Medical Hypotheses (1999) 53(4): 347-349 - Ó1999 Harcourt Publishers Ltd. - 
Article No. mehy. 1998.0780

Competition For Glutathione Precursors Between The Immune System And The Skeletal Muscle:  Pathogenesis Of Chronic Fatigue Syndrome

G. Bounous¹, J Molson²

¹Former Professor, Department of Surgery, McGill University, and career Investigation 
of the Medical Research Council of Canada²1994 Quebec Cycling Champion. Road and Time Trial

Summary - The chronic fatigue syndrome (CFS) is typically associated or follows a recognized or presumed infection.  Abnormalities of both humoral and cellular immunity have been demonstrated in a substantial proportion of patients with CFS. The most consistent findings are of impaired lymphocyte responses to mitogen. As an antioxidant, glutathione (GSH) is essential for allowing the lymphocyte to express its full potential without being hampered by oxiradical accumulation. Hence, protracted challenge of the immunocytes may lead to cellular GSH depletion. Because GSH is also essential to aerobic muscular contraction, an undesirable competition for GSH precursors between the immune and muscular systems may develop.  It is conceivable that the priority of the immune system for the survival of the host has drawn to this vital area the ever-diminishing GSH precursors, thus depriving the skeletal muscle of adequate GSH precursors to sustain a normal aerobic metabolism resulting in fatigue and eventually myalgia. © 1999 Harcourt Publishers Ltd. 

The Use of a Whey Protein Concentrate in the Treatment of Patients with metastatic Carcinoma: A Phase I-II Clinical Study

ABSTRACT -  Glutathione (GSH) concentration is high in most tumor cells and this may be an important factor in resistance to chemotherapy.  Previous in-vitro and animal experiments have shown a differential response of tumor versus normal cells to various cysteine delivery systems.  More specifically, an in-vitro assay showed that at concentrations that induce GSH synthesis in normal human cells, a specially prepared whey protein concentrate, [Immunocal]™, caused GSH depletion and inhibition of proliferation in human breast cancer cells.  On the basis of this information five patients with metastatic carcinoma of the breast, one of the pancreas and one of the liver were fed 30 grams of this whey protein concentrate daily for six months.  In six patients the blood lymphocyte GSH levels were substantially above normal at the outset, reflecting high tumor GSH levels.  Two patients (#1, #3) exhibited signs of tumor regression, normalization of haemoglobin and peripheral lymphocyte counts and a sustained drop of lymphocyte GSH levels towards normal.  Two patients (#2, #7) showed stabilization of the tumor, increased haemoglobin levels.  In three patients (#4, #5, #6) the disease progressed with a trend toward higher lymphocyte GSH levels.  These results indicate that whey protein concentrate might deplete tumor cells of GSH and render than more vulnerable to chemotherapy.

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ABSTRACT – On the basis of numerous animal experiments, a pilot study was undertaken to evaluate the effect of undenatured, biologically active, dietary whey protein in 3 HIV-seropositive individuals over a period of 3 months.  Whey protein concentrate was prepared so that the most thermosensitive proteins, such as serum albumin which contains 6 glutamylcysteine groups, would be in undenatured form.  Whey protein powder dissolved in a drink of the patient’s choice was drunk cold in quantities that were increased progressively from 8.4 to 39.2 g per day.  Patients took whey proteins without adverse side effects.  In the 3 patients whose body weight had been stable in the preceding 2 months, weight gain increased progressively between 2 and 7 kg, with 2 of the patients reaching ideal body weight.  Serum proteins, including albumin, remained unchanged and within normal range, indicating that protein replenishment per se was not likely the cause of increased body weight.  The glutathione content of the blood mononuclear cells was, as expected, below normal values in all patients at the beginning of the study.  Over the 3-month period, GSH levels increased and in one case rose by 70% to reach normal value.  The increase in body weight observed in these patients did not correlate with increase in energy or protein intake.

In conclusion, these preliminary data indicate that, in patients who maintain an adequate total caloric intake, the addition of “bioactive” whey protein concentrate as a significant portion of total protein intake increases body weight and shows elevation of glutathione (GSH) content of mononuclear cells toward normal levels.  This pilot study will serve as a basis for a much larger clinical trial 

ABSTRACT – This study compared the effects of different sources of whey protein concentrate (20 g/100 g diet) and of casein on the spleen, liver, and heart glutathione content of C3H/HeJ mice, and on the immune response of their spleen cells to sheep red blood cells.  Body weight curves were similar in all dietary groups.  Our data indicate that the humoral immune response is highest in mice fed a dietary whey protein concentrate exhibiting the highest solubility (undenatured conformation) and a greater relative concentration of the thermolabile cystine rich proteins.  In addition, the mice fed this type of whey protein concentrate exhibit higher levels of tissue glutathione.  The presence in the serum albumin fraction of glutamylcysteine groups (rare in food protein) and the specific intramolecular bond as related to the undenatured conformation of the molecule are considered to be key factors in the glutathione-promoting activity of the protein mixture.

ABSTRACT – Epidemiological and experimental studies suggest that dietary milk products may exert an inhibitory effect on the development of several types of tumors.  Some recent experiments in rodents indicate that the antitumor activity of the dairy products is in the protein fraction and more specifically in the whey protein component of milk.  We and others have demonstrated that whey protein diets result in increased glutathione (GSH) concentration in a number of tissues, and that some of the beneficial effects of whey protein intake are abrogated by inhibition of GSH synthesis.  Whey protein is particularly rich in substrates for GSH synthesis.  We suggest that whey protein may be exerting its effect on carcinogenesis by enhancing GSH concentration.

ABSTRACT – This study investigated the influence of two formula diets containing 20 g/100 g diet of either whey protein concentrate or casein, or Purina mouse chow on 1,2dimethylhydrazine (DMH)-induced colon carcinoma in A/J mice.  Four weeks after the 24^th DMH treatment the incidence of tumour and tumour area in the whey protein-fed mice was substantially less in comparison to either the casein or Purina groups.  The Purina group exhibited the greatest tumour burden.  At the end of the experiment all animals continuously fed the whey protein diet were found to be alive, whereas 33% of those on the casein or Purina diet had died.  Animals fed Purina diet for 20 weeks and then switched to either milk protein diet for a further 8 weeks exhibited a decrease in tumour burden as compared to those animals fed the Purina diet continuously.  Body weights were similar in all dietary groups.  In conclusion, a whey protein diet appears to significantly influence the development of chemically induced colon tumours and the short-term survival of mice.

ABSTRACT – This study compared the effects of a whey-rich diet (20 g / 100 g diet), with that of Purina mouse chow or casein-rich diet (20 g / 100 g diet), on the liver and heart glutathione content and on the survival of old male C57BL / 6 NIA mice.  The study was performed during a limited observation period of 6.3 months.  In mice fed the whey protein-rich diet between 17 months and 20 months of age, the heart tissue and liver tissue glutathione content were enhanced above the corresponding values of the casein diet-fed and Purina-fed mice.  Mice fed the whey protein diet at the onset of senescence, exhibited increased longevity as compared to mice fed Purina mouse chow over the 6.3 month observation period extending from the age of 21 months (corresponding to a human age of 55 years) to 26-27 months of age (corresponding to a human age of 80 years), during which time 55% mortality was observed.  The corresponding mean survival time of mice fed the defined casein diet is almost identical to that of Purina-fed controls.  Body weight curves were similar in all three dietary groups.  Hence, a whey protein diet appears to enhance the liver and heart glutathione concentration in aging mice and to increase longevity over a 6.3 month observation period.

ABSTRACT – The spleen cells immune response to sheep red blood cells of C3H/HeJ mice fed a 20 g whey protein/100 g diet is substantially higher than that of mice fed an equivalent casein diet of similar nutritional efficiency.  The present study indicates that the observed immunoenhancing effect of the whey protein mixture is dependent on the overall amino acid pattern resulting from the contribution of all its protein components.  Whey protein contains substantially more cysteine than casein.  Dietary cysteine is considered to be a rate limiting substrate for the synthesis of glutathione which is necessary for lymphocyte proliferation.  Our studies show that enhancement of host humoral immune response is associated with greater and more sustained production of splenic glutathione during the antigen driven clonal expansion of the lymphocyte in whey protein fed mice in comparison to mice fed the equivalent casein or the cysteine-enriched casein diet.  Hence the efficiency of dietary cysteine in inducing supernormal glutathione levels is greater when it is delivered in the whey protein than as free cysteine.  Administration of S-(n-butyl) homocysteine sulfoximine, which reduces splenic glutathione level by half, produces  a 4-5 fold drop in the humoral immune response of whey protein diet-fed mice.  This is further evidence of the important role of glutathione in the immunoenhancing effect of dietary whey protein.

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ABSTRACT - The plaque-forming cell response to sheep red blood cells was found to be enhanced in mice fed a formula diet containing 20 g lactalbumin /100 g diet in comparison to mice fed equivalent formula diets of similar nutritional efficiency containing 20 g / 100 g diet of either casein, soy, wheat or corn protein, egg albumin, beef or fish protein, Spirulina maxima, or Scenedesmus protein, or Purina mouse chow.  This effect was manifest after 2 weeks and persisted for at least 8 weeks of dietary treatment. Mixing lactalbumin with either casein or soy protein in a 20 g protein / 100 g diet formula significantly enhanced the immune response in comparison to that of mice fed diets containing 20% soy protein or casein.

ABSTRACT – This study investigates the influence of two formula diets containing 20 g/100 g diet of either whey protein concentrate or casein or Purina mouse chow, on the humoral immune responsiveness and dimethylhydrazine induced colon carcinogenesis in A/J mice.  After 20 weeks of dimethylhydrazine treatment, the number of plaque forming cells per spleen, following intravenous inoculation with 5 x 10⁶ sheep red blood cells, was nearly three times greater in the whey protein-fed group than in the casein-fed mice although both values were substantially below normal.  After 24 weeks of dimethylhydrazine treatment the incidence of tumors in the whey protein-fed mice was substantially lower than that in mice fed either the casein or Purina diet.  Similarly, the tumor area was less in the whey protein group in comparison to either the casein or Purina groups, with some difference between casein and Purina groups.  Body weight curves were similar in all dietary groups.

In conclusion, a whey protein diet appears to significantly inhibit the incidence and growth of chemically induced colon tumors in mice.

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ABSTRACT – The effect of 20 g/100 g dietary lactalbumin (L) or casein (C) diets or a nonpurified (NP) diet on the immune responsiveness of C57B1/6J, C3H/HeJ and BALB/cJ mice has been investigated by measuring the response to the T cell-independent antigen, TNP-Ficoll.  To investigate the possible influence of dietary protein type on the supply of B lymphocytes, bone marrow lymphocyte production has been examined by a radioautographic assay of small lymphocyte renewal and an immuno-fluorescent stathmokinetic assay of pre-B cells and their proliferation.  The humoral response of all mice fed the L diet was found to be higher than that of mice fed the C diet or non purified diet.  A similar pattern of dietary protein effect in (CBA/N x DBA/2J) F₁ mice carrying the xid defect was observed following challenge with sheep red blood cells (SRBC).  An even greater enhancing effect of dietary L was noted in normal (DBA/2J x CBA/N) F₁ mice after immunization with SRBC, but in contrast, the normal large-scale production of B lymphocytes in mouse bone marrow was independent of the type of dietary protein.  Dietary protein type did not affect blood level of minerals and trace metals.  The free plasma amino acid profile essentially conformed to the amino acid composition of the ingested protein, suggesting that the changes in plasma amino acid profile might be a crucial factor in diet-dependent enhancement or depression of the B-cell response.  The findings indicate that the observed effects of altered dietary protein type on humoral immune responsiveness are not exerted centrally on the rate of primary B-lymphocyte production in the bone marrow, but may reflect changes either in the functional responsiveness of the B lymphocytes themselves or in the processes leading to their activation and differentiation in the peripheral lymphoid tissues.

ABSTRACT – The effect of 20 g/100 g diet of lactalbumin (L), casein (C), soy (S) and wheat (W) protein on the immune responsiveness of C3H/HeN mice has been investigated by measuring the humoral immune response to the T cell-independent antigen, TNP-Ficoll.  The humoral immune response of mice fed the L diet was found to be higher than that of mice fed the C, S and W diets.  On the other hand, delayed-type hypersensitivity, and splenic cell mitogen responses to phytohemagglutinin and concanavalin A did not differ among mice fed the various diets.  Similarly, the type of diet did not appear to influence host resistance to Salmonella typhymurium.  It is postulated that the type of protein in the diet influences directly the intrinsic capacity of the B lymphocytes to respond to an immunogenic stimulus.

ABSTRACT – The effect of graded amounts of dietary lactalbumin (L), casein (C), soy (S), wheat (W) protein and Purina rodent chow (stock diet) on the immune responsiveness of C3H/HeN mice has been investigated by measuring the specific humoral immune response to sheep red blood cells (SRBC), and horse red blood cells (HRBC) as well as the nonspecific splenic cell responsiveness to phyto-hemagglutinin (PHA) and concanavalin A (Con A) after stimulation with Myco-bacterium bovis, strain BCG.  The nutritional efficiency of these diets was normal and similar.  The immune response of mice fed the L diets, was found to be almost five times higher than that of mice fed the corresponding C diets.  The humoral immune response of mice fed C, S, and W diets was substantially lower than that of mice fed stock diet, whereas that of mice fed L diet was higher.  The above-described immune effect of all tested proteins was obtained at 20 g/100 g concentration with no further increments with 30- and 40 g/100 g protein in the diet.  Mitogen responsiveness to PHA and Con A in L diet-fed mice was only slightly higher than that of C diet-fed mice.  Little difference in immune responses was noted among mice fed C, S or W protein diets.  The principal factor responsible for the observed immune effect does not appear to be the availability or concentration of single essential amino acids but rather the composite effect of the specific amino acid distribution in the protein.

A whey protein diet has been shown to enhance splenic immune response to sheep red blood cells (SBRC) in mice.  This study was designed to investigate the influence of the type of dietary protein on the biliary secretory IgA.  A/J mice were fed defined formula diets containing either 20% whey protein, or 20% casein.  Another group was fed Purina mouse chow.  After 3 weeks of dietary treatment the body weight of each mouse was recorded and the gall-bladder was removed and its whole content analyzed by ELISA to determine S-IgA secretion.  Body weight curves were similar in all dietary groups; higher biliary levels of S-IgA appeared in the whey protein fed mice than in the casein (p less than 0.025) or purine (p less than 0.025) fed mice.  Dietary protein type may have a direct influence on the immune response in the gastrointestinal tract, without affecting body weight.

SUMMARY - Oxidative stress, a known activator of HIV replication in vitro, has a potential role as a cofactor of HIV disease progression. Arguments supporting the role of oxidative stress as a cofactor in HIV activation are summarized in this review. The role of intracellular antioxidants such as glutathione (GSH), and drugs and nutriceutical agents promoting GSH synthesis, are discussed. The review also includes the early results of nutritional interventions based on a diet enriched with [Immunocal], a whey protein concentrate prepared in a proprietary manner.

ABSTRACT - The effect of graded amounts of dietary laetalbumin (L) and casein (C) hydrolyzates on the immune responsiveness of C3H/HeN and DBA/2 strain mice has been investigated by measuring both the specific humoral immune response to sheep red blood cells (SRBC) and the nonspecific splenic cell responsiveness to phytohemagglutinin, concanavalin A and Escherichia coli lipopolysaccharide after stimulation with Mycobacteriurn bovis, strain BCG. The nutritional efficiency of these diets was similar at both 12 and 28% amino acid levels. The immune responses of mice fed the L diets were found to be significantly greater than those of mice fed the corresponding C diets, especially at the 28% level. Furthermore in the mice fed L diet, increasing the concentration of amino acid in the diet from 12 to 28% greatly enhanced immune responsiveness by both parameters measured. In the C-fed mice, a comparable enhancement of mitogen responsiveness with increasing amino acid level of diet was seen, but there was no change in the humoral immune response. The enhancement of immune responsiveness observed in mice fed the 28% L diet was moderately reduced by the addition of phenylalanine to the diet, indicating that the lower level of this amino acid in the L protein may be of some significance. These dietary effects on immune responsiveness were remarkably similar in both mouse strains tested.

ABSTRACT – In the present study we investigated the effect of four weeks of treatment with a diet containing lactalbumin hydrolysate (LAH: Nestlé, Vevey, Switzerland) on the immune response of C3H/HeN mice.  Our data indicate that it was possible to increase the level of this type of protein in the diet above the minimum requirement (12% LAH) and thus produce augmented humoral immune responsiveness and resistance to salmonellosis. 

Lactalbumin = Whey Protein Concentrate

ABSTRACT: Oxidative stress contributes to muscular fatigue.  Glutathione (GSH) is the major intracellular antioxidant, whose biosynthesis is dependent upon cysteine availability.  We hypothesized that supplementation with a whey-based cysteine donor ([Immunocal] (HMS90)) designed to augment intracellular GSH, would enhance performance.  Twenty healthy young adults (10 m) were studied pre- and 3 months post-supplementation with either [Immunocal] (20 gm/day) or casein placebo.  Muscular performance was assessed by whole leg isokinetic cycle testing, measuring Peak Power and 30-sec Work Capacity.  Lymphocyte GSH was used as a marker of tissue GSH.  There were no baseline differences (age, ht, wt, % ideal wt, Peak Power, 30-sec Work Capacity).  Follow-up data on 18 subjects (9 [Immunocal], 9 placebo) were analyzed.  Both Peak Power (mean±se: 13±3.5%, p<0.02) and 30-sec Work Capacity (13±3.7%, p<0.03) increased significantly in the [Immunocal] group, with no change (2±9.0 and 1±9.3%) in the placebo group.  Lymphocyte GSH also increased significantly in the [Immunocal] group (35.5±11.04%, p<0.02) with no change in the placebo group (-0.9±9.6%).  This is the first study to demonstrate that prolonged supplementation with a product designed to augment antioxidant defenses resulted in improved volitional performance.

Treatment Of Obstructive Airway Disease With A Cysteine 
Donor Protein Supplement: A Case Report

ABSTRACT - Oxidant/antioxidant imbalance can occur in obstructive airways disease, as a result of ongoing inflammation.  Glutathione plays a major role in pulmonary antioxidant protection.  As an alternative or complement to anti-inflammatory therapy, augmenting antioxidant protection could diminish the effects of inflammation.  We describe a case of a patient with obstructive lung disease, responsive to corticosteroids, with low whole blood glutathione levels.  Following one month of supplementation with a whey-based oral supplement, designed to provide glutathione precursors, whole blood glutathione levels and pulmonary function significantly and dramatically increased.  The potential for such supplementation in pulmonary inflammatory conditions deserves further study.

In an open study, the clinical efficacy of whey protein ([Immunocal]: cysteine content; 7.6-fold that of casein) isolated from fresh milk and purified without being heated was evaluated based on liver function test, immunological parameters, plasma or lymphocyte GSH concentrations and hepatitis virus markers in 25 patients with chronic hepatitis B or C. [Immunocal] (12 g as protein) food (mousse) was given twice a day, in the morning and evening, for 12 weeks (test period).  Casein (12 g as protein) food (mousse) was given for 2 weeks prior to the start of -supplement with [Immunocal] food (induction period) and for 4 weeks after the end (follow-up period). The effects of [Immunocal] food on various clinical parameters were examined at 4-week intervals for 18 weeks to evaluate the efficacy of [Immunocal]. As a result, serum ALT activity decreased in 6 of 8 patients with chronic hepatitis B 12 weeks after the start of supplement with [Immunocal] food. Plasma GSH concentrations were increased in 5 of the 8 patients. Serum . concentrations of lipid peroxides significantly decreased 8 weeks after [Immunocal] food. Serum IL-2 levels began to increase 8 weeks and remained high even after supplement with [Immunocal] -food had ended. Furthermore, NK activity was significantly increased. However, an item correlating with reduced serum ALT activity could not be clarified. In 17 patients with chronic hepatitis C, there wore no significant [Immunocal]-related changes in liver function test or immunological parameters. These findings suggest that long-term supplement with [Immunocal] alone may be effective for patients with chronic hepatitis B, and a further clinical study that long-term combination therapy with [Immunocal] and other agents including interferon may be effective for those with chronic hepatitis C should be performed.

ABSTRACT - The glutathione (GSH) antioxidant system is foremost among the cellular protective mechanisms.  Depletion of this small molecule is a common consequence of increased formation of reactive oxygen species during increased cellular activities.  This phenomenon can occur in the lymphocytes during the development of the immune response and in the muscular cells during strenuous exercise.  It is not surprising that so much research has been done, and is still being done on this small tripeptide molecule. Whey protein concentrate has been shown to represent an effective and safe cysteine donor for GSH replenishment during GSH depletion in immune deficiency states.  Cysteine is the crucial limiting amino acid for intracellular GSH synthesis.  Animal experiments showed that the concentrates of whey proteins also exhibit anti-carcinogenesis and anticancer activity.  They do this via their effect on increasing GSH concentration in relevant tissues, and may have anti-tumor effect on low volume of tumor via stimulation of immunity through the GSH pathway.  It is considered that oxygen radical generation is frequently a critical step in carcinogenesis, hence the effect of GSH on free radicals as well as carcinogen detoxification, could be important in inhibiting carcinogenesis induced by a number of different mechanisms.  Case reports are presented which strongly suggest an anti-tumor effect of a whey protein dietary supplement in some urogenital cancers.  This non toxic dietary intervention, which is not based on the principles of current cancer chemotherapy, will hopefully attract the attention of laboratory and clinical oncologists.

ABSTRACT – To determine the enhancing effect of a whey protein isolate on the cytotoxicity of a potential anti-cancer drug. baicalein, human hepatoma cell line HepG2 was assigned to grow in different media for four days, followed by the investigation of cell growth and apoptosis. Excluding the control group with normal medium, other three treatment media included whey protein isolate (marketed as [Immunocal]) medium, baicalein medium, and combined medium containing both Irnmunocal and baicalein.  MTT assay indicated that cells grew in combined medium had a significantly lower survival rate compared to the cells grew in baicalein medium; in contrast, for the cells grew in [Immunocal] group, there was no significant difference on survival rate. In the investigation of apoptosis. compared to the cells in baicalein medium, cells in combined medium showed a higher phosphatidylserine exposure, lower rnitochondrial transmembrane potential and nearly 13 times more cells were detected undergoing apoptosis. We also demonstrated that [Immunocal] was able to reduce glutathione in HepG2 by 20% to 40% and regulated the elevation of glutathione, which was in response to baicalein. In conclusion, [Immunocal] seemed to enhance the cytotoxicity of baicalein by inducing more apoptosis, this increase in apoptotic cells may be in association with the depletion of GSH in HepG2. This is the first study to demonstrate, in vitro, that [Immunocal] may function as an adjuvant in cancer treatments. 

ABSTRACT – The biological activity of the proteins isolated from cow’s milk in [Immunocal] depends on the preservation of those labile proteins which share with the predominant human milk proteins the same extremely rare glutathione (GSH)-promoting components.  Cellular GSH depletion has been implicated in the pathogenesis of a number of degenerative conditions and disease states including Parkinson’s, Alzheimer’s, arteriosclerosis, cataracts, cystic fibrosis, malnutrition, aging, AIDS, and cancer.

This newly discovered nutriceutical modulation of GSH by the use of humanized native milk serum protein isolate of bovine origin in AIDS and cancer may well find other applications in disease where oxidative stress and pathology of GSH metabolism are largely implicated.  In a pilot study, this type of whey protein concentrate was found to be well tolerated in children with AIDS and wasting syndrome and was found associated with an improvement of the nutritional status of the patient.  Moreover, the GSH promoting activity on the peripheral blood lymphocyte of this protein concentrate was validated in patients with initial low GSH levels.  Extensive pharmaco-epidemiological study of GSH metabolism and standardized methods of measurement of intracellular GSH applicable in clinical trials are needed in order to better define the clinical application of this new type of therapy.