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Effect of Polymyxin B-Immobilized Fiber Hemoperfusion on Serum High Mobility Group Box-1 Protein Levels and Oxidative Stress in Patients With Acute Respiratory Distress Syndrome

Nakamura, Tsukasa*; Fujiwara, Nobuharu*; Sato, Eiichi*; Kawagoe, Yasuhiro*; Ueda, Yoshihiko†; Yamada, Shingo‡; Koide, Hikaru§

doi: 10.1097/MAT.0b013e3181a5290f
Clinical Critical Care

Acute respiratory distress syndrome (ARDS) is characterized by diffuse inflammation in the lung and resultant permeability edema. Polymyxin B-immobilized fiber (PMX-F) hemoperfusion is effective for sepsis-induced ARDS. High mobility group box-1 protein (HMGB1) is newly recognized as a proinflammatory cytokine. The aim of the study was to determine whether blood HMGB1 levels are increased in patients with ARDS and whether PMX-F treatment affects these levels. Subjects were 20 sepsis-induced patients with ARDS treated by PMX-F column and 20 age-matched healthy volunteers. Polymyxin B-immobilized fiber treatment was carried out twice at a rate of 100 ml/min for 2 hours. Systolic and diastolic blood pressures, the PaO2/FiO2 (PF) ratio and endotoxin, HMGB1, and urinary 8-hydroxy-2′-deoxyguanosine (OHdG) levels were measured before and after PMX-F treatment. Blood endotoxin levels, blood HMGB1 levels, and urinary 8-OHdG levels were significantly higher in patients with ARDS than in healthy volunteers. Systolic and diastolic blood pressures and the PF ratio increased significantly after PMX-F treatments. Polymyxin B-immobilized fiber treatment reduced blood endotoxin, blood HMGB1, and urinary 8-OHdG levels significantly. These data suggest that HMGB1 and oxidative stress play a role in the pathogenesis of ARDS and that PMX-F treatment may ameliorate increased blood HMGB1 and urinary 8-OHdG levels in patients with ARDS.

From the *Department of Medicine, Shinmatsudo Central General Hospital, Chiba; †Department of Pathology, Koshigaya Hospital, Dokkyo University School of Medicine, Saitama; ‡Central Institute, Shino-Test Corp., Kanagawa; and §Department of Medicine, Koto Hospital, Tokyo, Japan.

Submitted for consideration December 2008; accepted for publication in revised form February 2009.

Reprint Requests: Hikaru Koide, Department of Medicine, Koto Hospital, 6-8-5 Ojima, Koto-ku, Tokyo 136-0072, Japan. Email: hkoide@koto-hospital.or.jp.

Acute respiratory distress syndrome (ARDS) is characterized by severe acute hypoxemia caused by increased lung edema, atelectasis, intrapulmonary shunting, and hypoxemia.1 Although overall mortality seems to be decreasing because of recent improvements in supportive care, there are presently no proven, effective pharmacological therapies to treat ARDS or to prevent its associated complications.2 Exaggerated cytokine release from mononuclear cells has been observed in ARDS and ARDS is associated with endotoxemia.3

High mobility group box-1 protein (HMGB1) was originally as a DNA-binding nonhistone chromosomal protein implicated in diverse cellular functions such as stabilization of nucleosomal structure and regulation of transcriptional factors.4 On stimulation with lipopolysaccharides (LPS), HMGB1 is secreted from monocytes/macrophages and fosters inflammatory responses. HMGB1 is passively released from necrotic cells and mediates inflammation and immune responses.5 Recently, HMGB1 has been implicated as a putative danger signal involved in the pathogenesis of a variety of inflammatory conditions including septic shock, acute coronary syndrome, and disseminated intravascular coagulation.6 However, the regulation of HMGB1 levels in ARDS remained to be elucidated.

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) plays a crucial role in the initiation and progression of various diseases including septic shock, and it has been measured as a biomarker of oxidative DNA damage.7,8 However, little is known about HMGB1 and 8-OHdG regulation in patients with ARDS.

Polymyxin B-immobilized fiber (PMX-F) treatment is safe and effective in septic shock patients. We reported previously that PMX-F treatment is effective in reducing various inflammatory mediators.9,10 Tsushima et al.11 reported that PMX-F treatment is effective against ARDS induced by direct or indirect pulmonary injury. We hypothesized that blood HMGB1 and urinary 8-OHdG are abnormally secreted in patients with ARDS and that PMX-F treatment would be effective in ameliorating these changes.

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Patients and Methods

Patients

Our study group comprised 20 patients with ARDS (12 men and 8 women; mean age, 62 ± 8 years) who underwent PMX-F treatment and 20 age-matched healthy volunteers (12 men and 8 women; mean age, 60 ± 6 years). This study was approved by the local ethics committee for human research and was performed in the intensive care units of our hospitals. Informed consent was obtained from each patient and/or each patient's family. We excluded individuals with malignancy, ischemic heart disease, alcohol abuse, chronic respiratory failure including interstitial pneumonitis and tuberculosis, collagen disease, or liver disease. ARDS was defined according to the criteria of the American European Consensus Conference:12 acute onset of lung injury, diffuse bilateral infiltrates seen on chest radiography: a PaO2/FiO2 (PF) ratio of <200 torr, pulmonary artery occlusion pressure of <18 mm Hg, and no clinical evidence of left atrial hypertension. To rule out congestive heart failure, ultrasound cardiography was performed. For selection of patients, the condition underlying ARDS was pneumonia or another infection (Table 1). All patients were given several kinds of broad spectrum antibiotics on admission to other hospitals or clinics but were not given steroids before PMX-F treatment. On enrollment in the study, the same antibiotics were continued. The Acute Physiology and Chronic Health Evaluation (APACHE II) score,13 Sequential Organ Failure Assessment score,14 and lung injury score15 were determined at the time PMX-F treatment was started.

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PMX-F Treatment

PMX-F hemoperfusion was as described previously.16,17 PMX-F treatment was performed in patients twice, with a 24-hour interval. Access to the circulation for direct hemoperfusion with PMX-F was obtained by means of a double-lumen catheter (Arrow International, Inc., Reading, PA) inserted into the femoral vein according to the Seldinger method. Direct hemoperfusion was carried out for 2 hours at a flow rate of 100 ml/min. Nafamostat mesilate (Torii Pharmaceutical, Torii, Japan) was used as anticoagulant. Nafamostat mesilate is a serine protease inhibitor that exerts its anticoagulatory effects primarily by inhibiting thrombin. The half-life of nafamostat mesilate is 8 minutes, and its anticoagulatory effects were observed only in the extracorporeal circuit. Blood pressure, heart rate, the PF ratio, white blood cell count, C-reactive protein (CRP), blood endotoxin, blood HMGB1, and urinary 8-OHdG levels were measured before and after the first and second PMX-F treatments.

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Biomarkers

Blood endotoxin levels were determined by the Endospecy test (Seikagaku Corp., Tokyo, Japan) according to the perchloric acid method1,16 described previously. The upper limit of normal is 9.8 pg/ml. The urinary 8-OHdG levels were estimated at 11:00 A.M. Twenty-four-hour urine samples were obtained starting at 11:00 A.M. and ending at 11:00 A.M. the next day. Urine samples were obtained from all patients (there were no anuric patients). The urinary 8-OHdG levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit that uses a highly sensitive monoclonal antibody, as described previously (8-OHdG Check, Nikken Foods, Fukuroi, Shizuoka, Japan).18,19 Blood HMGB1 levels were measured by ELISA kit obtained from Shino-Test Corp. (Kanagawa, Japan) as previously described.20 Validated inter-assay and intra-assay coefficients of variation were <10% and the detection limit of the ELISA system was 0.3 ng/ml.21

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Statistics

Normally distributed values are presented as mean ± SD. Differences were analyzed by Student's t test. Differences in values obtained before and after PMX-F treatment were assessed by Wilcoxon's signed-rank test. To analyze the relations between endotoxin, HNGB1, and urinary 8-OHdG levels, linear regression was performed. p values <0.05 were accepted as statistically significant.

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Results

Twenty-eight-day survival of patients with ARDS after PMX-F treatment was 80%. Clinical and laboratory data of the 20 patients with ARDS are shown in Table 1. Blood endotoxin levels (20.6 ± 8.2 pg/ml), blood HMGB1 levels (26.5 ± 12.5 ng/ml), and urinary 8-OHdG levels (38.5 ± 19.2 ng/mg.Cr) were significantly higher in patients with ARDS than in healthy volunteers (2.2 ± 0.6 pg/ml, p < 0.001; 0.68 ± 0.22 ng/ml, p < 0.001; 4.6 ± 2.8 ng/mg.Cr, and p < 0.001, respectively). Clinical and laboratory markers before and after PMX-F treatment are shown in Table 2. Systolic and diastolic blood pressures and the PF ratio increased significantly after the first (p < 0.05) and second (p < 0.01) PMX-F treatments. White blood cell and CRP levels decreased significantly after the first (p < 0.05) and second (p < 0.01) treatments. Heart rate changed little after PMX-F treatment. Blood endotoxin levels decreased significantly after the first (10.8 ± 4.0 pg/ml, p < 0.01) and second (4.0 ± 1.2 pg/ml, p < 0.001) PMX-F treatments. Blood HMGB1 levels decreased significantly after the first (12.0 ± 6.0 ng/ml, p < 0.01) and second (2.8 ± 0.6 ng/ml, p < 0.001) treatments. Urinary 8-OHdG levels also decreased significantly after the first (19.0 ± 7.8 ng/mg.Cr, p < 0.01) and second (6.8 ± 2.2 ng/mg.Cr, p < 0.001) treatments. Relations between blood endotoxin, blood HMGB1, and urinary 8-OHdG levels in patients with ARDS before and after the first and second PMX-F treatments are shown in Table 3. Blood endotoxin levels correlated significantly with blood HMGB1 levels before (p < 0.01) and after the first (p < 0.01) and second (p < 0.01) PMX-F treatments. Blood endotoxin levels correlated significantly with urinary 8-OHdG levels before (p < 0.001) and after the first (p < 0.001) and second (p < 0.001) treatments. Blood HMGB1 levels correlated significantly with urinary 8-OHdG levels before (p < 0.001) and after the first (p < 0.01) and second (p < 0.01) PMX-F treatments.

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Discussion

In this study, blood endotoxin, HMGB1, and urinary 8-OHdG levels in patients with ARDS were significantly higher than those in healthy controls and PMX-F treatment markedly improved oxygenation and blood pressure, blood endotoxin, blood HMGB1, and urinary 8-OHdG levels. Uriu et al.22 reported that improvement of hyperdynamic circulation was directly related to endotoxin removal and that endotoxin plays an important role in the development of septic shock. Tsushima et al.11 reported that in patients with ARDS, PMX-F treatment improved circulatory disturbance and oxygenation despite the underlying disease and that mortality improved remarkably compared with that before the induction of PMX-F treatment.

HMGB1 mediates the induction of delayed endotoxin lethality and acute lung injury.23 The pathophysiology of ARDS involves resident lung cells, including epithelial cells, as well as neutrophils, monocytes/macrophages, and platelets.24 HMGB1 is released by activated macrophages as a late-phase mediator during prolonged inflammation.25 Vascular smooth muscle cells positive for HMGB1 were shown to express CRP and matrix metalloproteinase (MMP)-9.25 MMPs play an important role in pathogenic pulmonary processes, and MMP-9 is essential for remodeling of the basement membrane in various pulmonary inflammatory diseases including ARDS.26 HMGB1 binds LPS in a concentration-dependent manner, and this binding was shown to be inhibited by polymyxin B.27 A mixture of HMGB1 and LPS vs. HMGB1 or LPS alone, results in a higher increase in tumor necrosis factor-alpha production in peripheral blood monocytes.27 Thus, we propose that HMGB1 plays an important role in ARDS with endotoxemia. Recently, HMGB1 levels were shown to be higher in bronchoalveolar lavage (BAL) fluid obtained from the site of infection in patients with pneumonia than in lavage fluid from healthy control subjects, suggesting that HMGB1 release may occur predominantly at the site of infection.28 However, little is known about HMGB1 levels in BAL fluid from patients with ARDS. Measuring HMGB1 has been quite challenging because no ELISA was available until recently.20,21 Earlier studies used blotting methods for measuring HMGB1.29 Gaini et al.30 reported that median HMGB1 levels measured by ELISA were 1.54 ng/ml in non-infected patients, 2.41 ng/ml in infected patients without sepsis, 2.24 ng/ml in patients with sepsis, and 2.18 ng/ml in patients with severe sepsis. In this study, blood HMGB1 levels in patients with ARDS were extremely high (mean: 26.5 ng/ml) based on the same ELISA system. Our data may be due, in part, to disease severity or to the time of blood sampling (because HMGB1 is a “late-onset” cytokine). Recently, Sakamoto et al.31 reported that HMGB1 levels improved significantly after successful PMX-F treatment and that the circulation dynamics of patients with septic shock can be improved by reducing HMGB1 levels.

Oxidant-mediated tissue injury is probably important in the pathogenesis of ARDS.32 Oxidants are generated as a result of the inflammatory response by phagocytic cells such as mononuclear cells.33 Levels of reactive species correlate with both the disease outcome and the severity of the injury to the alveolar epithelium in acute lung injury.33 The most frequently detected and studied oxidized nucleoside in nuclear and mitochondrial DNA lesions is 8-OHdG. On DNA repair, 8-OHdG is excreted into the urine.34 Recently, investigators have reported the importance of urinary 8-OHdG in both adult and pediatric medicine.35,36 In this study, urinary 8-OHdG levels were significantly higher in patients with ARDS than in healthy volunteers. Oxidative stress is induced by administration of LPS. We first showed that urinary 8-OHdG levels in patients with ARDS correlated with blood endotoxin levels. Li et al.37 reported that the degrees of inflammation and oxidative stress were positively related to LPS. It may be important to study correlation between urinary 8-OHdG and other known oxidative stress markers including plasma malondialdehyde, plasma F2 isoprostanes, and plasma thiobarbituric acid reactant substances in patients with ARDS. Some investigators reported that trace elements and vitamins that support antioxidant functions are safe and may be associated with a reduction in mortality in critically ill patients.38 Nathens et al.39 reported that the early antioxidant supplementation with alpha-tocopherol and ascorbic acid reduces the incidence of organ failure in critically ill patients including patients with ARDS. In contrast, oxidative stress is increased in critically ill patients according to antioxidant vitamin intake, independent of disease severity.40 In this study, PMX-F treatment significantly inhibited urinary 8-OHdG levels in patients with ARDS.

In this study, we could not compare between patients with ARDS with PMX-F treatment and those who did not receive PMX-F treatment. Patients with ARDS were severe, therefore, randomized study was ethically difficult. However, it would be needed to compare patients with ARDS treated with PMX-F treatment or not in future. Sakamoto et al.41 reported the effectiveness of continuous hemodiafiltration (CHDF) after PMX-F treatment of septic shock patients with endotoxemia. Nakada et al.42 reported that cytokine-oriented critical care using CHDF may be an effective strategy for the treatment of septic shock. It may be needed to compare patients with ARDS with endotoxemia treated with PMX-F and those treated with CHDF or those treated with both combinations.

In summary, we showed that blood HMGB1 and urinary 8-OHdG levels are increased in patients with ARDS and that PMX-F treatment is effective in decreasing these levels.

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Acknowledgment

The authors thank Mr. Hisataka Shoji, Mr. Yoshihiro Nakamura, and Mr. Misao Hachiya, Toray Medical Co., Ltd., Tokyo, Japan, for their helpful suggestions. The authors also thank Mr. Yoshinobu Takahashi, Shinmatsudo Central General Hospital, Chiba, Japan, for his technical assistance.

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References

1. Tsushima K, Kubo K, Yoshikawa S, et al: Effects of PMX-DHP treatment for patients with directly induced acute respiratory distress syndrome. Ther Apher Dial 11: 138–145, 2007.
2. Bosma KJ, Lewis JF: Emerging therapies for treatment of acute lung injury and acute respiratory distress syndrome. Expert Opin Emerg Drugs 12: 461–477, 2007.
3. Buttenschoen K, Kornmann M, Beger D, et al: Endotoxemia and endotoxin tolerance in patients with ARDS. Langenbecks Arch Surg 393: 473–478, 2008.
4. Czura CJ, Wang H, Tracey KJ: Dual roles of HMGB1: DNA binding and cytokine. J Endotoxin Res 7: 315–321, 2001.
5. Voll RE, Urbonaviciute V, Herrmann M, Kalden JR: High mobility group box 1 in the pathogenesis of inflammatory and autoimmune diseases. Isr Med Assoc J 10: 26–28, 2008.
6. Klune JR, Dhupar R, Cardinal J, et al: HMGB1 endogenous danger signaling. Mol Med 14: 476–484, 2008.
7. Kimura S, Yamauchi H, Hibino Y, et al: Evaluation of urinary 8-hydroxydeoxyguanine in healthy Japanese people. Basic Clin Phamacol Toxicol 98: 496–502, 2006.
8. Nakamura T, Kawagoe Y, Ueda Y, et al: Polymyxin B-immobilized fibre haemoperfusion reduces urinary 8-hydroxy-2′-deoxyguanosine levels in patients with septic shock. Clin Intens Care 17: 95–100, 2008.
9. Nakamura T, Kawagoe Y, Suzuki T, et al: Changes in plasma IL-18 by direct hemoperfusion with polymyxin B-immobilized fiber in patients with septic shock. Blood Purif 23: 417–420, 2005.
10. Nakamura T, Suzuki T, Kawagoe Y, Koide H: Polymyxin B- immobilized fiber hemoperfusion attenuates increased plasma atrial natriuretic peptide and brain natriuretic peptide levels in patients with septic shock. ASAIO J 54: 210–213, 2008.
11. Tsushima K, Kubo K, Koizumi T, et al: Direct hemoperfusion using a polymyxin B immobilized column improves acute respiratory distress syndrome. J Clin Apher 17: 97–102, 2002.
12. Bernard GR, Artigas A, Brigham KI, et al: The American-European Consensus Conference on ARDS. Definition, mechanisms, relevant outcomes and clinical trial coordination. Am J Respir Crit Care Med 149: 811–824, 1994.
13. Knaus W, Draper E, Wagner D, Zimmerman J: APACHE II: A severity of disease classification system. Crit Care Med 13: 818–829, 1985.
14. Vincent JL, de Mendonça A, Cantraine F: Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: Results of a multicenter, prospective study. Working group on “sepsis-related problems” of the European Society of Intensive Care Medicine. Crit Care Med 26: 1793–1800, 1998.
15. Murray JF, Matthay MA, Luce JM, Flick MR: An expanded definition of the adult respiratory distress syndrome. Am Rev Respir Dis 138: 720–723, 1988.
16. Aoki H, Kodama M, Tani T, Hanasawa K: Treatment of sepsis by extracorporeal elimination of endotoxin using polymyxin B-immobilized fiber. Am J Surg 167: 412–417, 1994.
17. Shoji H, Tani T, Hanasawa K, Kodama M: Extracorporeal endotoxin removal by polymyxin B immobilized fiber cartridge: Designing and antiendotoxin efficacy in the clinical application. Ther Apher 2: 3–12, 1998.
18. Kanaya S, Ikeya M, Yamamoto K, et al: Comparison of an oxidative stress biomarker “urinary 8-hydroxy-2′-deoxyguanosine” between smokers and non-smokers. Biofactors 22: 255–258, 2004.
19. Saito S, Yamauchi H, Hasui Y, et al: Quantitative determination of urinary 8-hydroxydeoxyguanosine (8-OHdG) by using ELISA. Res Commun Mol Pathol Pharmacol 107: 39–44, 2000.
20. Yamada S, Inoue K, Yakabe K, et al: High mobility group protein-1 (HMGB1) quantified by ELISA with a monoclonal antibody that does not cross-react with HMGB2. Clin Chem 49: 1535–1537, 2003.
21. Yamada S, Yakabe K, Ishii J, et al: New high mobility group box 1 assay system. Clin Chim Acta 372: 173–178, 2006.
22. Uriu K, Osajima A, Hiroshige K: Endotoxin removal by direct hemoperfusion with an adsorbent column using polymyxin B-immobilized fiber ameliorates systemic circulatory disturbance in patients with septic shock. Am J Kidney Dis 39: 937–947, 2002.
23. Wang H, Bloom O, Zhang M, et al: HMG-1 as a late mediator of endotoxin lethality in mice. Science 285: 248–251, 1999.
24. Liu KD, Matthay MA: Advances in critical care for the nephrologists: Acute lung injury/ARDS. Clin J Am Soc Nephrol 3: 578–586, 2008.
25. Inoue K, Kawahara K, Biswas KK, et al: HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques. Cardiovasc Pathol 16: 136–143, 2007.
26. Lanchou J, Corbel M, Tanguy M, et al: Imbalance between matrix metalloproteinases (MMP-9 and MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in acute respiratory distress syndrome patients. Crit Care Med 31: 536–542, 2003.
27. Youn JH, Oh YJ, Kim ES, et al: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes. J Immunol 180: 5067–5074, 2008.
28. Van Zoelen MA, Laterre PF, van Veen SQ, et al: Systemic and local high mobility group box 1 concentrations during severe infection. Crit Care Med 35: 2799–2804, 2007.
29. Sunden-Cullberg J, Norrby-Teglund A, Rouhiainen A, et al: Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 33: 564–573, 2005.
30. Gaini S, Pedersen SS, Koldkjaer OG, et al: High mobility group box-1 protein in patients with suspected community-acquired infections and sepsis: A prospective study. Crit Care 11: R32, 2007.
31. Sakamoto Y, Mashiko K, Matsumoto H, et al: Relationship between effect of polymyxin B-immobilized fiber and high mobility group box-1 protein in septic shock patients. ASAIO J 53: 324–328, 2007.
32. Fukuto JM, Hobbs AJ, Ignarro LJ: Conversion of nitroxyl (HNO) to nitric oxide (NO) in biological systems: The role of physiological oxidants and relevance to the biological activity of HNO. Biochem Biophys Res Commun 196: 707–713, 1993.
33. Lang JD, McArdle PJ, O'Reilly PJ, Matalon S: Oxidant-antioxidant balance in acute lung injury. Chest 122: 314S–320S, 2002.
34. Wu LL, Chiou CC, Chang PY, Wu JT: Urinary 8-OHdG: A marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetes. Clin Chim Acta 339: 1–9, 2004.
35. Maeshima E, Liang XM, Goda M, et al: The efficacy of vitamin E against oxidative damage and autoantibody production in systemic lupus erythematosus: A preliminary study. Clin Rheumatol 26: 401–404, 2007.
36. Tsukahara H: Biomarkers for oxidative stress: Clinical application in pediatric medicine. Curr Med Chem 14: 339–351, 2007.
37. Li L, Shoji W, Takano H, et al: Increased susceptibility of MER5 (peroxiredoxin III) knockout mice to LPS-induced oxidative stress. Biochem Biophys Res Commun 355: 715–721, 2007.
38. Heyland DK, Dhaliwal R, Suchner U, Berger MM: Antioxidant mutriens: A systemic review of trace elements and vitamins in the critically ill patients. Intens Care Med 31: 327–337, 2005.
39. Nathens AB, Neff MJ, Jurkovich GJ, et al: Randomized, prospective trial of antioxidant supplementation in critically ill surgical patients. Ann Surg 236: 814–822, 2002.
40. Abiles J, de la Cruz AP, Castano J, et al: Oxidative stress is increased in critically ill patients according to antioxidant vitamins intake, independent of severity: A cohort study. Crit Care 10: R146, 2006.
41. Sakamoto Y, Mashiko K, Obata T, et al: Effectiveness of continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter after polymyxin B-immobilized fiber column therapy of septic shock. ASAIO J 54: 129–132, 2008.
42. Nakada TA, Oda S, Matsuda K, et al: Continuous hemodiafiltration with PMMA hemofilter in the treatment of patients with septic shock. Mol Med 14: 257–163, 2008.
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