***Bioctive whey protein and its role in enhancing glutathione levels
Bio-active whey protein refers to a mixture of globular proteins that are recovered from whey, the liquid by product obtained during cheese production (Smilkov, et al., 2013) . The protein composition in cow milk is 80% casein protein and 20% whey protein. Whey protein makes up 10% of whey’s dry weight. The protein is often made up of beta-lactoglobulin (65%), alpha-lactalbumin (25%), bovine serum albumin (8%), and immunoglobulins (Smilkov, et al., 2013) . A number of preclinical studies conducted in rodents have shown that whey protein might possess some anti-cancer and ant-inflammatory properties. Other studies have shown that the protein might have an effect on muscle growth, though this observation is debatable. Researchers are currently trying to understand if whey protein has any impact on human health (Smilkov, et al., 2013) .
Whey protein has been marketed as a dietary supplement under the name Immunocal (HMS-90). The name Immunocal is derived from the immune system effects. The supplement is globally marketed and sold as an all-natural non-prescription health product (Immunocal Health Benefits, 2013) . The protein holds several international patents and is medically recognized by several physician bodies around the globe, including the Physicians’ Desk Reference (“PDR” U.S.A) and the Compendium of Pharmaceutical Specialties (“CPS” Canada) (Immunocal Health Benefits, 2013) . Immunocal’s health benefits include providing the body with much needed amino acids, and raising glutathione levels (Immunocal Health Benefits, 2013) . According to unverified data, Immunocal provides cysteine, a glutathione precursor that is vital in the maintenance of a strong immune system (Immunocal Health Benefits, 2013) .
Glutathione (GSH) can be described as a tripeptide formed by gamma peptide linkages between the glutamate side-chain carboxyl group and the amine group of cysteine (Lupton, 2004) . Glutathione works as an antioxidant, and prevents damage to vital cell components as a result of reactive oxygen, including peroxides and free radicals. GSH is widely regarded as the master antioxidant in cellular defense (Lupton, 2004) . It also plays a vital role in the a number of cellular events including gene expression, cell proliferation and apoptosis, signal transduction, DNA and protein synthesis, cytokine production and immune response, and protein glutathionylation (Lupton, 2004) .
The tripeptide is produced by the liver and is able to function as an antioxidant in the lungs, Red blood cells, liver and the intestinal tract. It also helps in the removal of a wide range of toxins including those that come from heavy metals, cigarette smoking, alcohol, cancer chemotherapy and radiation (Lupton, 2004) .
In mammalian cells GSH concentrations range from 1 to 10mM, though reduced concentrations predominate over the oxidized form (GSSG). Studies indicate the maintenance of optimal ratios of GSH: GSSG is critical for survival, and the deficiency results into oxidative damage (Lupton, 2004) . GSH plays a critical role in the prevntion and control of a number of disorders, including autoimmune diseases, neurodegenerative diseases, diabetes and cancer (Lupton, 2004) .
Several molecular studies have been carried out to understand the interactions between specific whey proteins and tumor cells. In 1999, Svensson et al. conducted a molecular characterization of alpha-lactalbumin folding varriants that induce apoptosis in tumor cells but spare healthy cells (Svensson, et al., 1999) . Purification of the active fraction from other casein components was carried out by anion exchange chromatography, it was then retained by ion exchange and eluted after a high salt ingredient (Svensson, et al., 1999) . The active fraction was indicative of an N-terminal amino acid seqence identical to human milk alpha-lactalbumin, and post-translational modifications were ruled out by mass spectrometry (Svensson, et al., 1999) . Oligomers with high molecular weight exhibited kinetic stability and were found to have an essentially retained secondary structure but a less organized tertiary structure (Svensson, et al., 1999) . On comparison with native monomeric and molten globule alpha-lactalbumin, it was established that the active fraction contained oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state (Svensson, et al., 1999) . The findings of the study suggested that there might be differences in biological properties between folding variants of alpha-lactalbumin (Svensson, et al., 1999) .
There are various studies that indicate a role by bio-active whey protein on the level of glutathione. A study was conducted by Baruchel and Viau to investigate the in vitro selective modulation of cellular glutathione by humanized native milk protein isolate in normal cells and rat mammary carcinoma(1996) . The study utilzed immunocal to establish the inhibitory activity of whey protein on mammary cell carcinoma cells and Jurkat T cells (Baruchel & Viau, 1996) . This was compared to normal peripheral blood mononuclear cells and related to the inhibitory activity to a selective depletion of intracellular glutathione sysnthesis (Baruchel & Viau, 1996) . The findings of the study showed that the use of humanized whey as food supplementation may have a direct impact on a clinical trial with adjuvant chemotherapy
A review conducted by Ross et al. focused on Immunocal and preservation of glutathione as a novel neuroprotective strategy for degenerative disorders of the nervous system (2012) . The study was based on the background that oxidative stress and glutathione (GSH) depletion are both established as key contributing factors to the pathogenesis of many devastating neurodegenerative diseases as a of free radical generation (Ross, Gray, Winter, & Linseman, 2012) . According to the study, strategies that increase or preserve levels of intracellular glutathione have been demonstrated to act in a neuroprotective manner, indicating that the augmentation of available GSH pool may be a promising therapeutic target for neurodegeneration (Ross, Gray, Winter, & Linseman, 2012) . The review discussed the capacity of cystine-rich, whey protein supplement (Immunocal) to enhance the de novo synthesis of GSH in neurons, and highlighted its potential therapeutic use in the mitigation of oxidative damage which forms a key component in the pathogenesis of various neurodegenerative diseases (Ross, Gray, Winter, & Linseman, 2012) .
The increasing knowledge on the impact of whey protein on the levels of GSH, has prompted a number of clinical trials to investigate how this works in various neurodegenerative diseases. An open-label clinical trial was conducted by Kern et al. to investigate the effects of using a cysteine-rich protein isolate in children with autism (Kern, Grannenmann, Gutman, & Trivedi, 2008) . The study was partly based on findings that show low levels of glutathione in children suffering autism, and that non-denatured whey protein could positively impact on glutathione levels (Kern, Grannenmann, Gutman, & Trivedi, 2008) . A preliminary investigation showed that many children with autism could easily feed on non-denatured whey protein isolate (immunocal) without experiencing any problems (Kern, Grannenmann, Gutman, & Trivedi, 2008) .
During the study, a 6-week open-label trial was conducted , in which 10 children with autism or autism spectrum disorder (ASD), aged between 3-15 years, were supplemented with NWPI (Immunocal) (Kern, Grannenmann, Gutman, & Trivedi, 2008) . Several procedures that measured the frequency and intensity of various side effects were completed at baseline (Kern, Grannenmann, Gutman, & Trivedi, 2008) .The findings of the study showed that seven out of ten children who took the supplement tolerated it well (Kern, Grannenmann, Gutman, & Trivedi, 2008) . Two children were discontinued due to possibe side effects, while another was discontinued due to challenges experienced in the adminstration of the supplement. (Kern, Grannenmann, Gutman, & Trivedi, 2008) . The fiindings of the study suggested that Immunocal can be used in a number of autistic children without raising the risk of possible side effects. However, further investigations were recommended to establish the efficacy in larger populations (Kern, Grannenmann, Gutman, & Trivedi, 2008) .
Other studies have shown that Immunocal may cause the depletion of GSH in cancer cells and, therefore, facilitate chemotherapy. A study conducted by Kennedy et al. in 1995 seemed to suggest that whey protein causes depletion of high glutathione (GSH) levels in tumor cells. The study investigated on the use of whey protein concentrate in the treatment of patients with metastatic carcinoma (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . The study was based on the belief that the high levels of glutathione concentration in tumor cells may have a role in resistance against chemotherapy. According to the study, a number of in-vitro assays showed that concentrations that induce GSH synthesis in normal human cells, a specially prepared whey protein concentrate, Immunocal, caused GSH depletion and, therefore, caused the inhibition of the growth of human breast cancer cells (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . On the basis of this background, five patients suffering from metastatic breast carcinoma, one with metastatic carcinoma of the liver, and one with metastatic carcinoma of the pancreas, were fed on 30 grams of whey protein concentrate daily for a period of six months (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . In six of the patients included in the study, the blood lymphocyte GSH levels were found to be substantially high on the onset, indicating a high tumor GSH levels (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . Two of the patients showed signs of tumor regression, normalization of hemoglobin and peripheral lymphocyte counts and steep drop in the level of GSH in lymphocytes towards normal levels (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . Two patients showed a stabilized tumor and increased hemoglobin levels, while in three of the patients the disease was seen to progress with a trend toward higher levels of GSH in lymphocytes (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) . The results of the study seem to indicate that whey protein concentrate might deplete tumor cells of GSH and render them more vulnerable to chemotherapy (Kennedyy, Konok, Bounous, Baruchel, & Lee, 1995) .
Another study was conducted by Tsai et al. to investigate on the effect of patented whey protein isolate (Immunocal) on cytotoxicity of an anticancer drug (2000) . The human hepaloma cell line Hep G2 were left to grow in different media for four days, and cell apoptosis used to investigate the enhancing effect of whey protein on the cytotoxicity of a potential anticancer drug, baicalein (Tsai, Chang, Chen, & Lu, 2000) . The control groups were cultured in normal medium, while the other three groups were grown in Immunocal medium, baicalein medium, and a combination of Immunocal and baicalein (Tsai, Chang, Chen, & Lu, 2000) . The results showed that survival rates were much lower in cells grown in Immunocal and baicalein as compared to those grown in baicalein alone (Tsai, Chang, Chen, & Lu, 2000) . However, no significant differences were observed in the survival rates of cells grown in Immunocal. The researchers also demonstrated that immunocal reduced glutathione (GSH) in Hep G2 cells by a margin of between 20-40% and controlled the elevation of GSH, which was in response to baicalein (Tsai, Chang, Chen, & Lu, 2000) . The researchers concluded that Immunocal enhanced cytotoxicity of baicalein, and the increase in apoptotic cells was linked to the depletion of GSH in Hep G2 cells (Tsai, Chang, Chen, & Lu, 2000) .
Other studies have looked into the effect of Immunocal on glutathione levels in some disease states. A study was conducted by Grey et al. to establish how whey protein supplementation impacted on the status of glutathione in young adult patients suffering from cystic fibrosis (2003) . The study was based on the background that the lung disease of cystic fibrosis is associated with a chronic inflammatory reaction and an over abundance of oxidants to relative antioxidants and, therefore, glutathione was crucial in the prevention of oxidants build-up in the lungs (Grey, Mohammed, Smountas, Bahlool, & Lands, 2003) . Twenty-one patients in stable condition were randomly administered with 10 g of Immunocal twice a day or a casein placebo for period of three months. Peripheral GSH was then utilized as a marker for lung GSH (Grey, Mohammed, Smountas, Bahlool, & Lands, 2003) . At baseline, the researchers did not find any significant differences in age, height, weight, percent body weight or percent ideal height, however, a 46.6% was observed following supplementation(Grey, Mohammed, Smountas, Bahlool, & Lands, 2003) . The findings indicate that dietary supplementation with a whey-based product can enhance glutathione levels in cystic fibrosis (Grey, Mohammed, Smountas, Bahlool, & Lands, 2003) .
A study conducted by Micke et al. investigated on the effects of long-term supplementation with whey proteins on plasma glutathione levels in HIV-infected patients (2002) . The study was based on the background that HIV infections are characterized by an enhanced oxidant burden and systematic deficiency of glutathione (GSH), a key anti-oxidant (Micke, Beeh, & Buhl, 2002) . During the study, up to 30 patients were randomized to a daily dose of 45 g whey proteins of either Protectamin or Immunocal for a period of two weeks. In the patients, the pre-therapy GSH levels were lower than normal, however, following two weeks of whey supplementation, plasma total GSH levels increased in the Protectamin group by 44+/- 56%, while the difference in the Immunocal group remained below significance (Micke, Beeh, & Buhl, 2002) . As a result, all patients in the trial were switched to Protectamin, and six month analysis showed that the total GSH plasma levels were significantly elevated (Micke, Beeh, & Buhl, 2002). The study established that supplementation with whey proteins persistently led to increased plasma levels of GSH in patients with advanced HIV infection (Micke, Beeh, & Buhl, 2002) .
A study was carried out by Watanabe et al to investigate nutritional therapy of chronic hepatitis by non-heated whey protein (2000) . The clinical efficacy of whey (Immunocal) was investigated in 25 patients with chronic hepatitis B or C (Watanabe, et al., 2000) . Casein was given for two weeks prior to Immunocal supplementation. The supplement was provided twice a day, both in the morning and evening for a period of 12 weeks. Investigations showed that Serum alanine aminotransferase (ALT) activity was reduced, while glutathione levels increased in six and five of eight patients diagnosed with chronic hepatitis B respectively (Watanabe, et al., 2000) . The findings of this study suggest that long-term supplement with Immunocal might be effective in enhancing liver functions in patients with chronic hepatitis (Watanabe, et al., 2000) .
Apart from the glutathione use in enhancing the fight against disease states, the oxidant plays a key role in athletic activities. Athletes use muscles more and, therefore, generate a lot of free radicals. Glutathione plays a key role in the elimination of such radicals. A study conducted by Lands et al investigated on the effect of supplementation with a cysteine donor on muscular performance (1999) . The study was based on the knowledge oxidative stress contributes to muscular fatigue, and glutathione is the major intracellular antioxidant that is dependent on the availability of cysteine (Lands, Grey, & Smountas, 1999) . They hypothesized that by giving a whey based supplement ( Immunocal) that is designed to augment intracellular GSH, performance would be improved (Lands, Grey, & Smountas, 1999) . Twenty healthy young adults were studied both pre and post-supplementation with either Immunocal or placebo (Lands, Grey, & Smountas, 1999) . The results showed that GSH increased significantly in the immunocal group, with no change in the placebo group (Lands, Grey, & Smountas, 1999) .
Glutathione has been established as one of the most important intracellular antioxidant. Whey protein provides cysteine, which is critical in the biosynthesis of glutathione. The studies reviewed indicate that whey protein plays a positive role in enhancing glutathione levels both in healthy and various disease states.
Baruchel, S., & Viau, G. (1996). In vitro selective modulation of cellular glutathione by a humanized native milk milk protein isolate in normal cells and rat mammary carcinoma model. Anticancer Res , 16 (3A): 1095-9.
Grey, V., Mohammed, S. R., Smountas, A. A., Bahlool, R., & Lands, L. C. (2003). Improved glutathione status in young adult patients with cystic fibrosis supplemented with whey protein. J Cyst Fibrosis , 2(4):195-8.
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