF-β is able to regulate cell growth in both normal cells and tumor cells by suppressing prolif-

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eration or enhancing apoptosis (19). Given that TGF-β is unaffected by acidic stomach pH, it can reach intestinal cells where, after receptor binding, it can act through an antiproliferative fashion, and subsequently be absorbed and exert its action on other cellular compartments. Anticancer actions of WPC A preliminary study (20) and a more detailed version (21) reported the effects of formula diets containing 20 g/100 g diet of WPC, CAS or Purina mouse chow on dimethylhydrazine-dihydrochloride (DMH)-induced colon tumors in A/J mice. At 28 weeks, tumor incidence and tumor area were substantially reduced in WPC-fed mice, and in lower amounts, but always significant in CAS-fed mice. At the end of the experiment, the mice continuously fed WPC were alive, whereas 33% of the CAS- or Purina-fed mice had died. The Purina/WPC-fed mice were afforded greater protection from colon tumors than the Purina/CAS-fed mice. McIntosh et al (22) also demonstrated that dairy protein-based diets restricted DMH-induced tumor genesis, and soybean protein-fed at 20 g/100 g in AIN-76A diets resulted in a tumor incidence rate of 30, 45, 55 and 60% respectively, although the results did not reach statistical significance. Moreover, tumor burdens (i.e. the total number of tumors within each group) in WPC and CAS-fed groups were significantly (p<0.02) lower than for red meat or soybean protein-fed groups (Tab. I). The discovered immunoenhancing WPC activity inspired the first study of WPC feeding on the development of experimental colon carcinoma in mice (20). The positive results of this study (i.e. a reduction in tumor burden with WPC administration) were confirmed in rats (22) (Fig. 5, Tab. I) and extended to other types of malignancies such as mammary tumors in female rats (23).

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Fig. 5 - Results of Studies Demonstrating the Role of Specially Prepared Dietary wpc s in Cancer Prevention. Carcinogen was dimethylhydrazinedihydrochloride (DMH), which induces colon tumors similar to those found in humans (with regard to type of lesions1 and response to chemotherapy2). The diets were fed before and throughout the 24 weeks DMH-treatment period. No differential effect of diet on body weight was seen.

TABLE I - AMOUNTS OF SULPHUR AMINO ACIDS, LIVER GLUTATHIONE AND TUMOUR DATA FOR RATS FED VARIOUS DIETS1 Source of protein

Cysteine

Methionine

Total cysteine + methionine

Liver glutathione mmol/g (wet tissue)

Tumor incidence (%)

Tumor burden (tumors/group)

Soybean Meat Whey

0.70 0.50 2.30

1.30 2.20 2.10

2.00 2.70 4.40

2.45 4.16 5.21

60 55 30

26a 21b 7*

1

Adapted from McIntosh (1995).*p<0.02 vs. a, b

WPC effects on cell cultures Cell culture studies showed that whey protein or whey protein components were able selectively to inhibit cancer cell growth. The addition of whey protein to the medium of estrogen-responsive human breast cancer cell line MCF-7 and of a prostate cancer cell line significantly reduced cell growth (24). The growth in the breast cancer line MCF-7 was also demonstrated to be inhibited by bovine serum (25). Bovine serum albumin, but not total whey protein, β-lactoglobulin or soybean protein exhibited a strong anti-mutagenic effect against the mutagen 4-nitroquinoline-L-oxide in a Chinese hamster epithelial cell line (26). Finally, WPC stimulated the proliferation and GSH synthesis of normal lymphocytes whereas it inhibited the proliferation of rat mammary tumor or Jurkat T tumor cells (27). Anabolic effects Boirie et al (28) claimed recently, by analogy with carbohydrates, that slow and fast proteins exist, according to the speed at which they are digested and absorbed

as amino acids from the gut. In this report, the authors studied the effect of two milk proteins, CAS and WPC, on postprandial whole-body protein metabolism in humans. In order to achieve this, they combined oral and intravenous administration of 13C-leucine labeled and unlabeled specific proteins using a dual tracer methodology. The most exciting result of this study was that when they administered WPC to adult patients, a high, rapid and transient peak in plasmatic amino acids was observed. This event was associated with a significant increase (>68%) in protein synthesis, with no change in protein catabolism. On the contrary, after CAS administration, the plasma amino acid surge proved lower, slower but more prolonged. Therefore, protein synthesis was only moderately increased (>31%), whereas a marked inhibition in protein break down (i.e. leucine oxidation) was evidenced; notably, leucine intake was identical in both meals. Overall, the different behavior of these two proteins possibly reflects the different exit rate from the stomach into the duodenum, which is much faster for WPC, causing a rapid influx of amino acids analogous to the stimulation of protein synthesis observed after intravenous amino acid infusion. In addition, it must be

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NATURAL

PHARMACEUTICAL

WHEY PROTEIN CONCENTRATE

NAC (N-ACETYLCYSTEINE)

1. Bounous G et al. Clin Invest Med 1988; 11: 213-7 – The incidence and size of DMH induced colon tumors was lower in dietary-treated mice. 2. Papenburg R et al. Tumour Biol 1990; 11: 129-36 – Similar results as in 1, in addition, dietary treatment was effective also on established malignancy. Improved survival. 3. McIntosh G et al. J Nutr 1995; 125: 809-16 – Almost identical results as in 1 were obtained in rats. 4. Hakkar R et al. Cancer Epidemiol Biomarkers Prev 2000; 9: 1137 - In rats, the incidence of chemically induced mammary tumors was lower. 5. Bounous G et al. Anticancer Res 2000; 20: 4785-92 [1] - Cancer of the prostate. All patients had elevated PSA levels with biopsy confirmed cancer of the prostate. 13 out of 15 patients showed a progressive decline of PSA values during 3-12 months observation period. - Metastasis of renal carcinoma. In a 50-year-old woman: following 3 yrs, a significant decrease of metastatic disease in liver, resolution in lung and bone was seen. - Bladder cancer. During 1 yr observation, no recurrence of papillary transitional cell carcinoma.

1. De Flora S et al. Cancer Lett 1986; 32: 235-41 – Inhibition of urethan induced lung tumors in mice. 2. De Flora S et al. Am J Med 1991; 91: 1225-305 – Prevention of mutation and cancer by thiols is particularly useful in condition of GSH depletion. 3. De Flora S et al. J Cell Biochem 1995; (suppl 22): S33-41 – A study of the mechanisms contributing to NAC anticarcinogenesis. 4. De Flora S et al. Int J Cancer 1996; 67: 842-8 – Synergism between NAC and doxorubicin in the prevention of tumors and metastases in mice. 5. Delneste Y et al. Blood 1997; 90: 1124-32 – NAC exhibited potent anti lymphoma activity in mice. 6. D’Agostini F et al. Int J Oncol 1998; 13: 217-24 – In mice, NAC interact with a cytotoxic agent in inhibiting melanoma cell invasion and metastases. 7. Dröge W. Current Opin Clin Nutr Metab Care 1999; 2: 227-33 – Plasma albumin level and body cell mass in cancer patients are increased by NAC. 8. Estensen RD et al. Cancer Lett 1999; 147: 109-14 – In patients at risk of colon cancer, NAC produces a decrease of proliferation index in the crysts. 9. Morini M et al. Int J Biol Markers 1999; 14: 268-71 - Inhibition of neo angiogenesis and tumor progression in murine melanomas.

[1] The whey protein concentrate, specifically an isolate defined by protein grade, in non-instantized native form, marketed as Immunocal/HMS90, was obtained from Immunotec Research Ltd.

Fig. 6 - Anticancer effect of cyst(e)ine in natural and pharmaceutical compositions, cysteine delivery systems.

borne in mind that albeit the percentage of amino acids able to stimulate insulin secretion is similar in both proteins, WPC possess a higher proportion of branchedchain amino acids, which permits a synergistic effect with insulin on protein metabolism (i.e. anabolism). On the contrary, the percentage of amino acids that stimulate glucagone secretion is lower in WPC and, subsequently, the catabolic effect of this hormone is reduced after the ingestion of this protein (29). The same group (30) has recently extended previous data by analyzing the effects of dietary carbohydrates and lipids added to WPC and CAS. To verify this two test meals, which differed only in their protein composition (CAS vs. WPC) but not in leucine amount, were administered to healthy adult males. Each test meal was otherwise composed of identical amounts of carbohydrates and fat. Although preliminary, the results of the study suggested that (1) WPC was still more rapidly absorbed than CAS, as previously shown (28); (2) postprandial leucine balance was still lower with WPC-containing meals than with those containing CAS, i.e. proteolysis was more markedly inhibited by the latter “slow” protein, although the differences in digestion rate and leucine balance were less marked than when the pro-

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teins were administered alone. These results demonstrated that added non-protein energy sources to CAS and WPC reduced the differences in both the protein digestion rate and protein gain. Finally, this preliminary study showed that in contrast to the young, in elderly people a fast protein (i.e. WPC) induces a better postprandial leucine balance than that obtained with a slow protein (i.e. CAS). This latter finding suggests that in healthy elderly people, the optimization of protein retention could be achieved by changing the pattern of protein feeding and/or the rate of protein absorption (28-30). The potential to enhance protein synthesis and to reduce/inhibit protein breakdown improves the therapeutic possibilities for patients with wasting syndromes such as those with sepsis, burns, acquired immunodeficiency syndrome, skeletal trauma and multiple organ failure (29, 30). Mechanisms of cysteine delivering drugs/systems The effect of a cysteine pro-drug such as NAC on tumor development and carcinogenesis (Fig. 6) strongly suggests that WPC can act as a cysteine delivery system in inhibiting tumor growth. In discussing the effects of WPC on tumors, it is important to distinguish between

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the anti-carcinogenesis effect and the anti-tumoral effect. Our suggestion is that WPC can possess both; in particular the former effect could be obtained by the ability of WPC to provide high substrate levels for GSH synthesis in relevant tissues; therefore, enhancing the detoxification of potential carcinogens or free radicals in spontaneous carcinogenesis. The second property (i.e the anti-tumoral effect) could rely on the GSH-dependent stimulation of the immune system by WPC. Table I (22) illustrates the relationship between cysteine, GSH and the development of experimental tumors (19). The causative role of cysteine deficiency in immunological dysfunction developments in oncological patients is supported by the observation that additional cysteine sources can restore natural killer cell activity (31). It is interesting to note that oncological patients show an accelerated shift to more oxidized conditions, which could contribute to the loss of body cell mass (32). These data suggest that during cancer progression, the antioxidant buffer activity could progressively decline due to the depletion of the thiol (SH) group in the redox equation. Therefore, a natural cysteine donor such as WPC could inhibit/delay tumor growth by directly increasing intracellular thiol levels (33). Finally, cysteine itself could exert a direct anti-tumor effect in two different ways unrelated to GSH synthesis. Concerning the former, it was recently demonstrated that several sulfur-containing antioxidants such as NAC and dimercaptopropanol selectively induced a 5-10 fold increase in p53-dependent apoptosis in a transformed cell line and primary cultures, but not in normal cells. In particular, NAC elevated p53 expression post-transcriptionally by increasing the p53 mRNA translation rate rather than by altering protein stability. In contrast, antioxidants whose action is limited to scavenging radicals do not seem to possess this important property (33). The second feature of a cysteine delivery system is related to its inhibitory effect on neoangiogenesis and tumor progression and recurrence both in vitro and in vivo (rodents) (34). The promising anticancer effect of NAC is, unfortunately, hampered by its adverse effects at pharmacological doses. Therefore, long-term use of a natural cysteine donor such as WPC could be a promising option in the long-term nutritional support of oncological patients. In conclusion, WPC could be an interesting and alternative protein source with immunomodulating properties in clinical nutrition. Clinical trials are mandatory to verify the efficacy of whey protein to counteract the oxidative stress by the increase in GSH synthesis, and to verify the anabolic role as alternative protein substrates in malnutrition-inflammation syndromes.

RIASSUNTO Le proteine del siero del latte (WPC)rappresentano un gruppo eterogeneo di proteine (β-lattoglobuline, α-lattoalbumine, albumine seriche e immunoglobuline) derivate dal latte previa precipitazione delle caseine. Le WPC contengono anche sostanze bioattive quali ormoni, fattori di crescita (insulin-like growth factors (IGFs), transforming growth factor-β (TGF-β), platelet derived growth factor) e citochine, che presentano importanti ruoli fisiologici. Studi in vitro hanno dimostrato che le WPC presentano potere antimutageno e anticarcinogenetico mentre, in modelli sperimentali animali, sono in grado di inibire la crescita neoplastica. Le WPC mostrano inoltre proprietà immunostimolanti, associate ad un incremento della sintesi di glutatione linfocitario, potrebbe rappresentare un interessante strumento terapeutico nelle patologie correlate con lo stress ossidativo. Infine il consumo di WPC nell’uomo si riflette in un migliore effetto anabolico proteico rispetto al consumo di caseina in relazione al differente meccanismo di assorbimento enterico. I meccanismi fisiologici attraverso i quali le WPC interagiscono con il sistema antiossidante, con la carcinogenesi e con il metabolismo proteico, nonché il loro possibile ruolo immunonutrizionale sono discussi in questo articolo. In conclusione le WPC possono rappresentare una interessante fonte proteica integrativa in nutrizione clinica. È auspicabile che studi clinici controllati vengano condotti nell’uomo, al fine di definire l’efficacia clinica delle WPC nel contrastare lo stress ossidativo e nel favorire l’anabolismo proteico in corso di malnutrizione proteico-calorica.

Address for correspondence: Dr. S.G. Sukkar Clinical Nutrition Unit S. Martino University Hospital Largo R. Benzi, 10 16132 Genova, Italy e-mail: [email protected]

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