CLINICAL STUDY
Decreased oxidative stress in patients with idiopathic dilated cardiomyopathy one year after immunoglobulin adsorption
Ingolf Schimke, PhD*,
Johannes Müller, MD ,
Friedrich Priem ,
Ingrid Kruse*,
Birgit Schön*,
Julia Stein,
Rudolf Kunze, PhD||,
Gerd Wallukat, PhD and
Roland Hetzer, MD
* Medizinische Klinik, Berlin, Germany
Institut für Laboratoriumsmedizin, Humboldt-Universität zu Berlin, Berlin, Germany
Deutsches Herzzentrum Berlin, Berlin, Germany
Max-Delbrück-Centrum Berlin, Berlin, Germany
|| Affina GmbH, Berlin, Germany
Manuscript received November 21, 2000;
revised manuscript received March 7, 2001,
accepted April 2, 2001.
Reprint requests and correspondence: Dr. Ingolf Schimke, Medizinische Klinik, Humboldt-Universität zu Berlin, Schumannstr. 20/21, D-10098 Berlin, Germany
ingolf.schimke{at}charite.de
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Abstract
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OBJECTIVES
In a substudy to a recently reported investigation that demonstrated the benefit of immunoglobulin adsorption (immunoadsorption) for patients with idiopathic dilated cardiomyopathy (IDC), we tested whether this benefit is associated with a reduction of oxidative stress.
BACKGROUND
The progression of cardiomyopathy is believed to be related to the increase of oxidative stress. Therefore, reduction of oxidative stress could be one of the effects of immunoadsorption for improvement of cardiac performance and clinical status.
METHODS
Plasma markers for oxidative stress—thiobarbituric acid-reactive substances (TBARS), lipid peroxides (LPO), anti-oxidized low-density lipoprotein-autoantibodies (anti-oxLDL-AB), thiol groups and vitamin E—were compared in 31 patients, of whom 16 underwent immunoadsorption and 15 received conventional treatment (controls). All patients received a daily supplement of vitamins, minerals and trace elements.
RESULTS
After one year, TBARS (p = 0.026), LPO (p = 0.026) and anti-oxLDL-AB (p = 0.044) were decreased in the immunoadsorption group but not in the controls. Thiols were unchanged in the immunoadsorption group but were decreased in the controls (p = 0.001). Vitamin E accumulated in both groups (immunoadsorption: p = 0.001; controls: p = 0.031) with a trend for stronger accumulation after immunoadsorption (p = 0.09). Prior to the study, the anti-oxLDL-AB to left ventricular ejection fraction (LVEF) (p = 0.05) were inversely correlated. After one year, correlations with borderline significance were calculated for TBARS to New York Heart Association functional class (p = 0.081) and inversely for LPO to LVEF (p = 0.083).
CONCLUSIONS
Effective therapy in patients with IDC, such as immunoadsorption which improved cardiac performance and clinical status, is associated with a reduction of oxidative stress.
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Abbreviations and Acronyms
| | ACE | = angiotensin-converting enzyme | | ANOVA | = analysis of variance | | anti-beta1A-AB | = anti-beta 1-adrenoceptor antibodies | | anti-oxLDL-AB | = anti-oxidized low-density lipoprotein-autoantibodies | | IDC | = idiopathic dilated cardiomyopathy | | IgG | = immunoglobulin G | | LPO | = lipid peroxides | | LVEF | = left ventricular ejection fraction | | LVIDd | = left ventricular internal diameter in diastole | | LVIDs | = left ventricular internal diameter in systole | | NYHA | = New York Heart Association | | TBARS | = thiobarbituric acid-reactive substances |
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The benefit of immunoglobulin adsorption (immunoadsorption) for the elimination of cardiac-specific autoantibodies in patients with dilated cardiomyopathy was recently reported. Müller et al. (1) found that cardiac performance and clinical status improved one year after a cycle of
immunoadsorption, which was performed on five consecutive days. Improved hemodynamics after immunoadsorption, which was combined with subsequent immunoglobulin G (IgG) substitution, was demonstrated by Felix et al. (2), who performed immunoadsorption in four courses, at one-month intervals, until month 3.
However, heart failure is associated with increased oxidative stress (3–9), the extent of which correlates positively with the class of heart failure (6,7,9) and negatively with cardiac performance (4). Therefore, oxidative stress is believed to be involved in the development and progression of heart failure (10,11). Consequently, beneficial treatment, such as immunoadsorption, which improves cardiac performance and clinical status (1,2), could result in the reduction of oxidative stress that might contribute to the therapy benefit.
To test this hypothesis, which would substantiate a close relationship between oxidative stress and heart failure, we compared in a substudy to Müller et al. (1) the prestudy and one-year levels of plasma markers for oxidative stress in patients with idiopathic dilated cardiomyopathy (IDC) treated with immunoadsorption according to Müller et al. (1) versus conventionally treated patients.
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Methods
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Patient characteristics.
In the present study, we analyzed plasma samples that were collected from patients with IDC who were characterized by Müller et al. (1) with regard to the effects of immunoadsorption versus conventional treatment on cardiac performance and clinical status.
From the 32 patients with IDC who finished the Müller et al. study (1), we included 31 patients (mean age: 47.8 ± 10.3 years; range: 21 to 61 years, men: 29; women: 2) in our study. One patient was excluded owing to a hemolytic prestudy sample. All patients were accepted as candidates for heart transplantation and had New York Heart Association (NYHA) functional class II or worse, left ventricular ejection fraction (LVEF) <0.29, left ventricular internal diameter in diastole (LVIDd) >64 mm and evidence for cardiac-specific autoantibodies indicated by the presence of anti-beta1-adrenoceptor autoantibodies (anti-beta1A-AB). There was no evidence of coronary artery disease or any other cardiac disease. Exclusion criteria were atrial fibrillation, infectious diseases, alcohol-induced cardiomyopathy, previous allergic reaction to sheep protein and signs of malignancy. Sixteen patients (mean age 45.6 ± 10.0 years; range: 30 to 59 years; men: 14; women: 2) were treated with immunoadsorption and 15 (mean age: 50.1 ± 10.6 years; range: 21 to 61 years; men: 15) were conventionally treated. One year after immunoadsorption, the anti-beta1A-AB level (prestudy level: 5.9 ± 1.2 laboratory units) was reduced in the treatment group by approximately 90%. This was accompanied by a highly significant improvement (p < 0.0001) of cardiac performance (LVEF: 22.1 ± 3.3 vs. 37.8 ± 8.2%; LVIDd: 74.4 ± 7.3 vs. 63.8 ± 6.2 mm; left ventricular internal diameter in systole [LVIDs]: 66.0 ± 6.3 vs. 55.6 ± 6.2 mm) and clinical status (NYHA functional class I/II/III/IV: 0/2/13/1 vs. 7/9/0/0). By comparison, no significant changes of the controls in anti-beta1A-AB (prestudy level: 4.8 ± 1.0 laboratory units) or in cardiac performance (LVEF: 24.1 ± 3.0 vs. 25.2 ± 5.9%; LVIDd: 75.5 ± 5.3 vs. 73.7 ± 7.4 mm; LVIDs: 67.0 ± 5.4 vs. 65.1 ± 8.4 mm) and clinical status (NYHA functional class I/II/III/IV: 0/4/11/0 vs. 0/7/6/2) were seen after conventional treatment.
Written, informed consent was obtained from all patients. The study protocol was approved by the Human Ethics Committee of the Humboldt University of Berlin, Germany.
Medical treatment.
At baseline, medical treatment for heart failure was standardized in that all patients were administered maximal tolerated dosages of angiotensin-converting enzyme (ACE) inhibitors, digitalis, diuretics and oral anticoagulants. In addition, patients were treated with the beta-blocker bisoprolol for the first time. Dosage adjustments were made to achieve a systolic blood pressure of 100 to 110 mm Hg and a heart rate of 60 to 80 beats/min within three months. All patients received a daily supplement of a moderate dose of vitamins, minerals and trace elements (OrthoCorPlus; Orthomol, Langenfeld, Germany).
Extracorporeal immunoglobulin adsorption, measurement of anti-beta1A-AB and echocardiographic evaluation.
For the immunoadsorption, which was performed according to the established method of low-density lipoprotein (LDL) elimination, adsorption columns containing polyclonal anti-human immunoglobulin antibodies produced in sheep (12) were used.
Immunoadsorption was performed on five consecutive days, and serum IgG levels were monitored. To eliminate anti-beta1A-AB with an acceptable risk of infection, immunoglobulin G (IgG) reduction to <120 mg/dl was planned.
For the measurement of anti-beta1A-AB, a bioassay recently described by Wallukat et al. (13) was used.
Transthoracic echocardiography was performed according to the guidelines of the American Society of Echocardiography (14). (For more details, see Müller et al. [1]).
Sampling and measurement.
As markers for oxidative stress, we determined the levels of thiobarbituric acid-reactive substances (TBARS) (15), lipid peroxides (LPO) (PerOx; Immundiagnostik, Bensheim, Germany) (16), anti-oxidized low-density lipoprotein-autoantibodies (anti-oxLDL-AB) (oLAb Elisa; Biomedica, Wien, Austria), thiol groups (17) and vitamin E (18) in venous plasma samples (anticoagulated with 14,300 U/l heparin) collected prior to the study and after one year.
To avoid unspecific marker changes by in vitro oxidation, especially relevant to TBARS, LPO and thiol groups, and to guarantee high precision in the analytical procedures, adequate conditions for sampling, storage and measurement were ascertained in prestudy experiments. Using the findings of these experiments, samples and aliquots were prepared under a nitrogen atmosphere, immediately frozen and stored in liquid nitrogen until measurement. Both the prestudy and one-year sample of each subject of both groups were handled identically and—for high precision—analyzed in parallel.
Statistical analysis.
Data are expressed as mean ± SD. A log-transformation was applied to stabilize the variance. A two-way repeated-measures analysis of variance (ANOVA) was used to test for effect of time, group and their interaction followed by the Student t test for paired and unpaired data. Linear regression was performed using the SPSS software package (SPSS Inc., Chicago, Illinois).
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Results
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Oxidative stress in patients with IDC one year after immunoglobulin adsorption.
The prestudy and one-year levels of indicators for oxidative stress were widely scattered in both patient groups, as shown by their SD (Fig. 1). However, there were no significant group-specific differences prior to the study. Two-way ANOVA for analysis of the overall effect of group allocation and repeated measures on the markers for oxidative stress revealed significant time effects but no interaction effects for TBARS (p = 0.0001 [time effect p value] p = 0.99 [interaction effect p value]), thiol groups (p = 0.001; p = 0.1) and vitamin E (p = 0.001; p = 0.43). In contrast, no time effects but a trend to interaction was found for LPO (p = 0.14; p = 0.08) and anti-oxLDL-AB (p = 0.89; p = 0.09).
In addition to highly significantly reduced anti-beta1A-AB and improved cardiac performance and NYHA functional class in the patients who underwent immunoadsorption, as demonstrated by Müller et al. (1), a significant lowering was observed in these patients over the study period by 1.24 ± 0.5 µmol/l for TBARS (9.38 ± 2.48 vs. 8.14 ± 2.50 µmol/l; p = 0.026), by 110.6 ± 44.5 µmol/l for LPO (268.6 ± 186.1 vs. 157.9 ± 71.7 µmol/l; p = 0.026), and by 53.2 ± 24.0 U/l for anti-oxLDL-AB (323.1 ± 221.4 vs. 269.9 ± 153.1 U/l, p = 0.044). In contrast, TBARS decreased only by 0.87 ± 0.6 µmol/l (9.19 ± 3.36 vs. 8.33 ± 2.49 µmol/l, p = 0.165) in the controls. Both LPO (265.4 ± 198.9 vs. 321.4 ± 272.5 µmol/l, p = 0.364) and anti-oxLDL-AB (200.4 ± 78.7 vs. 222.6 ± 97.3 U/l; p = 0.334) increased by 56.0 ± 59.6 µmol/l and 22.2 ± 22.6 U/l, respectively, in the controls. However, these changes were insignificant.
For the thiol groups, there was only an insignificant change of 20.8 ± 12.4 µmol/l (341.1 ± 94.4 vs. 320.3 ± 105.1 µmol/l; p = 0.140) in the immunoadsorption group, but thiols decreased significantly in the controls by 53.2 ± 12.9 µmol/l (386.1 ± 129.0 vs. 332.9 ± 147.4 µmol/l, p = 0.001). Vitamin E increased significantly over the study period in the treated group by 20.56 ± 2.9 µmol/l (34.44 ± 18.64 vs. 55.0 ± 27.56 µmol/l, p = 0.001) as well as in the control group by 12.75 ± 5.2 µmol/l (22.85 ± 18.23 vs. 35.60 ± 34.42 µmol/l, p = 0.031) with a trend for stronger accumulation in the immunoadsorption group (p = 0.09).
Oxidative stress versus NYHA functional class and cardiac performance.
Prior to the study, a significant inverse correlation of anti-oxLDL-AB to LVEF was calculated for the complete population of patients (Fig. 2A). After one year (Fig. 2B, C) the best correlations were found for TBARS to NYHA functional class and inversely for LPO to LVEF. Additionally, the anti-oxLDL-AB changes over the study period correlated inversely to LVEF and directly to LVIDs changes.
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Figure 2 Relationships between markers of oxidative stress and clinical status and cardiac performance in the complete population of patients conventionally treated (solid diamonds) and those treated with immunoglobulin adsorption (open diamonds). (A) Anti-oxidized low density lipoprotein autoantibodies (anti-oxLDL-AB) versus left ventricular ejection fraction (LVEF) prior to the study. (B) Thiobarbituric acid-reactive substances (TBARS) versus New York Heart Association functional class one year after immunoadsorption. (C) Lipid peroxides (LPO) versus LVEF one year after immunoadsorption. Prestudy to one-year change of anti-oxLDL-AB (d anti-oxLDL-AB) versus change of (D) LVEF (d LVEF), and (E) LVIDs (d LVIDs).
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Discussion
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To our knowledge for the first time in humans, we demonstrated that improvement of cardiac performance and clinical status in heart failure, such as shown after immunoadsorption (1), is in parallel associated with a reduction of oxidative stress.
This was demonstrated by measurement of markers adaptable to clinical chemistry for analysis of oxidative stress in human plasma (19) such as TBARS, LPO, and anti-oxLDL-AB as indicators for lipid peroxidation; thiol groups, for which a decrease signals thiol oxidation; and vitamin E, an important nonenzymatic plasma antioxidant for which a decrease indicates consumption due to oxidative stress.
Oxidative stress in patients with IDC one year after immunoglobulin adsorption.
Decreased oxidative stress one year after immunoadsorption was indicated by a lowering of TBARS, LPO and anti-oxLDL-AB, which was not seen in the conventionally treated patients. With respect to anti-oxLDL-AB, immunoadsorption is not antigen selective and could—in addition to anti-beta1A-AB—eliminate other IgG class antibodies such as anti-oxLDL-AB. Therefore, a delayed reincrease of anti-oxLDL-AB could also explain the low anti-oxLDL level in the one-year samples. Müller et al. (1) have speculated that antioxidant supplementation could modulate the autoantibody reincrease. Although all patients received equal amounts of OrthoCorPlus, which supplied vitamin E, there was a trend to stronger accumulation of vitamin E in the plasma of the patients who underwent immunoadsorption. We attribute this effect to a lower consumption of vitamin E, due to decreased oxidative stress in these patients. The significant lowering of thiol groups over the study period, but only in the controls, is a further indication of different levels of oxidative stress in both groups.
Oxidative stress versus NYHA functional class and cardiac performance.
Close correlations for our patients between the intensity of oxidative stress and clinical status and cardiac performance would substantiate the relationship between oxidative stress and heart failure. Recently, such correlations were demonstrated in studies that enrolled patients with widespread data for cardiac performance and clinical status (4,6,7,9), which favor regression analysis. Regression analysis was limited by the small range of these parameters in our patients, especially prior to the study, and also by the relatively small number of patients in our study. However, the correlations for oxidative stress, clinical status and cardiac performance, demonstrated in Figure 2A–C, supplement the findings of the other studies (4,6,7,9). At present, it cannot be decided whether the correlations, which were calculated for the prestudy to the one-year changes of anti-oxLDL-AB and cardiac performance (Fig. 2D–E), support a close relationship between the intensity of oxidative stress and the severity of heart failure. For that, investigations must clarify the exact reasons—such as discussed above—for low anti-oxLDL-AB levels one year after immunoadsorption.
Potential reasons for reduced oxidative stress after immunoglobulin adsorption.
The reasons for reduced oxidative stress in patients with IDC one year after immunoadsorption are unknown. Predisposing events of heart failure, such as mechanical stress, ischemia, intoxication, viral myocarditis, reduced antioxidative defense due to malnutrition and neurohumoral hyperstimulation, are thought to drive myocardial oxidative stress (10,11), which can damage—via thiol oxidation and lipid peroxidation—myocardial structures and functions (20,21) and in this way join the initial pathophysiologic events and the development of heart failure. Because of stimulated myocyte growth, apoptosis and fibroblast proliferation during increased oxidative stress (22), progression of heart failure could be favored.
Reduced mechanical stress, probably reduced inflammation after immunoadsorption together with the vitamin E supplement, may minimize oxidative stress. Furthermore, hyperstimulation of the myocardial beta-adrenergic pathway, which is linked to heart failure (23), could increase the oxygen radical generation by blockade of the electron transport in the mitochondrial complex I (24). Therefore, immunoadsorption of anti-beta1A-AB, believed to be involved in the hyperstimulation of the myocardial beta-adrenergic pathway, could reduce oxidative stress.
Study limitations.
Decreases of plasma indicators for oxidative stress do not inevitably reflect reduced oxidative stress in the heart, which is favorable for an improvement of function. Apart from the myocardium, perhaps poorly perfused peripheral muscles and activated neutrophils may be responsible for the oxidative stress found in plasma. Future studies analyzing myocardial biopsies in parallel to plasma seem to be warranted.
Conclusions.
Improvement of clinical status and cardiac performance in patients with ICD due to immunoadsorption is associated with a reduction of oxidative stress. This could contribute to the benefit of immunoadsorption in these patients.
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Acknowledgments
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We are grateful to Diana Kendall and Anne Gale for their editorial assistance.
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Footnotes
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This study was supported, in part, by Orthomol, Langenfeld, Germany, and Immundiagnostik, Bensheim, Germany.
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References
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1. Müller J, Wallukat G, Dandel M, et al. Immunoglobulin adsorption in patients with idiopathic dilated cardiomyopathy. Circulation. 2000;101:385–391[Abstract/Free Full Text]
2. Felix SB, Staud A, Dörffel WV, et al. Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy. J Am Coll Cardiol. 2000;35:1590–1598[Abstract/Free Full Text]
3. McMurray J, McLay J, Chopra M, Bridges A, Belch JJE. Evidence for enhanced free radical activity in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol. 1990;65:1261–1262[CrossRef][Medline]
4. Belch JJ, Bridges AB, Scott N, Chopra M. Oxygen free radicals and congestive heart failure. Br Heart J. 1991;65:245–248[Abstract/Free Full Text]
5. Sobotka PA, Brottman MD, Weitz Z, Bimbaum AJ, Skosey JL, Zarling EJ. Elevated breath pentane in heart failure reduced by free radical scavenger. Free Radic Biol Med. 1993;14:643–647[CrossRef][Medline]
6. Diaz-Velez CR, Garcia-Castineiras S, Mendoza-Ramos E, Hernandez-Lopez E. Increased malondialdehyde in peripheral blood of patients with congestive heart failure. Am Heart J. 1996;131:146–152[CrossRef][Medline]
7. Keith M, Geranmayegan A, Sole MJ, et al. Increased oxidative stress in patients with congestive heart failure. J Am Coll Cardiol. 1998;31:1352–1356[Abstract/Free Full Text]
8. Yücel D, Aydogdu S, Cehreli S, et al. Increased oxidative stress in dilated cardiomyopathic heart failure. Clin Chem. 1998;44:148–154[Abstract/Free Full Text]
9. Mallat Z, Philip I, Lobret M, Chatel D, Maclouf J, Tedgui A. Elevated levels of 8-iso-prostaglandin F2 in pericardial fluid of patients with heart failure. A potential role for in vivo oxidant stress in ventricular dilatation and progression of heart failure. Circulation. 1998;97:1536–1539[Abstract/Free Full Text]
10. Ball AM, Sole MJ. Oxidative stress and the pathogenesis of heart failure. Cardiol Clin. 1998;16:665–675[CrossRef][Medline]
11. Singal PK, Khaper N, Palace V, Kumar D. The role of oxidative stress in the genesis of heart disease. Cardiovasc Res. 1998;40:426–432[Abstract/Free Full Text]
12. Reinke P, Brehme S, Baumann G, et al. Treatment of cardiomyopathy by removal of autoantibodies. International application published under the Patent Cooperation Treaty (PCT). International publication number WO 97/1798. International application number PCT/US96/18457.
13. Wallukat G, Wollenberger A, Morwinski R, Pitschner HF. Anti-beta 1-adrenoceptor antibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: mapping of epitopes in the first and second extracellular loops. J Mol Cell Cardiol. 1995;27:397–406[Medline]
14. Cheitlin MD, Alpert JS, Armstrong WF, et al. ACC/AHA Guidelines for the clinical application of echocardiography: a report of the American College of Cardiology/American Heart Association Task Force on Practical Guidelines (Committee on Clinical Application of Echocardiography) developed in collaboration with the American Society of Echocardiography. Circulation. 1997;95:1686–1744[Free Full Text]
15. Takeda K, Shimada Y, Amano M, Sakai M, Okada T, Yoshiya I. Plasma lipid peroxides and alpha-tocopherol in critically ill patients. Crit Care Med. 1984;12:957–959[Medline]
16. Roob JM, Khoschsorur G, Tiran A, Horina JH, Holzer H, Winklhofer-Roob BM. Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J Am Soc Nephrol. 2000;11:539–549[Abstract/Free Full Text]
17. Habeeb AF. Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymol. 1972;25:457–500[CrossRef]
18. Catignani GL, Bieri JG. Simultaneous determination of retinol and alpha-tocopherol in serum or plasma by liquid chromatography. Clin Chem. 1983;29:708–712[Free Full Text]
19. Armstrong D, Browne R. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory. Adv Exp Biol Med. 1994;366:43–58[Medline]
20. Ferrari R, Agnoletti L, Comini L, et al. Oxidative stress during myocardial ischemia and heart failure. Eur Heart J. 1998;19(Suppl B):B2–B11
21. Bolli R, Marban E. Molecular and cellular mechanisms of myocardial stunning. Phys Rev. 1999;79:609–634[Abstract/Free Full Text]
22. Colucci WS. Molecular and cellular mechanisms of myocardial failure. Am J Cardiol. 1997;80:15L–25L[CrossRef][Medline]
23. Bristow MR. Mechanism of action of beta-blocking agents in heart failure. Am J Cardiol. 1997;80:26L–40L[CrossRef][Medline]
24. Sawyer DB, Colucci WS. Mitochondrial oxidative stress in heart failure. "Oxygen wastage" revisited. Circ Res. 2000;86:119–124[Free Full Text]
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Abstract
Methods
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