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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 59-67
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
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.
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ABSTRACT |
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 µmol/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.
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INTRODUCTION |
LIKE 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. 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 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 protein-cysteine 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
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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.
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| Fig 1.
Correlation between plasma redox state and age in healthy
persons (study A). Postabsorptive plasma amino acid and acid-soluble thiol levels have been determined in the plasma from the cubital vein
of 205 randomly selected healthy human subjects of both sexes.
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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 × 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
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 = normal, no limitations;
1 = not normal, but stable enough to be up with fairly normal
activity; 2 = not feeling up to most things but in bed less than half
the day; 3 = able to do little activity and most of the day in bed or
chair; 4 = rarely out of bed).
Height, body mass, bcm, and body water were determined and blood
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 = 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 = 18) and
placebo group (n = 20). The dose of NAC was 2 × 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 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.
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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 age-dependent shift to a more
oxidized condition (Fig 1). Our analysis also confirmed the negative
correlation between plasma albumin concentration and age
(r = .49, P < 10 5, see also Fig
2). A possible linkage between the plasma
cystine/thiol ratio, the plasma albumin level, and the bcm index was
suggested by the strong correlation between these parameters (Fig 2,
left panel).
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| 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.
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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,
NO2 and NO3.
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 ± 1.1 years), the
cancer patients (mean age, 60.5 ± 1.1 years) had at baseline
examination significantly elevated plasma cystine/thiol ratios
(6.99 ± 0.57 v 5.53 ± 0.22; P < .05),
decreased plasma albumin levels (674 ± 9 v 760 ± 11 µmol/L; P < 107), and elevated plasma
glutamate levels (47.7 ± 2.9 v 30.6 ± 1.8 µmol/L;
P < 105). 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 = +.32 and +.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 = .08,
P = .37 and P = .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 ± 13 to 696 ± 20 µmol/L;
P < .03) and decrease in plasma glutamate (47.8. ± 5.1
to the essentially normal level 31.0 ± 3.6 µmol/L;
P = .002) if tested by the paired t-test. The
IL-2-treated group, in contrast, showed a decrease in plasma albumin
(713 ± 15 to 685 ± 16;P < .02), and a slight
increase in plasma glutamate (51.9 ± 5.5 to 53.2 ± 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.
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| 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).
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| 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 = normal, no limitations; 1 = not normal,
but able to be up with fairly normal activities; 2 = not feeling up
to most things, but in bed less than half the day; 3 = able to do
little activity and most of the day in bed or chair; 4 = 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 = .007).
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| 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 ( ) are shown in comparison with the combined
patients of the other two treatment groups ( ). The change in bcm was
statistically significant only if patients with observation periods
>100 days are being compared. The changes were defined by the first
and the last examination of each patient.
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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 = +.61;
P < .001, r =  .32; P = .05, and
r = .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 alone (21.5 ± 2.5
v 32.6 ± 3.5 µmol/L; P < .01) but not in the
group with IL-2 plus NAC (22.3 ± 3.1 v 24.6 ± 3.5
µmol/L). The analysis of 20 healthy subjects showed a concentration
of 23.1 ± 1.5 µmol/L. This effect of NAC on
NO2 and NO3 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
onco-osmotic 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 the decrease in plasma albumin levels below 680 µmol/L was associated with an increase in relative total body water (Fig 6).
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| 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 µmol/L (P < .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 <680 µmol/L.
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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 age-dependent 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 = .61; P < 104) and
(2) between the changes in the albumin level and corresponding changes
in the cystine/thiol ratio (r = .53;
P < 103). However, 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-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 = .07) for the correlation with VO2max, again
8.2 (P = .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.
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| 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 ± 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.
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DISCUSSION |
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
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 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 redox-oriented 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 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.
| |
FOOTNOTES |
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" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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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Abstract
Introduction
Abstract