The Redox State as a Correlate of Senescence and Wasting and as a Target for Therapeutic Intervention

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.

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-sidedt-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.

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.

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 

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.

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 

The statistical evaluation of the individual changes between baseline examination and terminal examination was performed by the pairedt-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.

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 Fig2). 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).

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 (Table1). 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 < 10⁻⁷), and elevated plasma glutamate levels (47.7 ± 2.9 v 30.6 ± 1.8 μmol/L;P < 10⁻⁵). 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).

The treatment groups showed similar survival curves (Fig3). 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 and5). 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.

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, andr = −.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 ± 2.5v 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).

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 (Fig6).

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 < 10⁻⁴) and (2) between the changes in the albumin level and corresponding changes in the cystine/thiol ratio (r = −.53;P < 10⁻³). However, neither of the two was significantly correlated with the corresponding changes in bcm in this study.

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.

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,31indicating 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 Fig7).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.

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.