European Journal of Clinical Investigation (2001) 31, 171±178

Oral supplementation with whey proteins increases plasma

glutathione levels of HIV-infected patients
P. Micke, K. M. Beeh, J. F. Schlaak and R. Buhl
University Hospital, Mainz, Germany

Abstract Background HIV infection is characterized by an enhanced oxidant burden and a systemic
deficiency of the tripeptide glutathione (GSH), a major antioxidant. The semi-essential
amino acid cysteine is the main source of the free sulfhydryl group of GSH and limits its
synthesis. Therefore, different strategies to supplement cysteine supply have been
suggested to increase glutathione levels in HIV-infected individuals. The aim of this
study was to evaluate the effect of oral supplementation with two different cysteine-rich
whey protein formulas on plasma GSH levels and parameters of oxidative stress and
immune status in HIV-infected patients.
Methods In a prospective double blind clinical trial, 30 patients (25 male, 5 female; mean
age (^ SD) 42 ^ 9´8 years) with stable HIV infection (221 ^ 102 CD4 1 lympho-
cytes L21) were randomized to a supplemental diet with a daily dose of 45 g whey proteins
of either Protectamin (Fresenius Kabi, Bad Hamburg, Germany) or Immunocal
(Immunotec, Vandreuil, Canada) for two weeks. Plasma concentrations of total, reduced
and oxidized GSH, superoxide anion (O2±) release by blood mononuclear cells, plasma
levels of TNF-a and interleukins 2 and 12 were quantified with standard methods at
baseline and after therapy.
Results Pre-therapy, plasma GSH levels (Protectamin: 1´92 ^ 0´6 mM; Immunocal:
1´98 ^ 0´9 mM) were less than normal (2´64 ^ 0´7 mM, P ˆ 0´03). Following two
weeks of oral supplementation with whey proteins, plasma GSH levels increased in the
Protectamin group by 44 ^ 56% (2´79 ^ 1´2 mM, P ˆ 0´004) while the difference in the
Immunocal group did not reach significance (124´5 ^ 59%, 2´51 ^ 1´48 mM, P ˆ 0´43).
Spontaneous O2 ± release by blood mononuclear cells was stable (20´1 ^ 14´2 vs.
22´6 ^ 16´1 nmol h21 1026 cells, P ˆ 0´52) whereas PMA-induced O2 ± release
decreased in the Protectamin group (53´7 ^ 19 vs. 39´8 ^ 18 nmol h21 1026 cells,
P ˆ 0´04). Plasma concentrations of TNF-a and interleukins 2 and 12 (P . 0´08, all
comparisons) as well as routine clinical parameters remained unchanged. Therapy was well
tolerated.
Conclusion In glutathione-deficient patients with advanced HIV-infection, short-term oral
supplementation with whey proteins increases plasma glutathione levels. A long-term
clinical trial is clearly warranted to see if this `biochemical efficacy' of whey proteins
translates into a more favourable course of the disease.
Keywords Cysteine, glutathione, HIV, whey protein.
Eur J Clin Invest 2001; 31(2): 171±178

III. (P. Micke, K. M. Beeh, R. Buhl) and I. (J. F. Schlaak) Medical
Department, University Hospital, D-55101 Mainz, Germany
Correspondence to: Dr med. Patrick Micke, Pulmonary Division,
III. Medical Department, Mainz University Hospital, D-55101
Mainz, Germany. Tel.: 1 49±6131±176850; fax: 1 49±6131±
176668; e-mail: p.micke@3-med.klinik.uni-mainz.de
Received 15 June 2000; accepted 11 October 2000
Introduction
The tripeptide glutathione (L-g-glutamyl-L-cysteinyl-gly-
cine; GSH) plays a pivotal role in metabolic and cell-cycle
related functions in virtually all cells. Its ability to directly
scavenge free radicals and to act as co-substrate in the
glutathione peroxidase catalyzed reduction of H2O2 and
lipid hydroperoxides [1] makes GSH central to defence
mechanisms against intra- and extra-cellular oxidative stress.

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172 P. Micke et al.

In addition, glutathione and glutathione transferases are milk products, drug addiction or relevant concomitant
major components among the mechanisms involved in the diseases. None of the patients were to consume excessive
metabolism of xenobiotics [2]. amounts of products likely to alter GSH levels (e.g. N-
HIV-infection is characterized by a systemic glutathione Acetylcysteine, alcohol, acetaminophen) for the 3 months
deficiency [3,4], which develops only weeks following before the study.
infection [5], and by increased oxidative stress [6,7].
Surprisingly, even given these facts, the role of the
glutathione system in the pathogenesis of HIV-associated Study design
disease and its impact on the clinical course of the
infection is yet unclear [8,9]. However, there is consensus A prospective, randomized, double blind clinical trial was
that HIV-infected individuals have subnormal GSH conducted between August 1998 and March 1999.
concentrations in plasma [3,4], lung epithelial lining Following a run-in period of one week to establish baseline
fluid [3] peripheral blood leukocytes [10] and CD4 1 levels for all study parameters, patients were randomized
lymphocytes [11]. Similarly, levels of cysteine and of on a 1 : 1 ratio to receive 45 g whey protein per day
glutamine, the second important glutathione precursor of two different formulas (Protectamin(r) [Fresenius] or
amino acid, are markedly decreased in blood and tissues of Immunocal(r) [Immunotec]; Table 1) for 14 days. The
HIV-infected individuals [12]. These findings raised hopes production processes are distinct, with a lower isolation
that therapeutic intervention with glutathione or gluta- temperature (, 728C) for Immunocal. Vanilla flavour was
thione precursors could stop or at least delay the normal added to both formulas. The protein content ranges from
progression of the disease [13,14]. Despite the attractive 75 to 95% with a fat content of 0±6%. The products were
theoretical background, evidence that augmentation of supposed to be taken in three equal portions of 15 g per
deficient glutathione levels really influences the course of day mixed with a nutritional adjuvant (e.g. milk, yoghurt,
the infection or even prolongs survival is scant. In a recent buttermilk).
double blind placebo controlled clinical trial, low gluta- Plasma glutathione levels were continuously monitored
thione levels in CD4 1 lymphocytes correlated strongly during the run-in phase (days 2 7, 2 5, and 2 3) and
with survival in HIV-infection. Oral administration of during the treatment period (days 1, 3, 5, 8, 10, 12, and
N-acetylcyteine, a glutathione precursor, restored intra- 15). Spontaneous and PMA-induced release of superoxide
cellular GSH levels and significantly increased survival anion by blood monocytes, concentrations of cytokines IL-
rates of patients in a two-year open-label follow-up study 2, IL-12 and TNF-a, peripheral blood leukocytes includ-
[15]. As a consequence, strategies to increase glutathione ing CD4 1 and CD8 1 lymphocytes, plasma immuno-
concentrations and to compensate for the oxidant-anti- globulins, and routine clinical parameters were quantified
oxidant-imbalance were again brought into focus of at screening visit (day 2 7), at the beginning (day 1) and at
clinical research and patients' interest. the end of the treatment period (day 15). The protocol was
Sufficient cysteine supply is essential for the main- approved by the Institutional Review Board of the
tenance of the glutathione pool [16]. Thus, highly purified University of Mainz and conducted in accordance with
cysteine-rich preparations of whey proteins have been the declaration of Helsinki. All subjects gave informed
proposed as ideal dietary supplements. A pilot study, consent for the study.
including three HIV-infected patients who were orally
treated with 40 g whey protein per day for three months,
Table 1 Amino acid content of the whey protein formulas
revealed an increase of GSH levels and body weight [17].
With this as background, the aim of this study was: (1) to
Amino acid Immunocal (g 100g21) Protectamin (g 100g21)
evaluate the influence of oral whey protein supplementa-
tion on the plasma GSH concentrations and the ex vivo Aspartatic 9´80 10´50
inflammatory homeostasis of HIV-infected individuals and Glutamic 16´37 17´48
(2) to compare two different whey protein formulas. Serine 3´56 5´58
Glycine 1´64 1´84
Histidine 2´04 1´73
Arginine 2´30 2´24
Threonine 4´63 6´97
Methods Alanine 4´8 4´87
Proline 3´76 6´01
Study population Tyrosine 3´76 1´97
Valine 4´53 6´06
The study population included HIV seropositive indivi- Methionine 2´13 2´27
duals aged . 18 years, with a diagnosis of HIV infection Isoleucine 6´41 6´32
confirmed by ELISA and Western blot analysis, a CD4 1 Leucine 12´56 9´96
lymphocyte count , 300 mL21, and a CD4/CD8 ratio Phenylalanine 4´00 3´15
Lysine 10´68 9´19
, 1´2. Patients had to be clinically stable, i.e. absence of
Tryptophane 2´86 1´53
severe infection, and life expectancy had to be . 6 month. Cysteine/Cystine 4´17 2´28
Exclusion criteria were severe diarrhoea, intolerance to

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Biologic samples
Venous blood was obtained by standard techniques, always
in the morning after overnight fasting. Extreme care was
taken to avoid haemolysis, and blood samples were
processed immediately.
Glutathione levels and form
Concentrations of total, reduced, and oxidized glutathione
in plasma were quantified with minor modifications of
standard methods, as previously described [3,18]. In
brief, to determine total glutathione levels (i.e. reduced
glutathione, glutathione disulfide (GSSG)), plasma was
mixed with an equal amount of 10 mM 5,5 0 -dithiobis
(2-nitrobenzoic acid) (DTNB) in 0´1 M potassium
phosphate, pH 7´5, that contained 17´5 mM ethylene-
diaminetetraacetic acid (EDTA). The samples were
centrifuged (2000 g, 10 min), and aliquots (50 ml) of
the supernatants were added to cuvettes containing
0´5 U of GSSG reductase in 0´1 M potassium phos-
phate, pH 7´5, that contained 5 mM EDTA. After
incubation for 1 min at room temperature, the assay
reaction was started by adding 220 nM of reduced
nicotinamide adenine dinucleotide phosphate (NADPH)
in 0´1 m potassium phosphate, pH 7´5, that contained
5 mM EDTA in a final volume of 1 mL. The rate of
reduction of DTNB was recorded spectrophotometri-
cally at a wavelength of 412 nm (Beckman DU-70
spectrophotometer, Beckman, Huenden, Germany).
Determination of the total glutathione concentration
was based on standard curves generated from known
concentrations of GSSG (0´125±4 mM) in phosphate
buffered saline, pH 7´4.
To quantify oxidized glutathione disulfide, the various
fluids were mixed, immediately after recovery, with an
equal volume of 10 mM N-ethylmaleimide (NEM) in
0´1 M potassium phosphate, pH 6´5, containing 17´5 mM
EDTA. The samples were then centrifuged at 2000 g,
10 min, and 250 ml of the supernatant was passed through
a SEP-PAK C18 cartridge (Waters Associates, Milford,
USA) that had been washed with 3 mL methanol followed
by 3 mL distilled water, and the effluent was collected.
GSSG was eluted from the column with 1 mL of 0´1 M
potassium phosphate, pH 7´5, that contained 5 mM
EDTA. An aliquot (750 ml) of the combined effluent and
eluate was mixed with 250 ml 0´1 M potassium phosphate,
pH 7´5, that contained 5 mM EDTA, 800 mM DTNB,
2 U mL21 glutathione reductase, and 1 mM NADPH,
and the rate of reduction of DTNB was recorded
spectrophotometrically at 412 nm. Standard curves were
derived from dilutions of known concentrations of GSSG
(0´125±4 mM) that had been mixed with 10 mM NEM
and chromatographed with SEP-PAK C18 cartridges as
described above.
The amount of reduced glutathione was obtained by
subtracting the amount of GSSG from the total glu-
tathione levels. If values above the range of the standard
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Whey protein increases glutathione levels 173
curve were observed, parallel runs of samples diluted with
0´9% NaCl were performed. All measurements were
carried out in duplicate.
Release of superoxide anion by blood monocytes
Peripheral blood mononuclear cells were isolated by
Ficoll-density gradient centrifugation [19]. The cells
were washed three times and resuspended in tissue culture
medium (macrophage serum free medium; Gibco, Karls-
ruhe, Germany). The percentage of blood monocytes
(34 ^ 9%) was determined by Giemsa staining. The cells
were allowed to adhere in 24-well tissue culture plates
(Falcon Labware, Heidelberg, Germany) at a concentra-
tion of 0´5  106 cells per well in DMEM for 1 h, 378C.
The supernatant was discarded and the adherent cells were
washed three times with HBSS (Biochom, Berlin, Ger-
many) at 378C. The adherent cells were then incubated in
the presence of 80 mM ferricytochrome C (Sigma,
Steinheim, Germany) for 30 min, 378C to assay for O2 ±
release [20]. Monocytes from each individual were
evaluated in duplicate. A negative control comprised of
cells incubated with 80 ñM ferricytochrome C and
2 mg mL21 superoxide dismutase (SOD; Sigma) was
evaluated in parallel for each individual. At the end of
the incubation period, the absorbency of the cell-free
supernatant was determined at 550 nm (Beckman DU-70
spectrophotometer). The amount of superoxide anion
spontaneously released by the monocytes was determined
by subtracting the absorbency values obtained in the
presence of SOD from those obtained for each sample in
the absence of SOD. All data are presented as the amount
of O2 ± released, as quantified in nmol ferricytochrome C
reduced hr21 1026 cells. To determine the overall capacity
of the cells to release O2 ±, parallel cultures of adherent
cells were incubated with phorbol 12-myristate 13-acetate
(PMA, 100 ng mL21) for 30 min, 378C and O2 ± release
quantified as described above.
Cytokine concentrations
Plasma levels of cytokines IL-2, IL-12 and TNF-a were
quantified using commercial enzyme immunoassay kits
(Coulter-Immunotech, Paris, France).
Statistical analysis
Statistical analysis was performed using REPORT 6´1 and
Smart Test 1´3 software. Evaluation of efficacy was done
by 'intention to treat' analysis. The change from baseline
plasma glutathione levels at day 15 was defined as the
primary end point. Secondary end points were levels of
glutathione disulfide (GSSG), CD4/CD8 ratio, superoxide
anion release by blood monocytes, plasma cytokine levels,
immunoglobulines (Ig) G, A, M and E, amount and severity
of infections and side-effects. Unless otherwise specified,
all data are given as mean ^ SD. The multivariate
174 P. Micke et al.

Wilcoxon±Mann±Whitney U-test was used for pre/post
analysis. Correlation coefficients were calculated by linear
regression analysis. A P-value of less than 0´05 was
considered statistically significant.
Results
Base-line characteristics of the study population
The study population included 25 male and 5 female HIV-
seropositive individuals (mean age 42´9 ^ 9´8 years.). All
patients belonged to defined risk groups (21 homosexuals,
4 former drug abusers and 5 with a history of sexual
contact with an infected partner). The majority of patients
(n ˆ 27) were under stable antiretroviral therapy. Most
patients received a combination of four (n ˆ 1), three
(n ˆ 17), or two (n ˆ 8) drugs. Usually, the combinations
consisted of one or two reverse transcriptase inhibitors and
a proteinase inhibitor. One patient received only mono-
therapy. Two patients (6´6%) of the Immunocal group and
one patient of the Protectamin group (3´3%) were without
antiretroviral therapy. In general antiretroviral treatment in
the two arms did not differ and was unchanged during the
study period.
Within the two treatment groups there were no relevant
differences in age, height, weight, body mass index (BMI),
smoking habits, performance status, routine clinical
parameters, and severity of the disease (Table 2). Due to
the small sample size, the proportion of female subjects
was larger in the Protectamin group. For comparison, 10
healthy individuals (7 male, 3 female; mean age
31´2 ^ 3´3 years; weight 72´2 ^ 9 kg) were evaluated.
All were HIV-seronegative by ELISA.
Plasma glutathione concentrations:
Baseline (day 1) total plasma glutathione levels of HIV-
infected patients were significantly lower than reference
Table 2 Study population (day 1)
values obtained from healthy blood donors (1´96 ^
0´8 mM vs. 2´64 ^ 0´74 mM, P ˆ 0´03). There were no
differences in baseline glutathione levels in the two treatment
arms (Immunocal: 1´99 ^ 0´9 mM, Protectamin: 1´92 ^
0´6 mM, P ˆ 0´83).
After 2 weeks of therapy (day 15) total plasma
glutathione levels were 2´51 ^ 1´48 mM in the Immunocal
group (124´5 ^ 59%, P ˆ 0´43) and 2´79 ^ 1´2 mM in
the Protectamin group (144 ^ 56%, P ˆ 0´004). The
same was true if all pretrial glutathione measurements
(days 2 7, 2 5, 2 3, 1) were compared with all measure-
ments performed during the treatment period (days 3, 5,
7, 10, 12, 15; Protectamin: 2´07 ^ 0´70 mM vs. 2´41 ^
0´99 mM, P ˆ 0´020; Immunocal: 1´93 ^ 1´09 mM vs.
2´35 ^ 1´59 mM, P ˆ 0´067).
The absolute change from baseline to day 15 was similar
in both treatment groups (Immunocal: 1 0´42 ^ 1´2 M,
Protectamin: 1 0´77 ^ 0´9 M, P ˆ 0´84; Figs 1 and 2).
As expected, levels of oxidized glutathione were mostly
near or beneath the lower limit of detection of the assay
and were not included in the statistical analysis.
Release of superoxide anion by blood monocytes:
There were no differences between the two treatment
groups regarding spontaneous and PMA-stimulated super-
oxide anion release by blood monocytes, neither at
baseline nor at day 15. PMA-induced O2 ± release by
blood monocytes decreased significantly in the Protecta-
min group (P ˆ 0´04, Table 3). With this exception, O2 ±
release did not change in either group during therapy
(P . 0´47, all comparisons; Table 3).
Plasma cytokine concentrations
Despite enormous variations of plasma concentrations of
cytokines IL-2, IL-12 and TNF-a, both from patient-to-
patient and from day-to-day, there were no differences

Immunocal (n ˆ 15) Protectamin (n ˆ 15)

Age (years) 42´7 ^ 10´3 43´4 ^ 9´2

Sex (male/female) 14/1 11/4
Height (cm) 176´0 ^ 5´21 174´5 ^ 8´9
Weight (kg) 71´8 ^ 10´1 70´0 ^ 9´9
BMI (kg m22) 23´0 ^ 3´4 22´5 ^ 2´1
Smoker (number) 8 7
CDC classification
C3 11 11
C2 1 0
B3 1 2
B2 2 2
Creatinine (mg dL21) 1´0 ^ 0´2 1´0 ^ 0´2
GOT (U L21) 30´6 ^ 13´5 33´6 ^ 15´4
GPT (U L21) 33´9 ^ 20´8 35´6 ^ 18´6
Plasma proteins (g dL21) 8´0 ^ 0´5 8´0 ^ 0´4
CD4 1 ± Lymphocytes (mL21) 226 ^ 122 241 ^ 95
CD4/CD8 ratio 0´30 ^ 0´20 0.41 ^ 0´37

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Figure 1 Total plasma glutathione levels (mean ^ SEM) were
determined pre-therapy (days 2 7, 2 5, 2 3), at the beginning of
the treatment period (day 1), during supplementation (day 3, 5, 8,
10, 12) and after the treatment (day 15) with Immunocal.
within or between treatment groups before and after
therapy (P . 0´08, all comparisons; Table 4). Plasma
concentrations of IL-2 were mostly under the detection
limit of the assay used and were not included in the
statistical analysis.
CD4 cell count and CD4/CD8 ratio
CD4 1 cell counts (243 ^ 102 mL21 vs. 217 ^ 90 mL21,
P ˆ 0´34) and CD4/CD8 ratio (0´35 ^ 0´27 vs. 0´31 ^
Figure 2 Total plasma glutathione levels (mean ^ SEM) were
determined pretherapy (days 2 7, 2 5, 2 3), at the beginning of
the treatment period (day 1), during supplementation (day 3, 5, 8,
10, 12) and after the treatment (day 15) with Protectamin.
Q 2001 Blackwell Science Ltd, European Journal of Clinical Investigation, 31, 171±178
Whey protein increases glutathione levels 175
0´29, P ˆ 0´84) were unchanged during the study period.
Interestingly, linear regression of baseline CD4-positve
lymphocyte numbers and total plasma glutathione
revealed a significant correlation (r ˆ 0´37, P ˆ 0´05).
Plasma concentrations of immunoglobulines A, E,
G, and M
Concentrations of immunoglobuline subclasses were
unchanged during the study period (P . 0´09, all
comparisons, data not shown).
Adverse events
26´6% of patients receiving Immunocal and 60% of
patients receiving Protectamin reported adverse events
during the study period. However, among these, only a few
moderate side-effects were potentially related to the study
diet. In this regard, four patients complained about slight
gastrointestinal disturbances, which they related to the
change in nutrition, and one patient in the Immunocal
group discontinued the study due to gastrointestinal
symptoms. There were no serious adverse effect. No
differences in frequency or severity of side-effects were
observed between treatment arms.
Discussion
A systemic glutathione deficiency is among the earliest
consequences of HIV infection [3,4]. The glutathione
system is therefore a potential target for adjuvant therapy
in HIV-infected patients. This prospective, randomized,
double-blind clinical trial demonstrates, for the first time,
that short-term oral treatment of adult glutathione-
deficient patients with advanced HIV-infection, with a
cysteine-rich nutritional supplement derived from whey
protein, significantly increases plasma glutathione levels.
Surrogate markers of oxidative stress and functional
parameters of the immune system were unchanged.
Therapy was well tolerated, no clinically meaningful
adverse effects were seen.
Several lines of evidence indicate that the glutathione
deficiency may be of clinical relevance in the course of
HIV infection: firstly, normal intra- and extracellular
glutathione levels are necessary for optimal function of
the cellular constituents of the immune system. This is of
particular importance in HIV infection, a disease char-
acterized by a profound dysfunction of the immune
system. Cell-culture studies demonstrated that lowering
intracellular GSH levels decreases cell survival of TNF-a
stimulated T-lymphocytes and alters T cell function
[21,22]. Secondly, addition of glutathione or the glu-
tathione precursor N-acetylcysteine (NAC) stopped HIV
replication in vitro via inhibition of NF-k-B-activation
[23,24]. Thirdly, glutathione and the enzymes of the
176 P. Micke et al.
Table 3 Spontaneous and PMA induced superoxide anion release of isolated blood monocytes
pre-therapy (day 1) and after treatment (day 15) with whey protein
Immunocal
Spontaneous O2 ± release (nmol hr 1026 cells)
PMA induced O2 ± release (nmol hr 1026 cells)
Protectamin
Spontaneous O2 ± release (nmol hr 1026 cells)
PMA induced O2 ± release (nmol hr 1026 cells)
glutathione redox cycle are central to the antioxidant
defenses of all tissues. The deficient antioxidant protection
secondary to the glutathione deficiency may be even more
consequential because HIV infection is also characterized
by marked oxidative stress. A beneficial effect of a therapeutic
correction of the imbalance between oxidants and anti-
oxidants has been demonstrated in vitro and in vivo
[25,26]. Fourthly, as previously observed [15], in the
study population GSH levels correlated with CD4 1 cell
counts. This may mainly reflect the fact that the
glutathione deficiency becomes more pronounced as the
disease progresses. Finally, however, intracellular glu-
tathione levels of CD4 1 lymphocytes predicted survival
in HIV infected individuals while restoration of intra-
cellular glutathione levels by oral NAC increased survival
rates of patients in a 2 year open-label follow-up study
[15]. The latter points in particular stress the clinical
significance of the systemic glutathione deficiency, and
the potential benefit of patients from a therapeutic
intervention directed at augmentation of glutathione
concentrations [13,14].
Different strategies have been suggested to increase
deficient glutathione concentrations in HIV-infected patients,
e.g. aerosol inhalation of GSH [27], oral GSH esters [23],
cysteine rich whey proteins [17], L-2-oxothiazolidine-4-
carboxylate (OTC, a prodrug of cysteine) [28], or most
often, oral NAC [29]. Any such adjuvant treatment to
conventional antiretroviral therapy should meet certain
requirements: (1) a potential to increase glutathione levels,
(2) no or least possible interference with established
treatment regimens, (3) comfortable application, and
(4) tolerable side-effects. Theoretically, highly purified
preparations of whey protein fulfil these criteria. They
are ideal dietary supplements to correct deficiencies of
glutathione or cysteine, its precursor amino acid. Firstly,
these preparations contain large amounts of cysteine,
glutamine and glutamyl-cysteinyl and therefore are a
convenient source of these amino acids. Secondly, dietary
peptide composition can regulate the intestinal home-
ostasis and lead to a lower stool frequency in HIV-infected
patients [30]. Thirdly, whey proteins showed in in vitro
experiments so called 'bioactivity' and were able to
enhance immune responses of leukocytes [31]. Further,
they are easy and convenient to apply as an adjuvant to the
daily meals, and they are virtually free of clinically relevant
adverse effects or interactions with other medication.
With this as background, this study was performed to
Q 2001 Blackwell Science Ltd, European Journal of Clinical Investigation, 31, 171±178
Day 1 Day 15
18´2 ^ 11 26´0 ^ 17
46´5 ^ 28 53´3 ^ 21
21´9 ^ 17 19´2 ^ 16
53´7 ^ 19 39´8 ^ 18
evaluate the efficacy and safety of whey protein supple-
mentation in HIV-infected individuals. As in former
studies [3,15] it was confirmed that HIV-infected patients
have plasma glutathione levels below the normal range,
which were significantly augmented by oral treatment with
whey proteins. If both treatment arms are analysed together,
plasma GSH-concentrations of patients increased by 32%
compared to baseline within just two weeks. This is true
despite the fact that only the results of the Protectamin
group reached statistical significance, and that a direct
comparison of the two whey protein formulas, with respect
to their effect on glutathione levels, did not reveal a
statistically significant superiority of one protein preparation.
This feasibility study was neither designed nor powered
enough to answer the latter question. The result is
interesting, however, given that the daily cysteine intake
in the Immunocal group was twice as high as in the
Protectamin group. It is tempting to speculate that
increasing glutathione plasma levels may suppress liver
glutathione synthesis by feedback inhibition, thereby
limiting any increase beyond a certain concentration
[32]. Supporting this hypothesis are the results of studies
aimed at augmenting glutathione levels in patients with
inflammatory lung disease, another condition character-
ized by a marked systemic glutathione deficiency [33,34].
These findings underline the necessity to choose optimal
dosing strategies in future long-term studies. Currently,
suggested daily cysteine doses range from low, individually
adjusted doses to avoid overdosing [29] to as high as 4 g
[15]. In this regard, it is postulated that protein isolation
protecting thermolabile amino acids and oligopeptides is a
crucial step for the biochemical efficacy of cysteine
supplementation [17,35]. The production of Immunocal
follows this recommendation. However, Immunocal was
definitely not superior to Protectamin in terms of its effect
Table 4 Plasma concentrations of interleukin-12 and TNF-a
before (day 1) and after treatment (day 15) with whey protein.
Day 1 Day 15
Immunocal
IL-12 (pg mL21) 31´6 ^ 29 25´9 ^ 17
TNF-a (pg mL21) 12´4 ^ 9´6 11´4 ^ 9´7
Protectamin
IL-12 (pg mL21) 31´7 ^ 24 47´5 ^ 31
TNF-a (pg mL21) 33´2 ^ 55 41 ^ 61

on the glutathione system. Therefore, the results of this
study do not support this hypothesis.
The significant increase of glutathione levels in HIV-
infected patients did not translate into a clinically relevant
treatment response during the treatment period. All
secondary study endpoints indicative of oxidative stress
and the patient's immune status remained unchanged,
most likely a consequence of the short duration of the
study of only two weeks and to the small patient sample.
Furthermore, it remains yet to be seen if oral whey
proteins influence glutathione concentrations in tissue
compartments relevant in the course of HIV infection (e.g.
lymphocyte subpopulations).
In general, oral whey protein supplementation was well
tolerated. Only one person quit the trial because of mild
gastrointestinal adverse effects. This type of unwanted
effect could possibly be related to the study medication,
but is also frequently observed secondary to regular
antiretroviral therapy. Most of the patients included in
this study (66%) chose to continue whey protein
supplementation after the trial was finished.
In conclusion, this study shows that whey protein
formulas are an effective and well tolerated way to increase
glutathione levels in HIV-infected individuals, and there-
fore, a convenient alternative to conventional glutathione
prodrugs such as NAC. This conclusion is justified by the
data presented, despite the fact that this trial did not
include a placebo arm. Until now, nearly all evidence in
favour of glutathione augmentation therapy in diseases
characterized by glutathione deficiency is based on either
in vitro experiments, animal models, or short-term clinical
trials. It remains yet to be seen if the `biochemical efficacy'
of therapeutic strategies, such as augmentation of glu-
tathione levels by oral whey proteins, translates into a more
favourable course of the disease assessable by key markers
of disease activity. A long-term randomized controlled trial
is clearly warranted to study the benefit of whey protein
therapy on survival and the course of the disease, especially
as long-term oral therapy with whey proteins has little
adverse effects. The results presented in this study support
this conclusion and underline the importance of the
selection of an appropriate dose of whey proteins.
Acknowledgements
Immunocal and Protectamin were provided by Fresenius
Kabi (Deutschland GmbH),Bad Homburg, Germany.
We greatly appreciate the support of Prof. Dr E. B.
Helm, III Medical Department, Frankfurt University
Hospital, Germany.
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