|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 59-67
By
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.
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.
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
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.
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.
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
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.
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.
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).
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 = 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)
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.
Submitted December 22, 1997;
accepted February 18, 1998.
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.
1.
Cohn SH,
Vartsky D,
Yasumura S,
Sawitsky A,
Zanzi I,
Vaswani A,
Ellis KJ:
Compartmental body composition based on total-body nitrogen, potassium, and calcium.
Am J Physiol
239:E524,
1980
2.
Brennan MF:
Uncomplicated starvation versus cancer cachexia.
Cancer Res
37:2359,
1977[Medline]
[Order article via Infotrieve]
3.
DeWys WD,
Begg C,
Lavin PT,
Band PR,
Bennett JM,
Bertino JR,
Cohen MH,
Douglass Ho Jr,
Engstrom PF,
Ezdinli EZ,
Horton J,
Johnson GJ,
Moertel CG,
Oken MM,
Perlia C,
Rosenbaum C,
Silverstein MN,
Skeel RT,
Sponzo RW,
Tormey DC:
Prognostic effect of weight loss prior to chemotherapy in cancer patients.
Am J Med
69:491,
1980[Medline]
[Order article via Infotrieve]
4.
Strain AJ:
Cancer cachexia in man. A review.
Invest Cell Pathol
2:181,
1979[Medline]
[Order article via Infotrieve]
5.
Pisters PW,
Pearlstone DB:
Protein and amino acid metabolism in cancer cachexia: Investigative techniques and therapeutic interventions.
Crit Rev Clin Lab Sci
30:223,
1993[Medline]
[Order article via Infotrieve]
6.
Long CL,
Crosby F,
Geiger JW,
Kinney JM:
Parenteral nutrition in the septic patient: Nitrogen balance, limiting plasma amino acids, and calorie to nitrogen ratios.
Am J Clin Nutr
29:380,
1976
7.
Ott M,
Lembcke B,
Fischer H,
Jager R,
Polat H,
Geier H,
Rech M,
Staszeswki S,
Helm EB,
Caspary WF:
Early changes of body composition in human immunodeficiency virus-infected patients: Tetrapolar body impedance analysis indicates significant malnutrition.
Am J Clin Nutr
57:15,
1993
8.
Kotler DP,
Wang J,
Pierson R:
Body composition in patients with the acquired immunodeficiency syndrome.
Am J Clin Nutr
42:1255,
1985
9.
Buchner DM,
Wagner EH:
Preventing frail health.
Clin Geriatr Med
8:1,
1992[Medline]
[Order article via Infotrieve]
10.
Rothschild MA,
Oratz M,
Schreiber SS:
Serum albumin.
Hepatology
8:385,
1988[Medline]
[Order article via Infotrieve]
11.
Cooper JK,
Gardner C:
Effect of aging on serum albumin.
J Am Geriatric Soc
37:1039,
1989[Medline]
[Order article via Infotrieve]
12.
Shibata H,
Haga H,
Ueno M,
Nagai H,
Yasumura S,
Koyano W:
Longitudinal changes of serum albumin in elderly people living in the community.
Age and Aging
20:417,
1991
13.
Baumgartner RN,
Koehler KM,
Romero L,
Garry PJ:
Serum albumin is associated with skeletal muscle in elderly men and women.
Am J Clin Nutr
64:552,
1996
14.
Tayek JA:
Albumin synthesis and nutritional assessment.
Nutr Clin Pract
3:219,
1988[Medline]
[Order article via Infotrieve]
15.
Naber THJ,
de Bree A,
Schermer TRJ,
Bakkeren J,
Bär B,
de Wild G,
Katan MB:
Specificity of indexes of malnutrition when applied to apparently healthy people: The effect of age.
Am J Clin Nutr
65:1721,
1997
16.
Brody JA:
Prospects for an aging population.
Nature
315:463,
1985[Medline]
[Order article via Infotrieve]
17.
Lamberts SWJ,
van den Beld AW,
van der Lely A-J:
The endocrinology of aging.
Science
278:419,
1997
18.
Campion EW:
The oldest old.
N Engl J Med
330:1819,
1994
19.
Fiatarone MA,
O'Neill EF,
Ryan ND,
Clements KM,
Solares GR,
Nelson ME,
Roberts SB,
Kehayias JJ,
Lipsitz LA,
Evans WJ:
Exercise training and nutritional supplementation for physical frailty in very elderly people.
N Engl J Med
330:1769,
1994
20.
Harman D:
Aging: A theory based on free radical and radiation chemistry.
J Geronterol
11:298,
1956
21.
Shigenaga MK,
Hagen TM,
Ames BN:
Oxidative damage and mitochondrial decay in aging.
Proc Natl Acad Sci USA
91:10771,
1994
22.
Stadtman ER:
Protein oxidation and aging.
Science
257:1220,
1992
23.
Gilchrest BA,
Bohr VA:
Aging processes, DNA damage, and repair.
FASEB J
11:322,
1997[Abstract]
24.
Manton KG,
Corder LS,
Stallard E:
Monitoring changes in the health of the U.S. elderly population: Correlates with biomedical research and clinical innovations.
FASEB J
11:923,
1997[Medline]
[Order article via Infotrieve]
25.
Tuten MB,
Wogt S,
Dasse F,
Leider Z:
Utilization of prealbumin as a nutritional parameter.
J Parent Ent Nutr
9:709,
1985
26.
Erstad BL:
Serum albumin concentrations: Who needs them?
Ann Pharmacother
26:1134,
1992[Abstract]
27.
Paluzzi M,
Meguid MM:
A prospective randomized study of the optimal source of nonprotein calories in total parenteral nutrition.
Surgery
102:711,
1987[Medline]
[Order article via Infotrieve]
28.
Finch JW,
Crouch RK,
Knapp DR,
Schey KL:
Mass spectrometric identification of modifications to human serum albumin treated with hydrogen peroxide.
Arch Biochem Biophys
305:595,
1993[Medline]
[Order article via Infotrieve]
29.
Kuwata K,
Era S,
Sogami M:
The kinetic studies on the intracellular SH, S-S exchange reaction of bovine mercaptalbumin.
Biochim Biophys Acta
1205:317,
1994[Medline]
[Order article via Infotrieve]
30.
Halliwell B,
Gutteridge MC:
The antioxidants of human extracellular fluids.
Arch Biochem Biophys
280:1,
1990[Medline]
[Order article via Infotrieve]
31.
Era S,
Kuwata K,
Imai H,
Nakamura K,
Hayashi T,
Sogami M:
Age-related change in redox state of human serum albumin.
Biochim Biophys Acta
1247:12,
1995[Medline]
[Order article via Infotrieve]
32.
Era S,
Hamaguchi T,
Sogami M,
Kuwata K,
Suzuki E,
Miura K,
Kawai K,
Kitazawa Y,
Okabe H,
Noma A,
Miyata S:
Further studies on the resolution of human mercapt- and nonmercaptalbumin and on human serum albumin in the elderly by high-performance liquid chromatography.
Int J Peptide Protein Res
31:435,
1988[Medline]
[Order article via Infotrieve]
33.
Hack V,
Stütz O,
Kinscherf R,
Schykowski M,
Kellerer M,
Holm E,
Dröge W:
Elevated venous glutamate levels in (pre)catabolic conditions result at least partly from a decreased glutamate transport activity.
J Mol Med
74:337,
1996[Medline]
[Order article via Infotrieve]
34.
Miles DW,
Thomsen L,
Balkwill F,
Thavasu P,
Moncada S:
Association between biosynthesis of nitric oxide and changes in immunological and vascular parameters in patients treated with interleukin-2.
Eur J Clin Invest
24:287,
1994[Medline]
[Order article via Infotrieve]
35.
Dröge W,
Gro
36. (suppl 3)
Ottery FD:
Supportive nutrition to prevent cachexia and improve quality of life.
Semin Oncol
22:98,
1995[Medline]
[Order article via Infotrieve]
37.
Kinscherf R,
Hack V,
Fischbach T,
Friedmann B,
Weiss C,
Edler L,
Bärtsch P,
Dröge W:
Low plasma glutamine in combination with high glutamate levels indicate risk for loss of body cell mass in healthy individuals: the effect of N-acetyl-cysteine.
J Mol Med
74:393,
1996[Medline]
[Order article via Infotrieve] |
Abstract
Introduction
Abstract
5, see also Fig
) 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).
) 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.
.32; P = .05, and
r = 
A,
Hack V,
Kinscherf R,
Schykowski M,
Bockstette M,
Mihm S,
Galter D:
Role of cysteine and glutathione in HIV infection and cancer cachexia. Therapeutic intervention with N-acetyl-cysteine (NAC).
Adv Pharmacol
38:581,
1997