United States Patent 5,456,924
Bounous , et al. October 10, 1995
Method of treatment of HIV-seropositive individuals with dietary whey proteins
Undenatured whey protein concentrate is administered to HIV-seropositive
individuals to elevate their blood mononuclear cells, glutathione (GSH) level,
body weight and sense of well being. In addition T-helper cells concentration
and their T-helper cells/T-suppressor cells ratio are slightly elevated.
Inventors: Bounous; Gustavo (Montreal, CA), Gold; Phil (Westmount, CA)
Assignee:Immunotec Research Corporation Ltd. (Montreal, CA)
[*] Notice: The portion of the term of this patent subsequent to July 27,
2010 has been disclaimed.
Appl. No.: 07/866,756
Filed: April 10, 1992
Related U.S. Patent Documents
Application NumberFiling DatePatent NumberIssue Date
Current U.S. Class:424/535 ; 426/72; 514/2; 514/21
Current International Class: A23L 1/305 (20060101); A61K 35/20 (20060101);
A61K 38/17 (20060101); A61K 39/395 (20060101); A61K 035/20 ()
Field of Search: 514/2,21 530/365,833 424/535 426/72
References Cited [Referenced By]
U.S. Patent Documents:
4880626 November 1989 McMichael
4983387 January 1991 Goldstein et al.
5030449 July 1991 Berzofsky et al.
5112373 June 1992 Eibl et al.
Foreign Patent Documents:
Bounous, G., Kongshavn, P., Gold, P., Clinical and Investigative Medicine,
vol. 11, No. 4, pp. 271-278 (Aug. 1988).
Bounous, G., Batist, G., Gold, P., Clinical and Investigative Medicine,
vol. 12, No. 3, pp. 154-161 (1989).
Fidelus, R. K., Tsan, M. F., Cellular Immunol., vol. 97, pp. 155-163
Gougerot-Pocidalo, M. A., et al., Immunology, vol. 64, pp. 281-288 (1988).
Noelle, R. J., Lawrence, D. A., Biochem. J., vol. 198, pp. 571-579 (1981).
Anderson, M. E., Meister, A., Proc. Natl. Acad. Sci.-USA, vol. 80, pp.
Meister, A., in Stranbury, J. B., eds, Metabolic Basis of Inherited
Diseases 4th Edn. McGraw Hill, pp. 328-335 (1978).
Bounous, G., Gold, P., Clinical and Investigative Medicine, vol. 14, pp.
Hirai, Y., et al., Evaluation of the Immunological Enhancement Activities
of Immunocol (Dec. 13, 1990) Osaka, Japan.
Buhl, R., et al., Lancet, pp. 1294-1297 (Dec. 2, 1989).
Anderson, M. E., C.R.C. Handbook of Methods for Oxygen Radical Research,
pp. 317-329 (1985).
Biological Abstracts, vol. 41, Abstract No. 84485, Voss et al. (Jun.
Biological Abstracts, vol. 41, Abstract No. 84488, Glauser et al. (Jun.
Primary Examiner: Naff; David M.
Assistant Examiner: Witz; Jean C.
Attorney, Agent or Firm: White; John P.
Parent Case Text
This application is a continuation-in-part of U.S. application Ser. No. 563,794,
filed Aug. 3, 1990, now U.S. Pat. No. 5,230,902, which is a continuation of U.S.
Ser. No. 289,971, filed Dec. 23, 1988, now abandoned and a continuation-in-part
of 417,246, now U.S. Pat. No. 5,290,571, filed on Oct. 4, 1989, which is a
continuation-in-part of Ser. No. 289,971, now abandoned, which in turn is a
continuation-in-part of U. S. Serial No. 188,271, filed Apr. 28, 1988, now
abandoned, all of which are incorporated by reference herein.
1. A method of treating HIV-seropositive individuals so as to increase their
blood mononuclear cell glutathione concentration and to maintain or increase
body weight which comprises administering to HIV-seropositive individuals a
substantially undenatured whey protein concentrate, wherein the substantially
undenatured whey protein concentrate comprises substantially all the heat labile
whey protein contained in raw milk, in an amount effective to increase their
blood mononuclear cell glutathione concentration and maintain or increase body
2. The method as in claim 1 in which the undenatured whey protein concentrate
has a solubility index at pH 4.6 of 99%.
3. The method as in claim 1 in which the undenatured whey protein concentrate
has a protein composition comprising beta-lactoglobulin 59.1.+-.4.0, alpha
lactalbumin 6.6.+-.0.7, serum albumin 9.7.+-.1.0, and immunoglobulin
4. The method as in claim 1 in which the amount of undenatured whey protein
concentrate administered daily is in the range of about 8 to 40 grams daily.
5. The method as in claim 4 in which the range is about 20 to 40 grams daily.
6. The method as in claim 5 in which the range is about 30 to 40 grams daily.
7. The method as in claim 2 in which the undenatured whey protein concentrate
has a serum albumin level of at least about 9.5%.
8. The method as in claim 1 in which the substantially undenatured whey protein
concentrate comprises substantially all the glutamyl-cysteine group containing
proteins contained in raw milk.
Reference is made to twelve articles listed at the end of this specification,
the contents of which are incorporated by reference herein.
Our studies showed that the humoral immune response (number of plaque-forming
cells formed in response to sheep red blood cells) is significantly higher in
mice fed a 20 g whey protein concentrate/100 g diet than in mice fed formula
diets of similar nutritional efficiency containing 20 g/100 diet of any other
type of commercially available semipurified food protein, such as casein, soy,
wheat, corn, egg white, fish, beef protein, Spirulina maxima, Scenedesmus algae
protein, or Purina mouse chow .
We have further shown that the immunoenhancing activity of dietary whey protein
concentrate (WPC) is related to greater production of splenic glutathione (GSH)
in the whey protein-fed animals during the oxygen-requiring antigen-driven
clonal expansion of the lymphocyte . It was then theorized that this might
reflect the ability of the lymphocytes of whey protein diet-fed mice to offset
potential oxidative damage, thus responding more fully to the antigenic
challenge [3,4]. In fact, the capacity of a cell to recover from an oxidative
insult is considered to be represented by its ability to regenerate
intracellular stores of glutathione .
Our studies also showed that administration of S-(n-butyl) homocysteine
sulfoximine, which reduces splenic glutathione in half, significantly reduced
the humoral immune response of whey protein-fed mice. This was taken as further
evidence for the important role of glutathione in the immunoenhancing effect of
dietary whey protein .
Tissue glutathione concentration may be increased by administration of
gamma-glutamyl-cysteine. Glutathione increased in the kidney by about 50%, 40-60
minutes after subcutaneous (s.c.) injection in mice, returning to control values
2 hours later . The administered gamma-glutamylcysteine is transported intact
and serves as a substrate for glutathione synthetase .
Advances in amino acid sequencing of food proteins allowed us to investigate the
occurrence of glutamylcysteine groups in whey protein and the possible relation
to glutathione promotion. Indeed, whey protein concentrate from bovine milk
contains substantial amounts of glutamylcysteine groups, unlike casein, which
does not increase tissue glutathione when fed to mice . The glutamylcysteine
groups are located primarily in the serum albumin fraction (six
groups/molecule). Glutamylcysteine groups are extremely rare in animal and plant
edible proteins. Extensive search of all available data on amino acid sequencing
of edible proteins reveals that the Gly-Cys group with a disulfide link is
indeed limited to some of the whey protein, and to the ovomucoid fraction of egg
white which contains 2 of these groups in a 30,000 mol.wt.molecule .
Our recent  data further indicate that the humoral immune response is highest
in mice fed a dietary whey protein concentrate exhibiting the highest solubility
(unclenatured conformation) and, more importantly, a greater relative
concentration of the thermolabile bovine serum albumin (.gtoreq.10%) and
immunoglobulins. 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
Recent experiments in Japan  showed that spleen cells of BALB/c male mice fed
25 g of our undenatured whey protein concentrate (WPC) (which we call
"Immunocal") per 100 g diet for 4 weeks had an increased immune response to SRBC
in vitro and a higher content of L3T4.sup.+ cells (12.58.times.10.sup.6
.+-.0.50) than mice fed an isocaloric diet with 25 g. pure casein/100 g. diet
(3.69.times.10.sup.6 .+-.0.50). Similarly, the spleen L3T4.sup.+ /LYt-2.sup.+
ratio was 1.36.+-.0.07 in undenatured WPC fed mice and 0.55.+-.0.07 in
casein-fed controls (P<0,001).
Materials and Methods
The whey protein concentrate (WPC) used in the examples was in undenatured form
prepared from milk treated in the most lenient way compatible with accepted
standards of safety with regard to bacterial contamination. The extremely high
solubility index indicates that the proteins present are essentially
undenatured, hence demonstrating the lineancy of the ultrafiltration process
. Although the proteins contained in the concentrates from the other
commercially available sources examined were mostly in undenatured form, as
indicated by the relatively high solubility of the concentrates, the content of
serum albumin and immunoglobulins in these mixtures is below the level
apparently necessary to produce a significant biological activity . These
very thermolabile proteins are denatured, hence precipitated and partially lost
from whey when high pasteurization temperatures are utilized. Conversely, the
relatively high concentrations of the thermosensitive serum albumin and
immunoglobulins resulting from the low degree of pasteurization of milk in our
WPC, may reflect more closely the pattern of raw milk. These data lend support
to the hypothesis that the thermolabile Gly-Cys containing proteins such as
serum albumin in undenatured conformation are crucial elements for the
biological activity of whey protein concentrate.
The bovine whey protein concentrate (WPC) was especially prepared by the
"Service de recherche sur les aliments du Ministers de l'agriculture du Quebec"
in St-Hyacinthe, Quebec, Canada, with the following characteristics: pure
protein content 75% (the rest mostly lactose, some fat and moisture); solubility
index: (ph 4.6); 99.5%. Protein composition as % of total whey protein measured
by polyacrylamide gel electrophoresis  was: beta-lactoglobulin 59.1.+-.4.0;
alpha-lactalbumin:6.6.+-.0.7; serum albumin: 9.7.+-.1.0; immunoglobulin
24.6.+-.2.6 (mean .+-.SD). The solubility index should preferably be above 99%.
The serum albumin of about 10% of the total whey protein was almost twice the
corresponding value found in other commercially available whey protein
concentrates that have been examined. It is believed that a serum albumin level
.gtoreq.10% is highly advantageous to improving the immune system.
Serum albumin includes a substantial amount of glutamyl cystsine which is a
substrate for glutathione synthesis in the body. The role of glutathione is
discussed in detail in "The Biological Activity of Undenatured Dietary Whey
Proteins: Role of Glutathione", Clin. Invest Med 14: 296-309, 1991, which is
incorporated by reference in its entirety.
Immunoglobulin in the range of about 25 to 30% of total whey protein is also
important. Pasteurization at 72.degree. C. for 13 seconds resulted in an
immunoglobulin level of 28.+-.2%. We have found it possible to achieve a serum
albumin level as high as 14.+-.1% with milk pasteurized at 72% for 13 seconds.
Upon bacteriological analysis no staph, salmonella, B cereus, or E coli were
isolated in either the WPC prepared by the "Service de recherche sur les
Aliments due Ministers de l'agriculture du Quebec" or in the sample pasteurized
at 72.degree. C. for 13 seconds.
The method used to prepare the WPC used in the examples is schematically
described below in Table 1.
TABLE 1 ______________________________________ SCHEMATIC REPRESENTATION OF THE
PROCESS TO PRODUCE OUR UNDENATURED WPC PRODUCT
______________________________________ Raw milk .dwnarw. Skimmed at 35.degree.
C. .fwdarw. cream .dwnarw. Skimmed milk pasteurized at 63.degree. C. for 30
minutes. .dwnarw. At 38.degree. C.: Addition of rennet (20 ml/100 kilos),
allowing the agitation to resolve at low speed. .dwnarw. .fwdarw. curd Whey
.dwnarw. Filtered with cheese cotton to remove debris. .dwnarw. At 40.degree.
C.: Ultrafiltration (Romecon UFSI, polysulphone membrane, cut off 50,000, pore
diameter 0.06 inch, surface 2-3 m.sup.2). Diafiltration to wash out salts and
lactose. Whey Protein Concentrate .dwnarw. At 40.degree. C: Lyophylization.
.dwnarw. Whey Protein Concentrate Powder ______________________________________
30 ml of heparinized blood are used to determine the glutathione content of
blood mononucleated cells. Counted mononuclear cells are resuspended in
phosphate buffered saline adjusted so that there are 10.sup.7 cells per tube.
After centrifugation 900 .mu.l of water is added to the pellet to lyse all the
cells. To each aliquot is added 30% sulfosalicylic acid for a final
concentration of 3% in i ml. After 15 minutes incubation, the samples are
centrifuged, and the clear supernatant is used for the biochemical assay
according to the method of Anderson . Values are expressed as nanomol (nMol)
of GSH/10.sup.7 cells. Blood lymphocyte subsets are determined by
The total serum protein, including the albumins and the immunoglobulins is
determined by the Biuret method. The level of Immunoglobulin A (IgA),
Immunoglobulin G (IgG) and Immunoglobulin M (lgM) are measured by
DESCRIPTION OF THE INVENTION AND EXAMPLES
There are two important subsets of lymphocytes in the blood; (1) CD4, also
called T-helper cells, because they help in the immune response and (2) CD8,
also called T-suppressors because they have the opposite effect, i.e. they
suppress the immune response. In HIV-seropositive individuals the number of CD4
(helper) cells is low, i.e. the ratio CD4/CD8 is down.
The object of this invention is to elevate the ratio of T helper cells to T
suppressor cells. This is accomplished by the administration of undenatured whey
The amount administered should be in the range of about 8 to 40 grams daily and
preferably 20 to 40 grams daily. It is particularly beneficial to the
glutathione level to administer 30 to 40 grams daily.
As more fully described in U.S. application Ser. No. 563,794 filed Aug. 3, 1990,
which is incorporated in its entirety by reference, raising the glutathione
level is beneficial to the immune system.
A whey protein concentrate as previously described was administered to 3 male
HIV-seropositive individuals identified in Table 2 as A, B and C. The product
was drunk cold daily in a liquid chosen by the patient. Originally a fourth
patient D was included but he took the WPC sporadically for lack of discipline
and none during the final days of the study. He therefore has been classified in
Table 2 as a control the other controls are E and F.
The daily intake of WPC was increased stepwise. During the first three weeks 8.4
grams were prescribed daily, in the following three weeks 19.6 g, in the next
three weeks 28 grams, and 39.2 grams in the final three weeks.
The observations made are shown below in Table 2.
Energy Ideal Patient Weeks (K Cal) Body Body Helper CD4 GSH Initials on Protein
g Weight Weight CD4 % Absolute CD8 n mol-10.sup.2 cells Serum Proteins in %
(age) Whey (1) (Kg) (Kg) (2) (3) (4) (5) Total Alb IgG IgA IgM
A 0 2180 86 102.5 23 368 0.38 9.75 77 42 20.8 1.54 1.14 (35) 87 6 1800 105 26
546 0.45 10.34 74 39 19.4 1.56 1.16 106 12 2111 108 24 480 0.41 13.9 80 42 20.1
1.61 1.05 100 B 0 1870 75.2 73.9 15 435 0.22 10.22 82 50 12.3 4.38 0.75 (32) 84
6 2035 74.5 19 532 0.28 9.6 79 45 14.3 4.53 0.76 140 12 2100 76 17 442 0.24
17.04 72 50 16.9 5.81 0.95 138 C 0 2400 78 76 24 672 0.39 10.38 82 52 12.4 3.59
0.76 (20) 100 6 2200 77.5 27 864 0.46 12.55 81 49 12.9 3.99 0.58 116 12 1400 78
26 676 0.45 7.06 80 50 12.8 3.8 0.7 98 CONTROL D 0 2940 95.5 27 540 0.47 12.36
75 47 15.6 1.63 1.34 107 6 2500 85 25 650 0.42 8.59 73 42 15 1.5 1.19 118 E 0
2500 70 69.5 17 420 10.3 81 44 (38) 98 6 2600 68.5 21 371 11 82 44 130 12 2480
68 18 359 9.8 81 44 108 F 0 2200 74 74 16 263 11.08 80 43 (37) 108 6 2100 73 12
222 10.3 83 43 120 12 2300 72 12.5 230 9 80 42 110
The energy and protein intake indicated represents the mean value for the
preceding weeks. (2) Normal range: 35-55 (3) Normal range: 580-1250 (4) Normal
range: 1.42-3.56 (5) GSH: Glutathione content of mononuclear blood cells. Normal
value in healthy HIV seronegative individual of corresponding age: 17.05
.sup..+-. 2.40 (mean .sup..+-. SD)
In Table 2:
CD 4% means the percentage of CD.sub.4 (helper cells) of the total lymphocytes
in the blood.
Helper absolute refers to the real number of CD4 cells per unit of blood (X
10.sup.3 per microliter (.mu.l))
CD4/CD8 refers to the ratio of the two types of lymphocytes in the blood, i,.e.
the T-helper cells and the T-suppressor cells.
Total refers to the total amount of protein in the serum which includes the
albumins and the imunoglobulins.
IgA means Immunoglobulin A
IgG means Immunoglobulin G
IgM means Immunoglobulin M
The data in Table 2 and other information gathered during the course of the work
indicates the following.
Three patients took the undenatured WPC daily for the 3-month period without any
adverse side effects. In all these patients body weight increased progressively
(from 2 to 7 kilos); and in fact two of them (C and B) reached 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.
In all three of these patients the blood T-helper cells concentration and the
T-helper/suppressor ratios were moderately but consistently higher during the
study than before undenatured WPC administration.
The blood mononuclear cells glutathione content was, as expected , below
normal values in all patients at the onset of the study. Over the three-month
period, however, glutathione levels increased and in one case (B) rose by 70% to
reach normal value.
Systemic glutathione deficiency in symptom-free HIV-seropositive individuals
 at page 1297 presents the re-establishment of normal extracellular
concentrations of glutathione as an unsolved problem, because GSH administered
intravenously has a half life of only 1-6 minutes. This problem is however
solved in accordance with this invention by the administration of undenatured
These objective changes were accompanied by a markedly improved sense of well
being in all three patients.
It is noteworthy that one patient over-concerned that the beneficial increase in
body weight could hamper his lean appearance, drastically reduced energy and
undenatured WPC intake during the second period of study. (C, Table 2). During
this time body weight increase was reduced, and glutathione and T-helper cells
failed to rise.
As indicated earlier, patient D. considered as control for his total lack of
submission to the protocol, exhibited a drop in body weight, T-helper/suppressor
ratio and glutathione on the 6-week end point and did not show up for the
12-week appointment. The other two controls (E. and F.) exhibited weight loss
and no change in GSH levels.
The example indicates that whenever patients essentially maintained their energy
intake at pre-study levels with only minor reductions in the prescribed WPC
intake, body weight increased and specific HIV indicators such as a blood cell
glutathione, T-helper cells concentrations and T-helper/suppressor ratios were
all moderately higher during the WPC administration.
The positive effects of undenatured WPC observed in this very limited number of
HIV-seropositive individuals acquires significance if viewed on the background
of a large number of animal experiments showing increased cellular GSH and
immune response by our WPC [1,2,8,9]. Animal studies emphasize the fact that the
immunoenhancing effect of undenatured WPC is not related to a greater systemic
nutritional efficiency, when compared to several other protein sources with
similar nutritional efficiency but no significant biological activity. Mice fed
undenatured WPC did not exhibit increased body growth nor any changes in serum
protein levels. Similarly in our patients undenatured WPC did not produce any
change in serum proteins which remained constant throughout the study. The
increase in body weight observed in our patients did not correlate with increase
in energy or protein intake throughout the study period but rather with improved
sense of well being and HIV specific blood parameters. The extra protein intake
through the undenatured WPC was generally compensated by reduced intake of
protein from other sources.
The presence of glutamylcysteine groups in the serum albumin component of the
whey protein concentrate is considered to be a key factor in the
glutathione-promoting and immunoenhancing activity of the protein mixture of the
undenatured WPC. Our laboratory studies indicate that whey protein concentrates
from other sources, did not produce significant biological activities while
exhibiting similar nutritional efficiency. The percent serum albumin
concentration in these products is (as mean .+-.SD) respectively: 4.+-.1 in
Promod (Ross Laboratories), 4.+-. in Alacen 855 (New Zealand Dairy), 4.8.+-. in
Lacprodan -80 (produced from 1989 by Danmark Protein), 4.8.+-.0.1 in Sapro
(Saputo, Montreal), 4.+-.1 in Savorpro -75 (Golden Cheese, CA), 5.+-.1 in
Bioisolate (Lesueur Isolates, Minneapolis)  and 4.3.+-.1 in Promix (Dumex,
Quebec). Similarly, the content of the other thermolabile protein,
immunoglobulin, was about half the value of the undenatured WPC used in this
The results indicate that undenatured whey proteins by providing specific fuel
for glutathione replenishment in the immunocytes could represent an adjuvant to
other forms of therapy.
Historically, and up until now, bacteria and spores in milk were reduced by
thermal treatment (pasteurization). In order to be effective, that method
inevitably produced denaturation, and hence subsequent precipitation and loss in
the curd of a substantial amount of the most thermolabile and presumed
biologically active fractions of serum albumin and immunoglobulin.
Our objective is to obtain a whey protein concentrate (w.p.c) containing the
proteins in proportion and conformation as close as possible to that of raw
milk, compatible with accepted safety standards of bacterial content. Up until
now we have utilized the lowest acceptable level of heat treatment of milk in
order to preserve thermolabile whey protein. From now on we will achieve this
objective with a new method based on membrane microfiltration.
Utilizing Bactocatch (Alfa-Laval Ltd. Scarborough, Ontario) we can obtain by
special membrane microfiltration of the skim milk a permeate whose bacteria
content has been reduced to less than 0.5% of original input levels.
This permeate is then treated with rennet and the proteins in the whey
supernatant concentrated by a lenient procedure to obtain the desired
undenatured whey protein concentrate. We believe that the membrane
microfiltration concept replacing heat treatment of milk will provide in the
future the appropriate way to preserve heat labile whey proteins, although
techniques and equipment may be improved in time.
1) Bounous G, Kongshavn P. A. L, Gold. P: The immunoenhancing property of
dietary whey protein concentrate. Clin Invest Med 11:271-8, 1988.
2) Bounous G, Batist G, Gold P: Immunoenhancing property of dietary whey protein
in mice: Role of glutathione. Clin Invest Med 12:154-61, 1989.
3) Fidelus R. K., Tsan M. F.: Enhancement on intracellular glutathione promotes
lymphocyte activation by mitogen. Cell Immunol 97: 155-63, 1986.
4) Gougerot-Pocidalo M. A., Fay M, Roche S: Mechanisms by which oxidative injury
inhibits the proliferative response of human lymphocytes to PHA, Effect of the
thiol compound 2-mercaptoethanol. Immunology 64: 281-8, 1988.
5) Noelle R. J., Lawrence D. A.: Determination of glutathione in lymphocytes and
possible association of redox state and proliferative capacity of lymphocytes.
Biochem J 198: 571-9, 1981.
6) Anderson M. E., Meister A: Transport and direct utilization of
gamma-glutamylcyst(e)ine for glutathione synthesis. Proc Natl Acad Sci
7) Meister A: 5-Oxoprolinuria and other disorders of glutathione biosynthesis.
In:Stranbury J. B., Wymgaarden J. B., Frederikson D. S., eds. Metabolic basis of
inherited diseases 4th edn. McGraw Hill, 1978:328-35
8) Bounous G, Gold P: The biological activity of undenatured dietary whey
proteins: role of glutathione. Clin Invest Med 14:296-309, 1991.
9) Hirai Y, Nakay S, Kikuishi H, Kawai K; Report: Evaluation of the
immunological enhancement activities of Immunocal. Otsuka Pharmaceutical Co.
Ltd: Cellular Technology Institute: Dec. 13, 1990, Osaka, Japan.
10) Buhl R, Holroyd K. J., Mastrangeli A, Cantin A. M., Jaffe H. A., Wells C,
Saltini C, Crystal R. G.:Systemic glutathione deficiency in symptom-free
HIV-seropositive individuals. Lancet(December 2): 1294-7, 1989.
11) Anderson ME: Tissue glutathione: In:C.R.C. Handbook of methods for oxygen
radical research. Boca Raton, Fla.:CRC Press, Inc., 1985:317-29
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