United States Patent 5,230,902
Gold , et al. July 27, 1993
Undenatured whey protein concentrate to improve active systemic humoral immune response
This invention provides a method of improving the humoral immune response or
increasing the concentration levels of glutathione in mammals, which comprises
administering orally to a mammal a therapeutically or a prophylactically
effective amount of undenatured whey protein concentrate which has a biological
activity based on the overall amino acid and associated small peptides pattern
resulting from the contribution of all its protein components. A method for
improving the humoral immune response in mammals also is disclosed which
comprises administering orally to a mammal the combination of a vitamin
supplement containing vitamin B.sub.2 in an amount in excess of minimum daily
requirements and an effective amount of undenatured whey protein concentrate.
This invention further provides a dietary supplement for a mammal which
comprises an effective amount of vitamin B.sub.1, and B.sub.2 and a
therapeutically or prophylactically effective amount of whey protein supplement.
Inventors: Gold; Phil (Westmount, CA), Bounous; Gustavo (Montreal, CA),
Kongshavn; Patricia A. L. (St. Lambert, CA)
Assignee:Immunotec Research Corporation (Montreal, CA)
Appl. No.: 07/563,794
Filed: August 3, 1990
Related U.S. Patent Documents
Application NumberFiling DatePatent NumberIssue Date
Current U.S. Class:424/535 ; 514/2; 530/365; 530/833
Current International Class: A23L 1/305 (20060101); A61K 35/20 (20060101);
A61K 38/17 (20060101); A61K 035/20 ()
Field of Search: 514/2,21,251,276,885 530/365,833 424/535 426/72
References Cited [Referenced By]
U.S. Patent Documents
4710387December 1987Uiterwaal et al.
4753926June 1988Lucas et al.
Foreign Patent Documents
Horwitt, M. K. in The Vitamins, 2d ed., vol. V, Academic Press, New York,
1972, pp. 88-96. .
Jansen, B. C. P. in The Vitamins, 2d ed., vol. V., Academic Press, New
York, 1972, pp. 156-164. .
Bounous et al. J Nutr 112(9):1747-1755 (1982). .
Bounous et al. J Nutr 113:1415-1421 (1983). .
Bounous et al. J Nutr 115(11):1409-1417 (1985)..
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Witz; Jean C.
Attorney, Agent or Firm: White; John P. Arnold; Craig J.
Parent Case Text
This application is a continuation of U.S. application Ser. No. 289,971, filed
Dec. 23, 1988, now abandoned, which is a continuation in part of U.S.
application Ser. No. 188,271 filed Apr. 27, 1988, now abandoned.
1. A method of improving the active systemic humoral immune response in a mammal
as measured by sheep red blood cell injection (SRBC), comprising administering
orally to the mammal an effective amount of undenatured whey protein concentrate
(WPC) obtained from bovine, goat or sheep milk and containing substantially all
the whey protein present in the raw milk, administered as a daily replacement
for up to all the protein consumed by the mammal, wherein the improved active
systemic humoral immune response is based on the overall amino acid and
associated small peptides pattern resulting from the contribution of
substantially all of the WPC protein components, the daily amount of WPC not
substantially exceeding the daily protein requirement for the mammal.
2. The method of claim 1, wherein the active systemic humoral immune response in
the mammal is characterized by a dose-response pattern with relation to the
amount of whey protein concentrate intake by the mammal.
3. The method of claim 1, wherein the improved active systemic humoral immune
response is enhanced resistance to pneumococcal infection.
4. The method of claim 1, wherein the whey protein concentrate is undenatured
bovine whey protein concentrate.
5. The method of claim 1, wherein the whey protein concentrate is a mixture of
bovine, goat or sheep whey protein concentrate.
6. The method of claim 1, wherein the improved active systemic humoral immune
response is associated with enhanced resistance of target cells against the
mutagenic and carcinogenic effect of dimethylhydrozine.
7. The method of claim 1, wherein the active systemic humoral immune response is
measured in splenic lymphocytes during the SRBC driven lymphocyte response.
8. The method of claim 1, wherein the whey protein concentrate has the amino
acid composition of
9. The method of claim 1, which further comprises orally administering to the
mammal about 1.2 to about 1.5 milligrams per day of vitamin B2.
10. The method of claim 1, which further comprises orally administering to the
mammal about0.5 to 0.6 milligrams of vitamin B2 pre 1000 calorie per day.
11. The method of claim 1, which further comprises orally administering to the
mammal about 0.5 milligrams per 1000 calorie of vitamin B1 per day.
12. The method of claim 1, which further comprises orally administering to the
mammal vitamins B1 and B2 in amounts in excess of the minimum daily requirement
for the mammal.
13. A method of producing a sustained increase of tissue concentration level of
glutathione in a mammal, which comprises orally administering to the mammal an
amount of undenatured whey protein concentrate obtained from bovine, goat or
sheep milk and containing substantially all the whey protein present in the raw
milk as a daily replacement for up to all the protein consumed by the mammal,
wherein the sustained increase of the tissue concentration level of glutathione
in the mammal is based on the overall amino acid and associated small peptides
pattern resulting from the contribution of substantially all the WPC protein
components, the daily amount of whey protein concentrate not substantially
exceeding the daily protein requirement for the mammal.
14. The method of claim 13, wherein the sustained increase of tissue
concentration level of glutathione in the mammal is characterized by a
dose-response pattern with relation to the amount of whey protein concentrate
intake by the mammal.
15. The method of claim 13, which further comprises orally administering to the
mammal vitamin B2 in an amount of about 0.5 to 0.6 milligrams per 1000 calorie
16. The method of claim 13, wherein the sustained increase of tissue
concentration levels of glutathione in the mammal comprises an elevated level of
glutathione above normal levels for at least three (3) months.
1. AREA OF INVESTIGATION
Effect of dietary whey protein on the immune response to sheep red blood cells,
host resistance to bacterial infections, development of tumors, and the process
Whey and whey protein have been utilized from time immemorial for nutritional
purpose. In addition, whey was recommended in folk and ancient medicine for the
treatment of various diseases and, in one instance, lifetime feeding of hamsters
with a whey protein diet has been shown to promote longevity with no explanation
given. We have shown, in controlled experiments, for the first time, that whey
protein feeding specifically enhances mice immune response to sheep red blood
cells (SRBC), resistance to pneumococcal infection, inhibits the development of
colon cancer and delays the process of aging independently of its nutritional
The search for the possible mechanism of immunoenhancement by whey protein
feeding has revealed to us the provocative possibility that whey protein may
contribute to a broader biological effect of a protective nature involving
susceptibility to cancer, diseases of aging and general detoxification of
environmental agents. All these conditions appear to be somehow related to
changes in glutathione which is a ubiquitous element exerting a protective
effect against superoxide radicals and other toxic agents. Our studies have
shown that the observed enhancement of the immune response is associated with
greater production of splenic glutathione in immunized mice fed whey protein in
comparison to mice fed a casein or cysteine enriched casein diet. The efficiency
of dietary cysteine in inducing supernormal glutathione levels is greater when
it is delivered in the whey protein than as free cysteine. Glutathione was found
at higher levels in the heart and liver of whey protein fed old mice in
comparison to mice fed the corresponding casein diet or Purina Mouse Chow. In
old mice dietary whey protein was found to delay the onset of the diseases of
(a) Whey Protein:
Whey proteins are the group of milk proteins that remain soluble in "milk serum"
or whey after precipitation of caseins at ph 4.6 and 20.degree. C. The major
whey proteins in cow's milk are beta-lactoglobulin (.beta.L), alpha-lactalbumin
(.alpha.L), immunoglobulin and serum albumin (SA) in order of decreasing
(c) SRBC=Sheep red blood cells;
(d) PFC Plaque forming cells (spleen): enumeration of PFC in spleen is used to
assess the humoral immune response to SRBC injection;
(e) GSH Glutathione (.gamma.-glutamyl-cysteinyl-glycine); and
(f) Unless otherwise specified, the defined formula diets tested varied only in
the type or protein.
(g) Whey of bovine milk contains approximately 6 g per liter protein, most of
the lactose, mineral and water soluble vitamins.
A suitable source of whey protein concentrate is the material known by the trade
mark PROMOD, which is a protein supplement provided in powder form by Ross
Laboratories, a Division of Abbott Laboratories U.S.A. This is a concentrated
source of high quality protein which is useful for providing extra protein to
persons having increased protein needs, or those who are unable to meet their
protein needs with their normal diet. It contains whey protein concentrate and
soy lecithin. It has the following nutrients:
______________________________________ NUTRIENTS: Per 5 g Protein (one scoop)
______________________________________ Protein 5.0 g Fat Does not exceed 0.60 g
Carbohydrate Does not exceed 0.67 g Water Does not exceed 0.60 g Calcium Does
not exceed 23 mg (1.15 mEq) Sodium Does not exceed 13 mg (0.57 mEq) Potassium
Does not exceed 65 mg (1.66 mEq) Phosphorus Does not exceed 22 mg Calories 28
It has the following typical amino acid composition per 100 g protein. 100 g
PROMOD protein yields approximately 105 g of aminoacids.
______________________________________ TYPICAL AMINO ACID COMPOSITION Per 100 g
Protein Essential ______________________________________ Amino Acids: Histidine,
1.9 g; Isoleucine, 6.2 g; Leucine, 10.8 g; Lycine, 9.3 g; Methionine, 2.2 g;
Phenylalanine, 3.6 g; Threonine, 7.3 g; Tryptophan, 1.9 g; Valine, 6.0 g.
Non-Essential Amino Acids: Alanine, 5.3 g; Arginine, 2.6 g; Aspartic Acid, 11.2
g; Cysteine, 2.6 g; Glutamic Acid, 18.2 g; Glycine, 2.1 g; Proline, 6.5 g;
Serine, 5.6 g; Tyrosine, 3.4 g. ______________________________________
The concentration of some vitamins in the defined formula diet used in (most of)
our experiments is given in Table 1 (Diet 1). Diets are prepared in the
following way: 20 g of selected pure protein, 56 g of product 80056 protein free
diet powder (Mead-Johnson Co. Inc., U.S.A.), 18 g cornstarch, 2 g wheat bran;
0.05 g Nutramigen vit-iron premix (Bristol-Myers, Ontario, Canada), 2.65 g KCl;
0.84 g NACl. Unless otherwise specified, the only variable in the various
purified diets was the type of protein
3. PREVIOUS WORK
Dairy products are widely used as a good source of nutrition. In addition claims
have been made to the effect that fermented wholemilk (yogurt) is beneficial in
the management of some types of intestinal infections. Certain dietary regimen
based on ill defined cultured dairy products are said to be associated with long
life expectancy in some regions of the USSR (Georgia etc).
Since time immemorable, serum lactis, latin for milk serum or whey, has been
administered to the sick for the treatment of numerous ailments. In 1603
Baricelli(7) reported on the therapeutic use of cow or goat milk serum,
sometimes mixed with honey or herbs. The spectrum of illness treated with whey
included jaundice, infected lesions of the skin, those of the genito-urinary
tract with purulent secretions, gonorrhea, epilepsy, quartan fever and other
febrile states of different origins. Indeed, the common denominator of most of
these illnesses appears to be a septic condition. Although physicians of both
Ancient Times and of the Middle Ages agreed that whey treatment should be
carried out over a period of several days, a difference of opinion appeared to
exist concerning the daily amount prescribed. Thus, Galen, Hippocrates and
Dioscoride insisted on a minimum daily amount of two 12 oz latin libras, and up
to five libras a day according to gastric tolerance. This would represent
between 1 to 2 liters of whey a day. Baricelli, on the other hand, reflected the
trend of his times, limited the amount prescribed to one libra a day, given in
fractionated doses on an empty stomach.
In the following year, Costaei(7) wrote about the virtues of whey in the
treatment of several unrelated syndromes including broncopneumonitis and
diarrhea with high fever. Unfortunately, in his long dissertation, the author
fails to clearly discriminate between milk and milk serum treatment.
Since then, numerous articles published in Europe throughout the 17th, 18th and
19th centuries have advocated the therapeutic use of whey. In an Italian
textbook published in the middle of the 19th century, at the dawn of scientific
medicine, an interesting distinction is made between whole milk and milk serum.
Milk is recommended firstly as a nutrient especially in patients with strictures
of the gastrointestinal tract. In this respect the author emphasizes that the
benefits of the then popular "milk therapy" of cachexia and tuberculosis are due
only to the nutritional property of milk. Secondly, milk was prescribed in the
treatment of poisoning because milk components would presumably neutralize
ingested toxic material. Thirdly, milk therapy was suggested for the alleged
capacity of this fluid to coat and sooth ulcers of the gastrointestinal tract.
Milk serum, on the other hand, was advocated in treatment of pneumonitis, acute
inflammatory diseases of the intestines and urogenital tract, in spite of its
recognized lower nutritional quality. Finally the author emphasized the
ineffectiveness of whey in the treatment of disorders of the nervous system.(7)
The prime difference between whey, (serum lactis) and whole milk is the near
absence in the former of the caseins, the casein-bound calcium and phosphate,
most of the fat and fat soluble vitamins. The actual concentration in whey of
"whey proteins" is only about 5% higher than that in milk. Hence quantitative
differences between whey and milk could not be construed to represent a key
factor in the alleged therapeutic effect of whey treatment because, if any, they
imply the lack, in whey, of some important nutrients. Our data (7,14) may
provide a scientific background to the presumed benefit of intensive treatment
with "serum lactis".
We have shown the importance of the characteristic amino acid profile of whey
protein concentrate in the immune enhancing effect of WPC. The caseins represent
80% of the total protein content of cow's milk while WPC is only 20%. Hence, it
is conceivable that it is the separation of WPC from the caseins in whey which
represents the crucial qualitative change, since this will render the amino acid
profile of whey proteins unaltered by that of the caseins, once the digestive
process has released free amino acids from all ingested proteins.(7)
Although no clinical trials have been reported, some interesting information can
be gathered from data on human subjects.
Whey Protein in Neonatal Nutrition
Infant feeding studies indicate that whey predominant formulas are metabolically
superior to casein predominant formulas (8, 9) in preterm babies.
Studies performed at the Eppley Cancer Center in Nebraska (10, 11) showed that
survival (resistance to spontaneous diseases) of female and male hamsters,
measured over a 20 week period of feeding from 4 weeks of age, was best with 20
g/100 g (grams per hundred grams) WPC diet, in comparison with a 20 g/100 g
methionine and cysteine supplemented casein diet. Body weight gains were similar
in both groups. In lifetime feeding studies, the mean and maximal longevity of
female and male hamsters fed 10, 20 and 40 g WPC/100 g diet was increased in
comparison with those fed commercial laboratory feed (estimated 24% protein from
various sources). Survival was best with the 20% WPC diet; in males, longevity
increased by 50%. No significant relationship was noted between food intake,
maximal weight and longevity.
4. OUR STUDIES
Our interest in the effect of amino acid intake upon the immune system was
prompted by an observation made several years ago (1). We fed mice a defined
formula diet containing a free amino acid mixture acid mixture duplicating
casein. Another group of mice was fed a similar diet but with moderate
restriction of phenylalanine and tyrosine compensated by a corresponding
increment in the non-essential amino acid mixture. The second group of mice
gained weight at the same rate as the mice fed the casein equivalent mixture of
Purina mouse chow. However, when challenged with sheep red blood cells, these
mice produced more antibodies and plaque forming cells against sheep red blood
cells than Purina or casein equivalent-fed mice.
A new concept thus emerged, namely, that changes in the amino acid profile of
the diet can influence the immune response independently of any systemic effect
on the nutritional status of the host. But, more importantly, changes in the
amino acid profile, i.e. protein type, could conceivably enhance the humoral
immune response beyond that which had traditionally been considered to represent
a "normal" response.
We subsequently assessed the effect on the immune response of different types of
proteins in nutritionally adequate diets. Mice fed formula diets containing 20%
or 28% lactalbumin (WPC) were found to produce more plaque forming cells to
sheep red blood cells than mice fed Purina mouse chow containing about 22%
protein from various sources and of similar nutritional efficiency. The immune
enhancing effect of lactalbumin was maximal at 20% concentration (2). A 20 g net
protein/100 g diet provides a good method to assess the effect of protein type
on the immune system. At this level most protein supplies the minimum daily
requirement of all indispensible amino acids for the growing mouse and this is
important because the amino acid distribution, and not adequacy, is the variable
In subsequent studies we have compared the effect of dietary lactalbumin (whey
protein) to that of other purified proteins in formula diets of similar
nutritional efficiency. 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 hosts red
blood cells (HRBC). The nutritional efficiency of these diets was normal and
The immune response of mice fed the WPC 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.(4)
Because L [whey protein (w.p.)] was tested in comparison to a limited number of
proteins, we could not ascertain at that time whether the enhancement of the
humoral immune response observed in five (5) unrelated strains of mice fed a
whey protein diet, was due to a real immunoenhancement, in absolute terms, by
whey protein feeding or immuno-depression by the other food protein tested.
Indeed, we can now state, in retrospect, that these few purified food proteins
(casein, soy and wheat) used as "control" for the whey protein mixture were
immunosuppressive when compared to all of the other purified food protein
subsequently tested, though nutritionally adequate and similar at 20%
concentration in diet.
In fact, we subsequently tested whey protein against most commercially available
purified food protein (casein, soy, wheat, corn, egg white, beef, fish protein,
gamma globulin, beta-lactoglobulin, alpha-lactalbumin, serum albumin, spirulina
maxima or scenedesmus algae protein) and found that indeed mice fed whey protein
exhibit the highest immune response to foreign antigen (SRBC) (12) (FIG. 1).
These proteins are nutritionally similar and adequate at the 20 g/100 g diet
concentration (Table 2).
We have concluded that our newly discovered immune enhancing and host resistance
promoting property of whey protein which we wish to protect by patient is not
related to the already known nutritional quality of this protein. In fact, the
nutritional property of whey protein at 20 g protein per 100 g diet
concentration as used in our experiment is similar to that of the other proteins
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings which form a part of this specification, FIGS. 1 and 2 show
plaque forming cells-spleen (PFC) on the day showing peak production of pfc
following immunization with SRBC.
FIG. 3 shows increased immune response noted with a 28% whey protein diet.
FIG. 4 shows the effects of whey protein on GSH, following immunization with
FIG. 5 and FIG. 6, respectively, show the heart and liver GSH levels which were
observed in old C-57 BL/GNIA mice fed the whey protein (WP) diet in comparison
with mice fed the equivalent C diet or Purina Mouse Chow.
FIGS. 7A and 7B illustrates survival curves showing the relative mortality of
mice fed Purina laboratory chow, casein and whey during a seven month period.
FIG. 8 shows the effect of 26 days dietary treatment on plaque forming cells
(PFC) response to sheep red blood cells (SRBC) in mice fed diets with various
levels of vitamines as indicated in Table 1.
As shown in reference 3 of the attached list of references showing current and
previous work, it can be demonstrated that although no significant differences
in body growth are seen between the 12% whey protein (lactalbumin) and 28% whey
protein diets, a dramatic enhancement of the immune response is noted with the
28% whey protein diet, unlike what happens with the casein diet where increasing
the protein content from 12% to 28% influences neither body growth nor the
immune response. In fact, we have found that the improved results appear to
reach a plateau at about 20%. FIG. 3 of the drawings illustrates the effect. In
FIG. 3 there is shown the effect of 3 weeks of dietary treatment with
lactalbumin (whey protein) hydrolysate (open bars) or casein hydrolysate
(hatched bars) on the number of plaque-forming cells (PFC) per spleen 5 days
after immunization with 5.times.10.sup.5 sheep red blood cells. Each value
represents the means of 10 mice.+-.SEM. 28% L diet vs. 12% L diet: P.ltoreq.0.01
by Student's t-test. By the two-way analysis of variance (F test) for both
strain of mice, the effect of the quality of protein (L vs. C) is highly
As shown in reference 12 on the list, it is clear that despite great differences
in immune response to SRBC no difference is seen in food consumption, final
weight, and serum proteins among mice fed the various purified proteins at 20
g/100 g diet concentration. (See Table 2).
In Table 2, in reference 3, data are presented on the nutritional efficiency of
the different diets. Mice fed Purina chow and the 12 or 28% C and L diets
increased in body weight by approximately the same amount with similar food
consumption ranging from 3.5 g to 3.8 g/24 hours. No significant differences
were observed between dietary groups in serum protein values and white cell
counts (data now shown).
We made the interesting observations that the delayed occurrence of spontaneous
death in mice fed the whey protein diet in comparison to those fed the casein
diet or Purin (Table 7), is not associated with any deference in food
consumption of body weight among the three dietary groups (Table 8).
FIG. 1 shows plaque forming cells-spleen (PFC) on the day showing peak
production of PFC following immunization with 10.sup.6 SRBC. Effect of 2 weeks
of dietary treatment with 20 g/100 g diet of either lactalbumin (L), casein (C),
Spirulina maxima protein (Sp), soy protein (S), wheat protein (W), Scenedesmus
protein (Sc), corn (Co) protein, egg albumin (E), beef protein (B), fish protein
(F), Purina mouse chow (P), or 20 g/100 g diet of a mixture containing 50% L and
50% S (L/S), or 80% L and 20% C, or 20% L and 80% C (L/C). Each value represents
the mean.+-.SD. See text (reference 12) for statistical significance of
differences. L=whey protein concentrate.
FIG. 2 shows plaque forming cells/spleen (PFC) on day showing peak production of
PFC following immunization with 10.sup.6 SRBC. Effect of 3 weeks of dietary
treatment with 20 g/100 g diet of either whey protein concentrate (WPC), casein
(C), whey protein concentrate hydrolysate, casein hydrolysate,
beta-lactoglobulin (.beta.L), alpha-lactalbumin (.alpha.L), gamma-globulin
(.gamma.G) or bovine serum albumin (SA). Each value represents the Mean .+-.SD.
Factors Responsible for the Immunoenhancing Effect of Whey Protein in Diet
Our studies show that the immunoenhancing effect of WPC in comparison to C is
maintained when these two proteins are replaced in formula diets by a pancreatic
hydrolysate (free amino acid and oligo peptides with MW less than 100) (FIG. 2)
(7,12). Our results also indicate that mice fed diets containing any one of the
four major protein components of the WPC mixture developed a PFC response to
SRBC inferior to that of mice fed the corresponding whey protein mixture. We can
thus conclude that the observed immunoenhancing effect of WPC is dependent upon
the contribution of all its protein components.
For these reasons we can assume that this phenomenon is not related to milk
protein allergy or some other manifestation of oral immunization.(14)
Mechanism Responsible for the Immunoenhancing effect of Whey Protein in Diet
Over the past few years we have attempted to identify the changes induced by
dietary protein type which might directly or indirectly affect the humoral
immune responsiveness. In mice not challenged with an immunogenic stimulus, the
type of protein in the diet was found to have little or no effect on a variety
of parameters examined. Thus, body growth, food consumption, serum protein,
minerals and trace metals, circulating leukocytes and more specifically, the
genesis of bone marrow B lymphocytes were all within normal limits (3-7). These
findings confirm that a 20 g/100 g diet concentration, the proteins provide an
adequate daily supply of essential amino acid for the growing mice. The only
significant effect of protein type was found to be in change in plasma amino
acid profile, which essentially conformed to the amino acid composition of the
ingested protein, with the notable exception of cysteine. (Tables 3, 4).
We were particularly intrigued by the finding that, in spite of an 8-fold higher
cysteine content in SPC, the plasma level of cysteine in WPC diet-fed mice was
not different from that in their C diet-fed counterparts. The fate of the excess
cysteine was a matter of interest. Dietary cysteine is a rate limiting substrate
for the synthesis of glutathione (GSH) which is necessary for lymphocyte
proliferation. The redox state of the lymphocyte can modulate the intracellular
concentration of cyclic GMP, which is known to be intimately involved in
Our studies have shown that the observed enhancement of the immune response is
associated with greater production of splenic glutathione in immunized mice fed
whey protein in comparison to mice fed a casein or cysteine enriched casein
diet. The efficiency of dietary cysteine in inducing supernormal glutathione
levels is greater when it is delivered in the whey protein than as free cysteine
(14) (See FIG. 4).
The search for the possible mechanism of immunoenhancement by whey protein
feeding has revealed to us the provocative possibility that whey protein may
contribute to a broader biological effect of a protective nature involving
susceptibility to cancer, diseases of aging and general detoxification of
environmental agents. All these conditions appear to be somehow related to a
drop in glutathione which is a ubiquitous element exerting a protective effect
against superoxide radicals and other toxic agents.
Increased heart and liver GSH levels were observed in old C-57 BL/GNIA mice fed
the whey protein (WP) diet in comparison to mice fed the equivalent C diet or
Purina Mouse Chow. (FIG. 5, 6), (reference 16)
In conclusion we have demonstrated that in all the experimental situations above
described the immune enhancing effect of whey protein does not depend upon its
Dietary Whey Protein and Bacterial Infection
Because our studies had shown that dietary protein type influences the humoral
immune response, we then proceeded to investigate the effect of WC in diet on
the resistance of mice to pneumococcal infection. Acquired immunity to this
infection is largely dependent on the humoral immune response. C3H/He3 mice fed
a diet containing 20 g WPC/100 g diet showed improved survival after i.v.
infection with Streptococcus pneumoniae type 3 as compared to mice fed a 20 g
C/100 g diet of similar nutritional efficiency (7) (Table 5).
On the basis of our various studies, it was shown that the enhanced resistance
of mice fed the whey protein diet to infection with Streptococcus pneumoniae
type 3 was independent of the weight of the animal at the time of infection and
the weight gained before infection (animals were fed the diets for 2 weeks prior
to infection). In this connection reference is made to reference 7 on the
enclosed list at page 22 and elsewhere.
DIETARY WHEY PROTEIN AND EXPERIMENTAL TUMORS
We have recently observed that a 20 g WPC/100 g diet significantly inhibits the
incidence and size of dimethylhydrazine (DMH) induced tumors in the murine colon
in comparison to a 20 g C/100 g diet or Purina mouse chow of similar nutritional
efficiency. This is a highly immunogenic type of tumors that develop after a
long term exposure to the carcinogen. DMH-induced colon tumors appear to be
similar to those found in humans as far as type of lesions and chemotherapeutic
response characteristics are concerned. The described enhancement of the humoral
immune response (heterologous erythorcytes) in WPC fed mice substantiate an
immunological mechanism for the observed resistance of WPC fed mice to this
immunogenic type of tumor (15) and Table 6.
It should be noted that the similarity of body weight curves among the three
dietary groups (whey protein, casein diets and Purina) is consistent with
studies in other mouse strains (2-7, 12-14). This effect is striking and appears
to rule out conventional nutritional factors for the observed differences in the
development of tumors. Reference is made to reference 15 on the enclosed list.
Tissue Glutathione (16)
Male C57/BL/6N1A mice were fed ad libitum either 20 g whey protein/100 g diet,
or 20 g casein/100 g diet or Purina mouse chow, from age 17 months until
sacrificed three months later at age 20 months. GSH (glutathione) content was
found to be higher in the liver and heart of whey protein fed mice in comparison
to the casein-diet or Purina fed counterparts (FIGS. 5 and 6). The GSH values in
heart and liver of mice fed Purina laboratory chow was almost identical at 17
and 20 months of age. Thus no age related decline is noticed during this period
of time. Moreover, the GSH values, at 17 and 20 months of age, of Purina fed
mice are similar to those of 10 week old mice. Indeed, the whey protein diet
appears to enhance, after 3 months, the GSH content of heart and liver above
"normal" values. The mean.+-.SD body weight changes over the three month period,
expressed as percentage of initial weight, of mice fed either the whey protein
diet, casein diet or Purina diet was 98.90.+-.17.7, 100.38.+-.15.99 and
99.30.+-.18.50, respectively. Thus no significant differences were noted in body
weight between the various dietary groups. Food consumption was also familiar,
varying from 3.4.+-.0.3 g/24 hr in the whey protein diet group to 3.8.+-.0.4
g/24 hr in the Purina fed mice.
Survival Studies (16)
Male C57BL/6N1A mice fed ad libitum at the onset of senescense a 20 g wpc/100 g
diet, in pathogen free environment, exhibit delayed mortality in comparison with
mice fed Purina laboratory chow over a 7 month observation period extending from
the age of 21 months (corresponding human age 55 years) to 28 months of age
(corresponding human age to 80 years) at which time 55% mortality is reached.
Mean survival time of mice fed the defined formula control diet differing from
the whey protein diet only in the type of protein (20 g casein/100 g diet) is
almost identical to that of Purina fed controls (FIG. 7), Table 7). NO
significant difference is noticed amongst dietary groups in average body weight
changes throughout the experiment (Table 8). Average food consumption in the
whey protein-diet group was 2.8.+-.0.4 g/24 hr and 3.0.+-.0.4 g/24 hr in the
casein-diet group. The greater amount of spillage of the Purina powder
substantially hampers any realistic appraisal of food consumption in this
particular group. Whereas other antioxidants, such as Vitamin E, have been shown
to be effective primarily in animals that are abnormally deficient in
antioxidant synthesis or absorption, dietary whey protein appear to represent an
important element in promoting higher tissue GSH levels and in delaying the
onset of the diseases of aging in normal wild-type animals. It is important to
emphasize that in these longevity studies, the effect of whey protein on the
diseases of aging and tissue glutathione is not related to the quality of whey
protein as a nutrient which appears to be similar, at 20 g wpc/100 g diet
concentration, to that of the other test proteins. A similar conclusion was
reached in our previous studies on the effect of a whey protein diet on PFC
response to SRBC, resistance to infections and development of tumors.
Synergistic Role of Vit. B.sub.2, B.sub.1 in the Immunoenhancing Effect of
Dietary Whey Protein Concentrate
In section 30 on page 13, page 14 and reference 14 are discussed the effect of
whey protein on splenic cell GSH and the relationship between splenic GSH and
PFC response to SRBC.
While whey protein represent an optimal source of cysteine, the rate limiting
substrate for the biosyntheses of GSH, Vit. B.sub.2 and B.sub.1 are important
element in the function of the GSH redox cycle.
Glutathione (GSH) status in tissues is maintained mainly in the reduced state
(GSH:GSSG, 250), which is achieved by the efficient GSH peroxidase and reductase
system coupled to the NADP+/NADPH redox pair. Endogenous toxic H.sub.2 O.sub.2
is reduced to H.sub.2 O through the oxidation of GSH to GSSG catalyzed by GSH
peroxidase. At the expense of cellular NADPH, GSSG is effectively reduced back
to GSH by NADPH:GSSG reductase, thus maintaining thiol balance. As a result,
GSSG reductase has a great capacity to protect cells against oxygen toxicity
from endogenous active oxygen species.
Vit. B.sub.1 (thiamine) is involved in the transketolase reaction of the pentose
phosphate shunt yielding NADPH and pentose.
Vit. B.sub.2 (riboflavin): The coenzyme derivatives of riboflavin, flavin
monomucleotide (FMN) and flavin adenin dinucleotide (FAD), are synthesized
sequentially from riboflavin. Vit. B.sub.2 deficient animals exhibit marked
decreases in activities of FMN and FAD-requiring enzymes, such as GSH reductase.
In this sense it is conceivable that all these water soluble vitamins naturally
present in whey, play an essential role for optimal function of the GSH redox
cycle particularly when whey protein intake, as shown in our experiments, has
produced higher level of GSH synthesis and storage in the tissues.
Our studies (FIG. 8) shows that dietary levels of Vit. B.sub.1, B.sub.2 slightly
above recommended allowance (Table 1, diets 5,6) significantly contribute to the
immunoenhancing effect of dietary whey protein concentrate. Whey protein, by
providing optimal bioavailability of the limiting substrate (cysteine) enhances
the synthesis and storage of GSH. On the other hand, higher than normal intakes
of Vit. B.sub.1 and B.sub.2 are necessary to maintain the GSH redox cycle at a
level higher than normal, thus allowing the development of a better than normal
immune response to SRBC. Individually the effect of each of the vitamins in whey
protein fed mice is limited; however their synergistic effect on the immune
response of whey protein fed mice is apparent (FIG. 8, diets 5,6 diet 1). The
same vitamins are ineffective on the immune response of casein diet-fed mice.
Although all these water-soluble vitamins are present in whey, it is interesting
to note that the main natural source of the single most effective vitamin,
riboflavin, is whey to which Vit. B.sub.2 given its characteristic color.
In conclusion, dietary intake of Vit. B.sub.1 and particularly B.sub.2 above
recommended daily allowance contribute to the development of enhanced immune
response in whey protein fed animals: Vitamin B.sub.2 +B.sub.1 appears to
produce the strongest effect. When intake of these vitamins is at or slightly
below these levels, body growth and animal appearance are normal, but the
response to immune challenge is below the maximum potential of whey protein fed
In the stomach, whey is separated from milk by the action of gastric juice. It
is conceivable that the transit and absorption of the water-soluble vitamins and
proteins of whey occur faster than those of the protein (casein) and vitamin
constituents of the milk coagulum (curd). Hence the whey protein and vitamins
including the vitamins B.sub.1, and B.sub.2 could enter the systemic circulation
at a different rate than that of other milk constituents and express their
synergistic effect on the immune system and the GSH redox cycle.
The immunoenhancing and the other specific biological properties of dietary whey
protein described in this application, are heat labile and dependant upon the
undenatured (native) state of the protein (which can also be affected by
vigorous shaking, solvents, extreme ph changes, etc.) and are independent of its
nutritional quality which is unaltered by the process of denaturation.
Unlike most other commercially available whey protein which are denatured, the
whey protein used in our experiments, produced in Denmark (Lacprodan--80) is 90%
undenatured (U.D. in FIG. 8). This protein displays the greatest tendency to
denature under heat thus exposing its free sulfhydryl group (17). When
experiments were done using a batch of w.p.c. received after a long surface
transport from Denmark through the U.S. in exceptionally hot and humid weather
(summer 1988), the immunoenhancing property of w.p.c. was lost (FIG. 8, 2d-8d).
These experiments, while indicating the synergistic role of vit B, and B2, in
the immunoenhancing effect of the diet, also show the negative effect of a
presumably partially denatured whey protein. Previous studies have shown (14),
that the immunoenhancing property of dietary whey protein is probably related to
an optimal intracellular transport and availability f the cysteine which is a
limiting precursor for glutathione synthesis. It is conceivable that partial
denaturation of this protein had brought about the loss of its specific
biological property by altering a cysteine bond crucial for intracellular
transport of cysteine and GSH synthesis, without any effect on its nutritional
In conclusion, the preparation of whey protein concentrate made as the "Danmark
Protein" lacprodan-80 was produced before 1985 or in a comparable appropriate
fashion, produced the biological activity sought. This activity correlates
directly with the PFC essay employed in our experiments.
TABLE 1 ______________________________________ Vitamin Content of Test Diets
VITAMINS REG. Diet Diet Diet Diet Diet Diet (mg/100 g Diet) (Diet 1) 3 4 5 6 7 8
______________________________________ VIT. B1 0.34 1.42 0.9 2.7 1.0 VIT. B2
0.38 1.47 0.9 2.7 0.6 VIT. B6 0.26 0.7 AC. FOLIC 0.063 0.1 VIT. C 53.3 118.3
Effect of 19 days dietary regimen on food consumption, body growth, total serum
protein and development of spleen..sup.h Avg. Consumption Initial Weight Final
Weight Serum Protein Averge Spleen Protein type (g/mouse/day .+-. SEM).sup.a
(g).sup.b (% initial wt.).sup.c (g/100 ml).sup.d Wt(mg).sup.3 # cells(10.sup.6)
Lactalbumin.sup.j 2.8 .+-. 0.1 22.6 .+-. 0.6 118.0 .+-. 3.2 5.8 .+-. 0.2 117
.+-. 2.1 194 .+-. 4.0 Casein 2.9 .+-. 0.2 23.0 .+-. 0.8 117.8 .+-. 4.6 6.1 .+-.
0.3 113 .+-. 3.6 150 .+-. 4.1 Spirulina maxima 2.9 .+-. 0.3 19.8 .+-. 0.9 121.0
.+-. 1.8 5.4 .+-. 0.5 104 .+-. 3.4 138 .+-. 6.0 protein Soy protein 3.1 .+-. 0.2
21.2 .+-. 0.3 114.1 .+-. 1.3 6.0 .+-. 0.4 107 .+-. 3.8 144 .+-. 4.3 Wheat
protein 2.9 .+-. 0.2 20.0 .+-. 0.3 115.0 .+-. 2.2 5.9 .+-. 0.3 109 .+-. 2.6 139
.+-. 8.0 Scenedesmus 3.1 .+-. 0.4 23.0 .+-. 0.3 113.0 .+-. 3.0 6.1 .+-. 0.1 107
.+-. 4.0 152 .+-. 10.0 protein Corn protein 3.1 .+-. 0.2 22.8 .+-. 1.1 115.5
.+-. 5.4 5.6 .+-. 0.2 118 .+-. 3.2 162 .+-. 7.0 Egg albumin 3.0 .+-. 0.1 20.7
.+-. 0.6 116.0 .+-. 2.9 5.8 .+-. 0.3 114 .+-. 3.0 157 .+-. 6.0 Fish protein 2.8
.+-. 0.4 20.9 .+-. 0.3 117.1 .+-. 1.3 5.5 .+-. 0.1 105 .+-. 2.4 152 .+-. 6.0
Beef protein 2.9 .+-. 0.4 22.0 .+-. 0.3 113.0 .+-. 1.9 5.7 .+-. 0.3 109 .+-. 1.8
150 .+-. 5.0 Lactalbumin/Soy 2.9 .+-. 0.3 20.7 .+-. 0.5 121.0 .+-. 4.7 5.8 .+-.
0.5 110 .+-. 8.0 180 .+-. 7.0 (50:50) Lactalbumin/Casein 2.7 .+-. 0.4 23.6 .+-.
0.4 121.0 .+-. 2.0 5.6 .+-. 0.4 112 .+-. 4.0 148 .+-. 4.9 (80:20)
Lactalbumin/Casein 3.0 .+-. 0.2 23.4 .+-. 0.5 116.0 .+-. 2.0 6.0 .+-. 0.3 118
.+-. 4.0 145 .+-. 5.0 (20:80) Nonpurified diet.sup.g 3.2 .+-. 0.3 21.1 .+-. 0.5
114.7 .+-. 1.8 5.8 .+-. 0.2 114 .+-. 1.9 189
6.0 .sup.a The average food consumption over the 18 days feeding period was not
considered different by ANOVA. .sup.b,c,d,e,f The average initial body weight
(b), increase in body weight (c), total serum protein (d) and spleen weight (e)
were not considered different by ANOVA. The numbers of cells per spleen (f) in
lactalbumin and Purina fed groups were higher by ANOVA (p:0.0001) than th
corresponding values in the casein, wheat, soy and fish protein groups. .sup.g
Purina mouse chow, Ralston Purina Company, St. Louis, MO., (estimated 22 g
protein from various sources per 100 g diet). .sup.h Mice received 5 .times.
10.sup.6 SRBC on day 14. .sup.j Lactalbumin = Whey Protein Concentrate.
TABLE 3 ______________________________________ AMINO ACID COMPOSITION OF TEST
PROTEINS.sup.(a) (g/100 g protein) WHEY PROTEIN AMINO ACID CASEIN CONCENTRATE
______________________________________ Phenylalanine 5.3 .+-. 0.2 3.4 .+-. 0.3
Tryptophan 1.4 .+-. 0.2 2.1 .+-. 0.0 Glycine 2.0 .+-. 0.1 2.0 .+-. 0.2 Serine
6.2 .+-. 0.5 5.2 .+-. 0.4 Leucine 10.0 .+-. 0.4 10.4 .+-. 0.7 Isoleucine 6.0
.+-. 0.6 6.1 .+-. 0.8 Valine 7.1 .+-. 0.3 5.8 .+-. 0.8 Methionine 2.9 .+-. 0.2
2.1 .+-. 0.3 Cysteine 0.3 .+-. 0.1 2.3 .+-. 0.3 Aspartic acid 7.3 .+-. 0.1 10.7
.+-. 0.7 Glutamic acid 22.9 .+-. 0.3 18.8 .+-. 0.7 Histidine 3.0 .+-. 0.1 2.0
.+-. 0.2 Tyrosine 6.0 .+-. 0.1 3.0 .+-. 0.4 Proline 11.6 .+-. 0.4 6.1 .+-. 0.7
Arginine 4.0 .+-. 0.1 2.8 .+-. 0.3 Alanine 3.1 .+-. 0.3 4.9 .+-. 0.4 Lysine 8.2
.+-. 0.1 9.2 .+-. 0.5 Threonine 4.6 .+-. 0.3 6.8 .+-. 1.3
______________________________________ .sup.(a) Value expressed as Mean .+-.
S.D. of data from reliable sources (Reference 13).
TABLE 4 ______________________________________ EFFECT OF DIETARY PROTEIN TYPE ON
PLASMA AMINO ACID PATTERNS Lactalbumin 20 g % (whey protein Casein Amino Acid
nmol/ml concentrate) 20 g % P-value ______________________________________
Isoleucine 90 .+-. 5 95 .+-. 8 -- Leucine 125 .+-. 5 113 .+-. 4 -- Valine 232
.+-. 10 278 .+-. 13 0.025 Methionine 72 .+-. 3 92 .+-. 6 0.025 Cystine 37 .+-. 3
37 .+-. 3 -- Phenylalanine 51 .+-. 1 75 .+-. 4 0.0005 Tyrosine 55 .+-. 2 83 .+-.
5 0.005 Threonine 310 .+-. 7 223 .+-. 2 0.0005 Tryptophan -- -- -- Lysine 301
.+-. 6 323 .+-. 7 -- Histidine 50 .+-. 1 64 .+-. 4 0.005 Arginine 61 .+-. 4 92
.+-. 6 0.005 Glycine 142 .+-. 7 144 .+-. 7 -- Serine 120 .+-. 8 132 .+-. 4 --
Alanine 437 .+-. 18 382 .+-. 19 0.05 Proline 52 .+-. 5 117 .+-. 10 0.0005
Aspartic Acid 24 .+-. 2 16 .+-. 1 0.005 Glutamic acid 65 .+-. 2 44 .+-. 4 0.005
______________________________________ Mean .+-. SD.
TABLE 5 ______________________________________ SUSCEPTIBILITY TO TYPE 3 S.
PNEUMONIAE OF THREE SERIES OF MICE FED DIETS OF VARIOUS PROTEIN TYPES.sup.1 Days
Experiment Ratio of alive:dead mice Experiment Post- 1 Experiment 2 3
Infection.sup.2 C L C L C L ______________________________________ 0 (10.sup.2)
8:0 8:0 10:0 10:0 10:0 10:0 2 8:0 8:0 10:0 10:0 10:0 10:0 3 7;1 8:0 10:0 10:0
10:0 10:0 4 7:1 8:0 9:1 10:0 9:1 10:0 9 (10.sup.3) 7:1 8:0 9:1 10:0 9:1 10:0 11
7:1 8:0 9:1 10:0 9:1 10:0 12 7:1 8:0 5:5 9:1 8:2 10:0 13 6:2 8:0 4:6 9:1 8:2
10:0 14 5:3 8:0 4:6 9:1 7:3 9:1 40 5:3 8:0 4:6 9:1 7:3 9:1
______________________________________ .sup.1 Mice were infected after 2 wk
treatment with casein diet (C) (20 g casein/100 g diet), or lactalbumin diet (L)
(20 g/100 g). .sup.2 Injected i.v. in 1% FCSRinger; 9 days after infection with
10.sup. pneumococci the surviving mice were infected with a dose of 10.sup.3
pneumococci. C = Casein L = Lactalbumin = Whey Protein Concentrate. Overall
mortality is 36% in the C fed groups and this is significantly higher (P =
0.002) than that of the L fed mice which is 7.1%.
TABLE 6 ______________________________________ Effect of Dietary Protein Regimen
on Body Growth and Tumor Development in 1,2-Dimethylhydrazine treated A/J
mice.sup.a. Dietary Treatment Whey Variable Protein Casein Purina
______________________________________ Body weight.sup.b Initial(g) 21.06 .+-.
1.32 23.94 .+-. 2.49 22.13 .+-. 1.36 Final(% 108.0 .+-. 7.7 108.9 .+-. 10.2
110.7 .+-. 9.70 initial) Number of Tumours.sup.c 8.50 .+-. 3.87 13.8 .+-. 4.83
16.9 .+-. 9.85 Tumour Area.sup.d 32.18 .+-. 13.69 47.35 .+-. 14.02 78.15 .+-.
31.19 ______________________________________ .sup.a Mean of 10 mice per group
.+-. Standard Deviation. .sup.b Among dietary groups there was no statistically
significant difference in initial body weight or in the body weight reached
after 28 weeks. .sup.c Whey Protein versus Casein P = .0138 Whey Protein versus
Purina P = .0208 .sup.d Whey Protein versus Casein P = .0236 Whey Protein versus
Purina P .ltoreq. .001 Purina versus Casein P = .0104
TABLE 7 ______________________________________ Time at which 55% of mice fed
either of three dietary regimen (from 21 months of age to 28 months of age) were
dead. Dietary treatment Days of feeding.sup.(a)
______________________________________ Casein 92.2 .+-. 55.2.sup.(b) Whey 125.0
.+-. 41.6.sup.(c) Purina 92.7 .+-. 31.7.sup.(d)
______________________________________ .sup.(a) Mean of 10 mice per group .+-.
standard deviation. Survival time for d < c (p < 0.05). If the two control diet
groups with near identical survival time are pooled together: b,d < c (p <
TABLE 8 ______________________________________ VITAMIN CONTENT/100 g DIET Diet
Diet Diet Diet Diet 1 2 3 4 5 ______________________________________ Ascorbic
acid 65.0 (N).sup.a 47.0 140.0 47.0 47.0 (vitamin C), mg Niacin, mg 9.2 -- -- --
-- Riboflavin (vitamin 0.69 (0.60).sup.b 0.54 0.54 1.00 0.54 B.sub.2), mg
Thiamin (vitamin B.sub.1), 0.63 (0.60).sup.b 0.54 0.45 0.45 1.00 mg Folic acid,
mg 0.12 -- -- -- -- Vitamin B.sub.6, mg 0.36 -- -- -- -- Biotin, mg 0.058 -- --
-- -- Pantothenic acid, mg 3.38 -- -- -- -- Choline, mg 76.0 -- -- -- -- Vitamin
B.sub.12, mg 0.55 -- -- -- -- Phylloquinone 1.8 -- -- -- -- (vitamin K), mg
Inositol, mg 34.39 -- -- -- -- Retinyl palmitate 1800 -- -- -- -- (vitamin A),
I.U. Ergocalciferol 360 -- -- -- -- (vitamin D.sub.2), I.U. Dl-tocopheryl
acetate 24.0 -- -- -- -- (vitamin E), I.U.
______________________________________ The mineral content of ions or cations
(expressed in milligrams per 100 g diet) and the actual chemical compounds fed
are: Ca, 378 (CaHPO.sub.4.2H.sub.2 O and Ca.sub.3 (C.sub.6 H.sub.5
O.sub.7).sub.2.4H.sub.2 O); P, 208 (K.sub.2 HPO.sub.2.2H.sub.2 O); Fe, 7.
(FeSO.sub.4.2H.sub.2 O); Mg, 44 (MgO); Cu, 0.38 (CuSO.sub.4.5H.sub.2 O); Zn, 2.5
(ZnSO.sub.4.7H.sub.2 O); Mn 0.63 (MnSO.sub.4); Cl, 840 (C.sub.5 H.sub.14 CINO);
K, 1050 (K.sub.2 HPO.sub.4.2H.sub.2 O); Na, 245 (NaCl). .sup.a N = Not required.
.sup.b = Values between brackettes are the vitamin concentrations of an adequate
mouse purified diet. (AIN 76: KNAPKA J. Jr. in "The mouse in biomedical
research". Eds. H. L. Foster, J. D. Small, J. G. Fox, Academic Press, New York,
p. 58, 1983).
References Related to Previous and Current Work (Incorporated by Reference)
1) Bounous G., Kongshavn P. A. L. "The effect of dietary amino acid on immune
2) Bounous G., Stevenson M. M., Kongshavn P. A. L. Influence of dietary
lactalbumin hydrolysate on the immune system of mice and resistance to
Salmonelloss--I. of Infect Diseases 144/281/1981
3) Bounous G., Kongshavn P. A. L. Influence of dietary proteins on the immune
system of mice--J. Nutr. 112/1747-1755/1982
4) Bounous G., Letourneau L., Kongshavn P. A. L. Influence of dietary protein
type on the immune system of mice--J. Nutr. 113/1415-1421/1983
5) Bounous G., Kongshavn P. A. L. Differential effect of dietary protein type on
the B-Cell and T-Cell immune responses in mice--J. Nutr. 115/1403-1408/1985
6) Bounous G., Shenuda, N., Kongshavn P. A. L., Osmond D. G., Mechanism of
altered B-Cell response induced by changes in dietary protein type in mice--J.
7) Bounous G., Kongshavn P. A. L. Influence of protein type in nutritionally
adequate diets on the development of immunity (In press in) "Absorption and
utilization of amino acids" Editor, N. Friedman publisher CRC press Fall 1988.
8) Raiha N. C. R., Heinonen K., Rassin D. K., Gaull G. E. Heindnen, Mild protein
quantity and quality in low-birth weight infants: 1: Metabolic responses and
effects on growth--Pediatric 57/659-674/1976
9) Darling P., Lepage G., Tremblay P., et al Protein quality and quantity in
preterm infants receiving the same energy intake. Am. J. Dis. Child
10) Birt D. F., Baker P. Y., Hruza D. S. Nutritional evaluation of three dietary
levels of lactalbumin throughout the lifespan of two generations of syrian
hamsters--J. Nutrit 112/2151-2160/1982
11) Birt D. F., Schuldt G. H., Salmasi S. "Survival of hamsters fed graded
levels of two protein sources"--Lab Animal Sci 32/363-366/1982
12) Bounous G., Kongshavn P. A. L., Gold P. "The immunoenhancing property of
dietary whey protein concentrate" "clinical and Investigat. Med.
13) Bounous G., Kongshavn P. A. L., Taveroff A., Gold P. "Evolutionary traits in
human milk proteins"--"medical hypothesis", 27/133-140/1988.
14) Bounous G., Batist G., Gold P. Immunoenhancing effect of dietary whey
proteins in mice: role of glutathione. Accepted for publication in Clin. Invest.
15) Bounous G., Papenburg R., Kongshavn P. A. L., Gold P, Fleiszer D. Dietary
whey protein inhibits the development of dimethylhydrazine induce
16) Bounous G., Gervais F., Batist G., Gold P. Effect of dietary whey protein
and tissue glutathione on the diseases of aging. Submitted to "clinical
17) Farrell H. M., Douglas F. W. Effect of ultra-high-temperature pasteurization
on the functional and nutritional properties of milk proteins. Kieler
Milch-wirtschfliche Forsch. 35/365-56/1983
* * * * *