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The Redox State as a Correlate of Senescence and Wasting
and as a Target for Therapeutic Intervention
By Volker Hack, Raoul Breitkreutz, Ralf Kinscherf, Helmut Röhrer, Peter Bärtsch, Friedemann Taut,
Axel Benner, and Wulf Dröge
The loss of body cell mass (bcm) in senescence and wasting
is poorly understood. We now show that the plasma cystine/
acid soluble thiol ratio, ie, an indicator of the redox state, is
increased in old age and cancer patients and correlated with
a decrease in bcm and plasma albumin. A cause/effect
relationship was suggested by two independent studies
with N-acetyl-cysteine (NAC). NAC caused an increase in the
bcm of healthy persons with high plasma cystine/thiol
ratios, and treatment of cancer patients with NAC plus
interleukin-2 caused an increase in bcm, plasma albumin,
and functional capacity. Albumin levels below 680 mmol/L
were associated with an increase in body water. Our studies
suggest that the shift in the redox state may contribute to
the loss of bcm and may provide a quantitative guideline for
therapeutic intervention. Treatment of cancer patients with
thiol-containing antioxidants may improve the quality of life.
r 1998 by The American Society of Hematology.
L
Because the acid-soluble sulfhydryl groups represent mostly
cysteine, the cystine/thiol ratio is essentially an indicator of the
equilibrium between cysteine disulfide (cystine) and cysteine,
ie, of the redox state of the cysteine/cysteine disulfide redox
couple in the plasma. This is analogous to the glutathione/
glutathione disulfide (GSH/GSSG) ratio that is widely used as
an indicator of the intracellular redox state. The effect of
N-acetyl-cysteine (NAC) on the plasma albumin level and bcm
has been studied to show a cause/effect relationship. Correlations between biochemical and biophysical parameters have
been analyzed to test the strength of hypothetical linkages. The
resulting pool of data illustrates the normal and pathological
ranges of redox states and suggests in addition that certain redox
states within the normal range might be associated with certain
risks.
To determine the pathological role of the redox state in cancer
patients, we investigated the hypothesis that treatment with the
cysteine derivative NAC may have a positive effect on bcm and
functional capacity of cancer patients. Prospectively defined
secondary outcome measures were the survival time, the
intracellular levels of glutathione (GSH) and glutathione disulfide (GSSG) in peripheral blood mononuclear cells (PBMC),
and the plasma levels of amino acids and acid-soluble thiol. In
addition, the plasma albumin level was determined because the
albumin level of cachectic patients is a strong predictor of
survival and cost of treatment,10 and because several previous
attempts to increase the albumin level by nutritional therapy
were not successful.10,25-27 Also, in elderly subjects a low
albumin level was found to be correlated with a low 10-year
IKE APOPTOTIC cell death, senescence and wasting are
largely autonomous and biologically meaningful processes associated with an increased probability of death. The
hallmarks of these processes include the massive loss of body
cell mass (bcm) and muscle function, decreased resistance to
infections, frailty (increased probability of disability), and
organ failure.1-9 A biochemical correlate of senescence and a
quantitative measure of cachexia is the decrease in the plasma
albumin level.10-15 Wasting is a common phenomenon in
malignancies,2-5 sepsis, trauma,6 and certain infectious diseases
including human immunodeficiency virus (HIV) infection.7,8
Wasting and death of an individual organism (ie, a competitor
for food and space) may be advantageous for the species, just as
apoptosis of an individual cell is typically advantageous for the
organism. However, for the individual human subject the loss of
bcm and muscle strength in senescence and wasting is often
associated with psychological stress and financial burden. Its
medical and social relevance may increase even further because
the average lifespan is increasing progressively.16,17 Most of the
years beyond age 70 are years of compromised physical and
social function,16-18 and 45% of the people in the United States
over the age of 85 years require assistance.16 The loss of muscle
strength in the elderly is mostly related to loss of muscle mass.19
One of the prevailing hypotheses states that senescence may
result from the accumulation of oxidative damage20-22 and that
dietary antioxidants may slow the degenerative process.20,22,23
In support of this paradigm, vitamin E was shown to ameliorate
age-related health problems,24 and certain age-related degenerative changes were even found to be reversed by antioxidant
treatment.22,23 However, the available information fails to tell us
what type and quantity of antioxidant we need and how we can
monitor that we have received enough. In this report we now
show (1) that senescence and wasting are associated with an
easily demonstrable change in the redox state of the blood
plasma, and (2) that important consequences of changes in the
redox state can be shown in human subjects within a few weeks
or months. In view of these relatively short observation periods,
our findings may provide, for the first time, a quantitative
guideline for a redox-oriented prophylactic therapy.
To increase the weight of the evidence, we performed several
independent and complementary studies. To study the hypothetical role of the redox state in wasting, senescence, and albumin
degradation, we determined in healthy elderly subjects and
cancer patients the ratio of plasma cystine and acid-soluble
sulfhydryl groups as a measure of the thiol/disulfide redox state.
Blood, Vol 92, No 1 (July 1), 1998: pp 59-67
From the Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg; Onkologische Schwerpunktpraxis, Ihringen; the
Department of Sports Medicine, University of Heidelberg, Heidelberg;
the Department of Anesthesiology, University of Heidelberg, Heidelberg; and the Biostatistics Unit, Deutsches Kresbsforschungszentrum,
Heidelberg, Germany.
Submitted December 22, 1997; accepted February 18, 1998.
Address reprint requests to Wulf Dröge, PhD, Division of Immunochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld
280, D-69120 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9201-0012$3.00/0
59
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60
HACK ET AL
survival rate12 and loss of muscle mass (sarcopenia).13 Human
albumin and bovine albumin contain a single unpaired cysteine
residue (Cys34) with antioxidative function.13,28-30 In the blood,
albumin exists largely in its reduced form (mercaptalbumin,
MA) and to a lesser extent in the oxidized form (nonmercaptalbumin, NA). The latter consists mainly of a mixed proteincysteine disulfide or protein-glutathione disulfide and increases
in proportion with age.30-32 It has been reported that redox
processes mediate the conversion of albumin into an aged form
with a threefold higher catabolic rate.29 The plasma glutamate
level was of interest because the process of skeletal muscle
wasting is commonly associated with a strong increase in the
plasma glutamate level as a consequence of a decreased
glutamate uptake capacity of the skeletal muscle tissue.33
Nitrate and nitrite levels were also included retrospectively to
monitor the effects of interleukin-2 (IL-2) on nitric oxide
production. High doses of IL-2 were previously found to induce
an increase in nitric oxide production as manifested by increased plasma nitrate levels, hypotension, and a capillary leak
syndrome associated with an increase in body water and body
weight.34
SUBJECTS, MATERIALS, AND METHODS
Study on healthy human subjects (study A). Plasma amino acids
and acid-soluble thiols have been determined in the venous blood of 205
randomly selected healthy human subjects. The age distribution can be
seen in Fig 1. Plasma albumin levels have been determined in 86
subjects and the bcm index in 93 subjects.
Prospectively designed randomized clinical trial for the comparison
of the effect of NAC plus IL-2 versus IL-2 on cancer patients (study B).
Included were adult patients with different types of inoperable cancer
who had previously failed to respond to standard therapy. Not eligible
were patients with anorexia, a life expectancy of less than 2 months, and
any type of cancer therapy during the preceding 6 weeks. Fifty patients
were recruited initially. One patient who wanted a specific treatment
was excluded. Twenty-seven patients were treated with IL-2 only (4 of
these patients died and 1 left the study before the second examination)
and 23 patients were treated with IL-2 plus NAC (3 of these patients
died before the second examination). Randomization was performed by
the attending physician (H.R.) (ie, by tossing a coin) and was stratified
according to the type of tumor; treatment was known to both the
physician and the patient (unblinded study). The sample size was
estimated on the basis of preliminary information about intracellular
GSH/GSSG ratios and plasma glutamate levels. It was estimated that 25
patients were needed for each treatment group to detect desirable
changes with a power of at least 80% with a one-sided t-test and 5%
significance level.
An additional group of 20 patients was recruited subsequently for
treatment with standard chemotherapy. Six of the 20 patients died and
eight left the study before the second examination. This may be
explained by their previous failure to respond to standard therapy.
IL-2 was administered at a dose of 6 3 106 IU subcutaneously twice a
week. NAC was taken orally 3 times per day. To avoid excessive plasma
concentrations of cystine and glutamine,35 NAC was used at variable
doses ranging between 0.6 and 4.2 g depending on the latest measurements of plasma cystine and glutamine levels. A daily dose of 4.2 g
NAC was assigned to patients with a plasma glutamine (gln) level ,550
µmol/L and cystine (cys2) ,60 µmol/L, 3.6 g NAC for patients with gln
,550 µmol/L and cys2 . 60 µmol/L and for patients with gln 550 to 700
µmol/L and cys2 , 50 µmol/L, 2.4 g NAC for gln 550 to 700 µmol/L and
cys2 . 60 µmol/L, and 0.6 g NAC for patients with gln . 700 µmol/L.
With these dose schedules, none of the patients under NAC therapy
Fig 1. Correlation between plasma redox state and age in healthy
persons (study A). Postabsorptive plasma amino acid and acidsoluble thiol levels have been determined in the plasma from the
cubital vein of 205 randomly selected healthy human subjects of both
sexes.
showed a plasma cystine level . 100 µmol/L and glutamine level . 910
µmol/L. On the basis of established knowledge, neither of the three
treatment protocols, ie, low-dose IL-2 alone, IL-2 plus NAC, or
standard therapy could be expected to have a priori an obvious
advantage or disadvantage for these patients with respect to the sum of
therapeutic benefits and potential side effects.
The primary and secondary outcome measures of study B have been
described in the introduction. The functional capacity index as defined
in Table 3, point 4 of ref 36 was taken as a measure of how the patient
viewed her/his own quality of life (0 5 normal, no limitations; 1 5 not
normal, but stable enough to be up with fairly normal activity; 2 5 not
feeling up to most things but in bed less than half the day; 3 5 able to do
little activity and most of the day in bed or chair; 4 5 rarely out of bed).
Height, body mass, bcm, and body water were determined and blood
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REDOX STATE IN SENESCENCE AND WASTING—TARGET FOR THERAPY
samples were taken at baseline examination before the start of therapy.
The second measurements were performed about 4 weeks after baseline
examination and start of the therapy. Additional measurements followed
at larger time intervals. Blood samples were taken from the cubital vein
in the postabsorptive period. Acid-soluble thiol levels have been
determined in all samples from the two groups with NAC plus IL-2 and
IL-2 only, but only in one sample from the standard treatment group
(n 5 51).
The studies were conducted according to the principles of the
Declaration of Helsinki. Before entering one of the treatment programs,
each person was given a detailed explanation of all testing procedures
and signed an informed consent. Each patient agreed to be assigned to
the chosen treatment protocol.
Longitudinal study on a single healthy individual (study C). Blood
samples were obtained from a single healthy male subject in the sixth
decade of life at randomly distributed time points over an observation
period of 2 years.
Study on the effects of NAC on bcm of healthy subjects and its
correlation with plasma amino acid levels (study D). The study was
designed as a randomized double-blind trial. Healthy and moderately
well-trained men between 20 and 60 years old were recruited into the
study and randomly assigned to the verum group (n 5 18) and placebo
group (n 5 20). The dose of NAC was 2 3 200 mg orally per day on 3
days per week for 4 weeks. During this period both groups were also
subjected to a program of anaerobic physical exercise. For details of this
program see the report of Kinscherf et al.37
Determination of bcm, bcm index, and body water. The bcm is
defined as the sum of the oxygen-consuming, potassium-rich, and
glucose-oxidizing cells. In practical terms it is the total body mass
minus body fat and extracellular mass (bone and extracellular water).
Bcm and body water were computed from the body weight and the
electrical resistance and reactance of the body to weak alternating
current (ie, by bioelectrical impedance analysis) with a commercial
computer program as described previously.37 The biological and
technical variability of longitudinal bcm measurements has been
assessed previously.37 In analogy to the body mass index (body
weight/height2), the bcm index was defined as the ratio of body cell
Fig 2. Thiol redox state,
plasma albumin, and bcm index
of healthy subjects and cancer
patients. Each point represents
an individual person. The bcm
index was defined as the bcm/
height2 in analogy to the body
mass index. Note that the bcm
index and the plasma albumin
level have not been determined
for all subjects of study A and
study B (see Subjects, Materials,
and Methods). Horizontal and
vertical lines indicate the window that contains most of the
healthy young subjects less than
35 years old.
61
mass/height2 (kg/m2). The relative total body water was defined as the
ratio of total body water/bcm.
Determination of plasma levels of acid-soluble thiol, albumin, nitrate
and nitrite, and amino acids including cystine, glutamine, and glutamate. Plasma amino acid levels (including cystine, glutamine, and
glutamate concentrations) were determined with the amino-acid analyzer, and acid-soluble thiol was determined with a photometric assay as
described previously.37 Albumin was determined with a commercial kit
(Sigma, Steinheim, Germany),38 and the sum of plasma nitrate and
nitrite was measured colorimetrically by the Griess reaction.39
Statistical analysis. The statistical evaluation of the individual
changes between baseline examination and terminal examination was
performed by the paired t-test for dependent samples (two-tailed). The
data from different treatment groups were compared statistically by the
Kruskal Wallis or Wilcoxon rank-sum tests, or by Student’s t-test for
independent samples as indicated. The Trend test40 was used to compare
the functional activity data. Arithmetic means and standard errors of the
means were used as descriptive statistics. Correlations between parameters were described graphically by scatter plots and linear regression
lines. The strength of relationship was assessed either by Spearman’s
rho or by Pearson’s product correlation coefficient r as indicated. The
result of the statistical test was judged by its P value. A P value ,.05
was regarded as statistically significant.
RESULTS
Senescence is associated with a change in the redox state.
Correlation between bcm index, redox state, and plasma
albumin level in healthy human subjects (study A). Our study
on healthy human subjects (study A) showed a significant
age-dependent increase of the plasma cystine level and a
decrease of the plasma thiol level indicative of an agedependent shift to a more oxidized condition (Fig 1). Our
analysis also confirmed the negative correlation between plasma
albumin concentration and age (r 5 .49, P , 1025, see also Fig
2). A possible linkage between the plasma cystine/thiol ratio,
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62
HACK ET AL
the plasma albumin level, and the bcm index was suggested by
the strong correlation between these parameters (Fig 2, left
panel).
Changes of the redox state, albumin level, and bcm in cancer
patients. Baseline characteristics of the treatment groups in
study B. To determine the role of the redox state in cancer
cachexia, we determined intracellular GSH and GSSG levels
and plasma levels of cystine and thiol and studied the effect of
NAC on bcm, functional capacity, and albumin level of cancer
patients.
Seventy patients with inoperable cancer were recruited. Fifty
patients were randomly assigned to treatment with IL-2 alone or
IL-2 plus NAC. In addition, 20 patients received standard
therapy. The most frequent tumors were carcinoma of the breast
(3/5/3), pancreas (5/3/1), colon (3/2/1), prostate (2/2/1), and
hematological tumors (4/2/0). (The data in brackets indicate the
number of patients treated with IL-2 plus NAC, IL-2 alone, and
conventional chemotherapy, respectively.) Primary outcome
measures were changes of body cell mass and functional
capacity. Secondary endpoints were survival time, intracellular
GSH and GSSG levels, and plasma cystine/thiol ratios as
indicators of pathological redox changes, the plasma glutamate
level as an indicator of skeletal muscle function, plasma
albumin, NO22 and NO32.
The results showed first of all that cancer patients have, on
the average, an altered redox state as manifested by increased
plasma cystine/thiol ratios (Fig 2), decreased intracellular
glutathione levels, and decreased intracellular GSH/GSSG
ratios in the PBMC (Table 1). In comparison with 82 healthy
40- to 70-year-old subjects from study A (mean age, 57.7 6 1.1
years), the cancer patients (mean age, 60.5 6 1.1 years) had at
baseline examination significantly elevated plasma cystine/thiol
ratios (6.99 6 0.57 v 5.53 6 0.22; P , .05), decreased plasma
albumin levels (674 6 9 v 760 6 11 µmol/L; P , 1027), and
elevated plasma glutamate levels (47.7 6 2.9 v 30.6 6 1.8
µmol/L; P , 1025). The bcm indices, albumin levels, and redox
states of the cancer patients were again significantly correlated
with each other and showed a pattern similar to that of healthy
subjects with an age .75 years but different from that of young
or age-matched subjects (Fig 2). Unexpectedly, the two studies
B and A showed only a weak correlation between intracellular
GSSG/GSH ratio and plasma cystine/thiol ratio (r 5 1.32 and
1.23, respectively).
Two of the prognostic factors, ie, plasma albumin and bcm
index, showed an imbalance among the three treatment arms of
study B in favor of the groups treated with IL-2 alone or with
standard therapy if tested by the Kruskal Wallis rank-sum test
(P , .002). The plasma cystine/thiol ratio, plasma glutamate
level, and the functional capacity index, in contrast, were not
significantly different between treatment groups if tested by the
Wilcoxon and Kruskal Wallis rank-sum tests and the Trend test,
respectively (P 5 .08, P 5 .37 and P 5 .09).
Bcm, plasma albumin, plasma glutamate, and the functional
capacity of cancer patients are improved by treatment with NAC
(study B). The treatment groups showed similar survival
curves (Fig 3). However, the IL-2 plus NAC–treated group
showed a significant improvement of functional capacity,
plasma albumin, plasma glutamate level, and cystine/thiol ratio,
if compared with the other two treatment groups (Figs 4 and 5).
Also, in comparison with the individual baseline levels, the 20
IL-2 plus NAC–treated patients showed, on the average, a
significant increase in plasma albumin (651 6 13 to 696 6 20
µmol/L; P , .03) and decrease in plasma glutamate (47.8. 6 5.1
to the essentially normal level 31.0 6 3.6 µmol/L; P 5 .002) if
tested by the paired t-test. The IL-2–treated group, in contrast,
showed a decrease in plasma albumin (713 6 15 to 685 6 16;
P , .02), and a slight increase in plasma glutamate (51.9 6 5.5
to 53.2 6 5.2). A significant increase in bcm was detectable in
the IL-2 plus NAC–treated group after a lag phase. Therefore,
we showed in Fig 5 the subgroup of patients with observation
periods .100 days.
The data from all patients together showed a significant
correlation of the change in functional capacity with the change
in plasma albumin (P , .05) and the change in plasma glutamate (P , .05) if tested by Spearman’s rho test. The change in
albumin level, in turn, was correlated with the relative changes
in bcm, plasma glutamate, and cystine/thiol ratio (r 5 1.61;
P , .001, r 5 2.32; P 5 .05, and r 5 2.48, P , .02, respectively), if tested by Pearson’s r-test.
The relatively low dose of IL-2 caused no increase in body
water or hypotension (data not shown). The plasma levels of
nitrate plus nitrite increased only in the group treated with IL-2
Table 1. Effect of NAC and IL-2 on the Intracellular Glutathione Level in PBMC of Cancer Patients
A
Conventional
Chemotherapy
B
C
D
Significance Between
Groups (P values)
IL-2
NAC/IL-2
Healthy Subjects
n (F/M)
Age
Total intracellular glutathione
(nmol/mg protein)
b
t
6 (4/2)
56.5 6 2.5
22 (9/13)
59.7 6 1.7
20 (12/8)
60.2 6 2.4
82 (34/48)
57.5 6 1
11.6 6 1.75
10.0 6 1.58
12.7 6 1.2
18.9 6 1.9
P , .01
13.5 6 1.4
19.8 6 1.6
P , .005
24.4 6 1.1
24.4 6 1.1
,.03
,.005
GSH/GSSG ratio
b
t
9.85 6 2.80
4.43 6 0.93
8.7 6 1.4
17.5 6 2.1
P , .005
7.8 6 1.6
18.0 6 2.2
P , .001
17.7 6 1.7
17.7 6 1.7
,.005
,.005
Bold face indicates values for which significant changes occur.
Abbreviations: b, baseline examination; t, terminal examination.
A-B
A-C
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REDOX STATE IN SENESCENCE AND WASTING—TARGET FOR THERAPY
63
the decrease in plasma albumin levels below 680 µmol/L was
associated with an increase in relative total body water (Fig 6).
Longitudinal changes in albumin are correlated with changes
in the plasma cystine/thiol ratio during short observation
periods (study C). In view of the relatively slow agedependent changes between the third and tenth decade of life
(see Figs 1 and 2), we performed a longitudinal study on a
single healthy individual in the sixth decade of life to determine
whether a correlation between longitudinal changes in plasma
albumin level and plasma cystine/thiol ratio may be demonstrable also in a healthy person within a relatively short
observation period. Plasma cystine/thiol ratios, albumin levels,
and bcm were determined at 39 randomly chosen time points
during a 2-year observation period. The resulting data (not
shown in detail) showed considerable variations in cystine/thiol
ratios (3.3 to 9.4) and albumin levels (684 to 884 µmol/L) and
showed significant correlations (1) between the albumin level
and plasma cystine/thiol ratio (r 5 2.61; P , 1024) and (2)
between the changes in the albumin level and corresponding
changes in the cystine/thiol ratio (r 5 2.53; P , 1023). How-
Fig 3. Survival time of the two groups treated with IL-2 plus NAC
and IL-2 only (study B). (Upper panel) The survival time of the 48
patients with at least two examinations. (Lower panel) The survival
curves of the entire group of 70 recruited patients. (—) The IL-2 plus
NAC–treated group. (---) The combined groups treated with either
IL-2 alone or standard therapy. Among the patients that were
indicated as alive was one patient with the last observation point at
319 days (IL-2 plus NAC group) and one patient with the last
observation at 291 days (control group).
alone (21.5 6 2.5 v 32.6 6 3.5 µmol/L; P , .01) but not in the
group with IL-2 plus NAC (22.3 6 3.1 v 24.6 6 3.5 µmol/L).
The analysis of 20 healthy subjects showed a concentration of
23.1 6 1.5 µmol/L. This effect of NAC on NO22 and NO32 may
be useful for any type of IL-2 therapy. A significant increase in
the glutathione levels and GSH/GSSG ratios of the PBMC was
found in both IL-2–treated groups but was not further increased
by the additional treatment with NAC (Table 1).
Most of the adverse events in study B could be explained by
the underlying malignant disease and to a lesser extent by the
treatment. Mild fever has been observed about 6 to 8 hours after
IL-2 injection, and patients with a daily dose of .3 g NAC
complained frequently about heartburn and nausea. These
symptoms disappeared after treatment with OMEPRAZOL
(Astra Chemicals, D 22876, Wedel, Germany; oral doses of 20
to 40 mg/d).
The decrease in plasma albumin below 680 µmol/L is
associated with an increase in relative body water. Because
albumin plays an important role in the control of the oncoosmotic pressure and prevention of edema,10,41 we determined
also the relative body water of the cancer patients as defined by
the ratio of total body water per bcm. The analysis showed that
Fig 4. Functional capacity of cancer patients (study B). The figure
shows frequency histograms. The scales on the x-axis have been
reversed to account for the fact that a higher functional capacity
index means a lower quality of life according to the definition by
Ottery36: 0 5 normal, no limitations; 1 5 not normal, but able to be up
with fairly normal activities; 2 5 not feeling up to most things, but in
bed less than half the day; 3 5 able to do little activity and most of the
day in bed or chair; 4 5 rarely out of bed. Two patients in the
IL-2–treated group did not complete the questionnaire. The changes
of the functional capacity were different between the two treatment
groups by the Trend test (P 5 .007).
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64
HACK ET AL
producing capacity, and, in line with the correlation in Fig 2, a
lower bcm index than the rest of the group (Fig 7, upper panels).
Using maximally selected two-sample tests to search for
possible structural changes and to select an optimal cutoff
value46 we obtained a cystine/thiol cutoff ratio of 8.2 (P 5 .07)
for the correlation with VO2max, again 8.2 (P 5 .03) for the
correlation with the bcm index, and 7.2 (P , .02) for the
correlation with the lactate level. Treatment with NAC induced
in comparison with the placebo group a relative increase in bcm
in persons with baseline cystine/thiol ratio .6.28 (ie, .median).
For this group, ie, for the upper two quartiles together, the
relative increase in bcm was statistically significant (P , .05).
However, in line with the results of Table 1, NAC caused in
persons with cystine/thiol ratios . 6.28, on the average, no
increase in the intracellular GSH level of PBMC.
DISCUSSION
Fig 5. Effect of NAC treatment on the cystine/thiol ratio, bcm,
plasma glutamate, and plasma albumin (study B). The changes in the
IL-2 plus NAC–treated group (h) are shown in comparison with the
combined patients of the other two treatment groups (J). The
change in bcm was statistically significant only if patients with
observation periods G100 days are being compared. The changes
were defined by the first and the last examination of each patient.
ever, neither of the two was significantly correlated with the
corresponding changes in bcm in this study.
NAC causes an increase in bcm but not in intracellular
glutathione of healthy volunteers with high plasma cystine/thiol
ratio (study D). To obtain additional evidence for a cause/
effect relationship between redox state and bcm in healthy
subjects, we investigated in a placebo-controlled study the
effect of NAC treatment in the context of the endogenous
cystine/thiol ratio on the change in bcm. The healthy volunteers
were additionally subjected to a program of anaerobic physical
exercise to generate a condition similar to that of cancer patients
who are known to express a high rate of glycolytic activity in
muscle tissue.42 (Another reason for this study design was that
physical exercise has been considered as a therapeutic tool to
increase body cell mass17,43 and that strong physical exercise
was shown to cause the oxidation of glutathione in the
blood.44,45 This oxidation was previously shown to be ameliorated by treatment with NAC.45)
When the group of 38 volunteers of study D was divided into
quartiles according to their cystine/thiol ratio at baseline
examination, it was seen that persons with a cystine/thiol
ratio . 8.7 had a significantly lower VO2max, lower lactate-
Taken together, our studies show a substantial change in the
plasma thiol/disulfide redox state in human senescence and
wasting and suggest strongly that this change may be a
causative factor and a potential target for therapeutic intervention. The weight of the evidence for a cause/effect relationship
is mainly based on the effects of NAC on the bcm in the two
independent studies B and D. It is additionally supported by the
correlation between bcm index and redox state in the two
independent studies A and B. Treatment of the cancer patients
with IL-2 plus NAC improved the functional capacity, bcm,
albumin level, and the glutamate level. It must be emphasized
that the changes in bcm in Figs 5 and 7 are based on the
differences between longitudinal impedance measurements and
body mass data of individual subjects. In view of the relative
constant geometry of the individual subject, intraindividual
differences in bcm can be determined with much greater
Fig 6. Correlation between relative body water and plasma albumin in cancer patients (study B). Each point indicates a single person
at baseline examination. The albumin cutoff level of 679.6 mmol/L
(P F .001) has been computed by maximally selected two-sample
tests designed to search for possible structural changes and to select
an optimal cutoff value as described by Lausen et al.46 The figure
indicates also the correlation coefficient r and the corresponding P
value for the group of persons with albumin F680 mmol/L.
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REDOX STATE IN SENESCENCE AND WASTING—TARGET FOR THERAPY
Fig 7. Baseline performance and response to NAC treatment of
healthy subjects with different plasma redox states (study D). The
design of this study has been described in detail.37 The total group of
38 volunteers has been subdivided into quartiles according to their
baseline cystine/thiol ratio (upper left panel). The data for VO2max,
bcm index, and plasma lactate after exercise to exhaustion (see ref
37) indicate the mean 6 SEM of the two treatment groups in the four
quartiles. The lower two panels show the relative changes of bcm
and total intracellular glutathione in PBMC during the 5-week observation period including 4-week anaerobic physical exercise and
treatment with NAC or placebo. The significance between the indicated groups has been determined by the t-test for independent
samples.
precision than interindividual differences.37 Moreover, the oxidative metabolic capacity (VO2max) and the capacity to produce lactate (which is linked to VO2max47) were significantly
lower in healthy persons with high cystine/thiol ratio than in
persons with lower cystine/thiol ratio (Fig 7). Our data suggest
(1) that a ratio .8.2 which corresponds, according to Fig 2, to
an albumin level of about 680 µmol/L may be a risk factor for
loss of bcm and muscle function even among otherwise healthy
persons, and (2) that persons with a ratio .6.3 may already
benefit from treatment with a thiol-containing antioxidant.
Persons with a ratio ,6.3, in contrast, did not appear to benefit
from NAC-treatment, and persons with a ratio ,4.34 showed
even a negative effect of NAC. This negative effect was not
65
statistically significant, but in combination with the profile in
the upper right panel of Fig 7, it suggested the possibility that
the cystine/thiol ratio of approximately 4.3 to 6.3 may be
superior to both higher and lower cystine/thiol ratios. These
findings may provide a guideline for prophylactic redoxoriented therapy.
The strong correlation between the plasma cystine/thiol ratio
and albumin level in three independent studies, and the effect of
NAC on the albumin level (Fig 5) are again strong indications
for a cause/effect relationship. It may be noted that the
cystine/thiol ratio of the young and old healthy subjects is
approximately 4 and 8, respectively (Fig 1), whereas the ratio of
oxidized/reduced albumin was reported to be approximately 0.3
and 1, respectively,31 indicating that the plasma cystine/thiol
ratio is approximately 10-fold higher than the ratio of oxidized
albumin/reduced albumin. The plasma albumin level may be
limited, in principle, either by the rate of albumin degradation or
by the rate of hepatic albumin biosynthesis. NAC was previously shown to improve liver cell functions in patients with
paracetamol intoxication, ie, in persons with a strong hepatic
glutathione deficiency,48 but there is no evidence that the
patients and healthy subjects in our present studies had a priori
abnormal hepatic glutathione levels. Nevertheless, the possibility that NAC may increase the albumin level by enhancing the
rate of hepatic albumin biosynthesis rather than by decreasing
its rate of degradation has not been formally excluded.
The effect of NAC treatment on the albumin level is
particularly important in view of earlier unsuccessful attempts
to improve the albumin level by nutritional therapy14,25-27 and
because the albumin level is a strong predictor of hospital
survival and cost of hospitalization.14 One of the important
functions of albumin is the maintenance of the colloid oncotic
pressure and prevention of edema.10,14,26,41 We found that a
decrease in plasma albumin below 680 µmol/L was associated
with a strong increase in body water. The biphasic concentration
dependency (Fig 6) resembles the correlation between blood
pressure and albumin concentration.49 However, it may be an
oversimplification to assume that the linkage between redox
state and bcm or functional capacity is entirely mediated by
changes in the albumin level. Numerous proteins appear to be
tagged for degradation by oxidation,50 and changes of the
extracellular redox state may also induce intracellular changes,
including the induction of immediate/early genes.51
Unexpectedly, the albumin level, bcm, and functional capacity were significantly increased by NAC in the absence of
detectable changes in the glutathione status (studies B and D).
The increase in the intracellular glutathione level in response to
IL-2 treatment alone (Table 1) was in line with earlier studies in
vitro,52 but was not accompanied by an improvement of other
biochemical or clinical parameters. The additional treatment
with NAC, in contrast, caused an improvement of several
clinically relevant parameters but no additional increase in the
glutathione level or GSH/GSSG ratio (Table 1). The observation that NAC failed to increase the intracellular glutathione
level or GSH/GSSG ratio despite its significant effects on the
thiol redox state in the plasma (Fig 5, upper left panel) is
puzzling but is consistent with earlier studies from other
laboratories. An increase in intracellular glutathione levels by
NAC was previously seen only in the liver of patients with
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
66
HACK ET AL
paracetamol intoxication, ie, in persons with a strong hepatic
glutathione deficiency,48 but not in other cell types and/or other
conditions (see Fig 7).53,54
NAC has been proposed as a drug for cancer prevention.55 A
direct antitumoral activity has been shown in mice56 but not in
humans. NAC has also been proposed for the treatment of HIV
infection with the aim to reconstitute the abnormally low
plasma cystine, glutamine, and arginine levels.35,57,58 In contrast
to cancer patients and elderly subjects, HIV-infected persons
frequently have cystine/thiol ratios that are lower than normal
(H.P. Eck, R. Breitkreutz, W. Dröge, unpublished observation,
1988-1998). These relatively reducing conditions in the plasma
of HIV-infected persons may result from the increase in the
plasma level of thioredoxin that has been reported by others and
which may be induced by IL-6.59
The positive effect of NAC on the bcm and functional activity
of the cancer patients (study B) was not associated with
improved survival (Fig 3). However, cachexia is often a limiting
factor in standard chemotherapy, and any treatment that ameliorates the wasting syndrome may possibly enable the physician
to apply a more aggressive chemotherapy. Therefore, this report
may be useful for the design of new clinical trials. However, it
may be important to design individually guided doses of NAC
(or related drugs) according to the individual needs. NAC is
generally described as a relatively safe drug,52,55 but should be
used with caution.
ACKNOWLEDGMENT
The statistical advice of Dr L. Edler for the design of the randomized
trials, and the technical assistance of N. Erbe and M. Schykowski and
the assistance of I. Fryson in the preparation of this manuscript are
gratefully acknowledged.
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1998 92: 59-67
The Redox State as a Correlate of Senescence and Wasting and as a Target
for Therapeutic Intervention
Volker Hack, Raoul Breitkreutz, Ralf Kinscherf, Helmut Röhrer, Peter Bärtsch, Friedemann Taut, Axel
Benner and Wulf Dröge
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