- Email: [email protected]
Levels of oxidized low-density lipoproteins are increased in patients with severe sepsis
Journal of Critical Care (2008) 23, 537–541
Levels of oxidized low-density lipoproteins are increased in patients with severe sepsis☆ Michael Behnes MS a , Martina Brueckmann MD a , Volker Liebe MD a , Claudia Liebetrau a , Siegfried Lang PhD a , Christian Putensen MD b , Martin Borggrefe MD a , Ursula Hoffmann MD a,⁎ a
First Department of Medicine, Faculty of Medicine Mannheim, University of Heidelberg, 68167 Mannheim, Germany Department of Anesthesiology, University of Bonn, 53127 Bonn, Germany
b
Keywords: Drotrecogin α (activated); Oxidative stress; Oxidized low-density lipoprotein; Sepsis
Abstract Background: It was hypothesized that the inflammatory response of patients with severe sepsis may result in changes of plasma levels of oxidized low-density lipoproteins (ox-LDLs) and that drotrecogin α (activated) (DAA) (Xigris, Eli Lilly and Company [Indiana 46285, USA]) may influence ox-LDL levels. Materials and Methods: The ox-LDL levels were measured in severe septic patients on day 1, 4, and 7 of severe sepsis. Patients were treated either with or without DAA. Results: The ox-LDL levels increased significantly (P b .05) from day 1 to day 7 (day 1, mean ± SEM, 25.4 ± 1.8 U/L; day 4, mean ± SEM, 34.3 ± 2.1 U/L; day 7, mean ± SEM, 38.3 ± 2.1 U/L) in all patients (n = 68). The ox-LDL levels increased significantly from day 1 to day 7 both in patients treated with (n = 31) and without DAA (n = 37) (P b .001) (DAA-group: day 1, mean ± SEM, 24.4 ± 2.8 U/L; day 4, mean ± SEM, 35.5 ± 2.9 U/L; day 7, mean ± SEM, 40.7 ± 3.2 U/L) (control group: day 1, mean ± SEM, 26.3 ± 2.8 U/L; day 4, mean ± SEM, 33.2 ± 2.9 U/L; day 7, mean ± SEM, 36.4 ± 2.9 U/L). No significant differences of ox-LDL levels were observed between both groups at any point of time (P N .05). Conclusions: The ox-LDL concentrations increase significantly during the first week of severe sepsis and are not affected by administration of drotrecogin α (activated) (Xigris). © 2008 Elsevier Inc. All rights reserved.
1. Introduction Sepsis is defined as a clinical syndrome including infection and the systemic inflammatory response [1,2]. Sepsis emerges to severe sepsis when one or more organ ☆
This work was supported by a grant of the Faculty of Medicine Mannheim, University of Heidelberg, Mannheim, Germany. ⁎ Corresponding author. Tel.: +49 621 383 2030; fax: +49 621 383 2030. E-mail address: [email protected] (U. Hoffmann). 0883-9441/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jcrc.2008.09.002
dysfunctions reemerge. Individual factors (eg, genetic factors or premorbid health status), extent of infection, and complicating organ dysfunction have a substantial impact on the disease process, host response, and mortality. Several inflammatory and procoagulant markers have been identified indicating this response as well as the disease severity in severe septic patients (eg, procalcitonin [PCT], interleukin 6 [IL-6], D-dimer) [1-3]. Free radical generation represents one of many proinflammatory reactions produced along the cytokine cascade in a septic organism [4]. Oxidative stress mediated by reactive
538 oxygen intermediates (ROIs) leads to oxidation of DNA, proteins, and lipids of the endothelium [5]. It has been demonstrated that plasma redox status—mostly measured by levels of lipoperoxides or myeloperoxidase—relates to the severity of the disease in critically ill patients [6-8]. Moreover, oxidation of low-density lipoproteins (LDLs) has been shown to occur in diseases associated with acute or chronic infection and inflammation [9,10] as well as in arteriosclerosis [11-14]. Several mediators and enzymes have been identified to initiate LDL oxidation (eg, metal ions, lipoxygenase, thiols, superoxides, myeloperoxidase, or inducible nitric oxide synthase) [8]. Drotrecogin α (activated) has been demonstrated to reduce mortality in patients with severe sepsis within the recombinant human activated protein C worldwide evaluation in severe sepsis trial [15]. However, the time course of ox-LDL levels in severe sepsis as well as its possible modulation by the treatment with drotrecogin α (activated) has not been evaluated yet. It was hypothesized that the inflammatory response of patients with severe sepsis may result in changes of plasma levels of ox-LDL and that drotrecogin α (activated) might influence ox-LDL levels. Therefore, the aim of the present study was to investigate the time course of ox-LDL levels in septic patients treated with or without drotrecogin α (activated).
M. Behnes et al. 80 000/mm3, or an unexplained metabolic acidosis with pH of less than 7.3 and lactate levels of more than 1.5 times of the upper limit of normal. Sepsis-induced organ failures in these patients were strongly connected to infection and were present for less than 24 hours. Severity of sepsis was defined by the Acute Physiology and Chronic Health Evaluation II (APACHE II) score [16]. Thirty-one septic patients received a 96-hour infusion of drotrecogin α (activated) (Xigris, Eli Lilly and Company [Indiana 46285, USA]: 24 μg/kg body weight per hour). Thirty-seven patients with severe sepsis (control subjects) did not receive drotrecogin α (activated) because of contraindications for drotrecogin α (activated) such as platelet count less than 30000/mm3, need for therapeutic anticoagulation with heparin, increased risk of bleeding, recent gastrointestinal bleeding, or stroke within the last 3 months [15]. Blood samples were obtained by venipuncture within the first 24 hours after the diagnosis of severe sepsis (defined as day 1). Additional blood samples were obtained on day 4 and 7 of severe sepsis. Baseline characteristics, such as APACHE II scores, creatinine levels, white blood cell count, platelet count, C reactive protein, bilirubin, international normalized ratio, activated partial thromboplastin time, antithrombin III, and D-dimer as well as central venous pressure were determined on day 1 of severe sepsis.
2.2. Plasma samples and ox-LDL assay
2. Materials and methods 2.1. Study population A total of 68 patients having severe sepsis were prospectively enrolled for this study from March 2001 until October 2003. Respectively, 31 patients were treated with drotrecogin α (activated) and constituted the treatment group. The remaining 37 patients not treated with drotrecogin α (activated) were defined as controls. The study was performed at the Department of Medicine at the University Hospital Mannheim, Germany, and at the Department of Anesthesiology at the University Hospital Bonn, Germany. The study was carried out according to the principles of the declaration of Helsinki and was approved by the local ethics committees. Written informed consent was obtained from all participating patients or their legal representatives. The diagnosis of severe sepsis was based on criteria established by the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference 1992 [1]. Patients presented with a proven infection, 3 or more criteria of the systemic inflammatory response syndrome criteria and at least 1 of the following newly developed, sepsis-induced organ failures: cardiovascular organ failure with need for vasopressors, pulmonary organ failure defined as PaO2/FIO2 less than 250, renal organ failure with urine output of less than 0.5 mL/kg per hour, hematologic organ failure with platelet count of less than
Blood samples were obtained by venipuncture into EDTA plasma tubes. Within 30 minutes, all blood samples were centrifuged at 2700g at 4°C for 10 minutes. The EDTA plasma was separated, frozen, and stored at −80°C. Measurements of ox-LDL were performed with a solid phase 2-site enzyme-linked immunosorbent assay (ELISA) (Mercodia AB, Uppsala, Sweden; Mercodia oxidized LDL ELISA). Based on the direct sandwich technique, 2 monoclonal antibodies (ie, murine monoclonal antibodies mAb-4E6) are directed against antigenic determinants on the human ox-LDL molecule. After incubation and washing, a peroxidase-conjugated antibody recognizes the ox-LDL bound to the solid phase. After a second incubation and washing, the bound conjugate is detected by reaction with 3′,5,5′-tetramethylbenzidine. Reaction is stopped by adding acid to result in a colorimetric end point. The colored product is measured spectrophotometrically at a wavelength of 450 nm. The lower detection limit of the Mercodia oxidized LDL ELISA is less than 1 mU/L of ox-LDL within a measurable range up to 117 U/L.
2.3. Statistical analysis For data with normal distribution, the paired or unpaired Student t test was applied. Otherwise, the Mann-Whitney U test was used as a nonparametric test. Deviations from a Gaussian distribution were tested by the Kolmogorov-
Oxidized LDL in severe sepsis
539
Smirnov test. More than 2 groups were compared using paired t tests adjusted for multiple comparisons using the Holm-Bonferroni method. Pearson's correlation was used for normally distributed data. Spearman's rank correlation was used for nonparametric data. Noncontinuous variables were analyzed using a 2 × 2 table and Fisher exact test. Data are presented as mean ± SD or SEM as indicated. Values of P b .05 (2-tailed) were considered statistically significant. The calculations were performed with InStat (GraphPad Software Inc. [San Diego 92130, USA]) and SPSS software (SPSS Inc. [Chicago 60606, USA]).
3. Results Baseline characteristics of 68 patients having severe sepsis are given in Table 1. Age of patients treated with drotrecogin α (activated) (n = 31, treatment group) was 55 years compared to a mean age of 63 years of patients not treated with drotrecogin α (activated) (n = 37, control group) (P = .02). The most common primary site of infection in both groups was the lung (n = 14; n = 21), followed by intraabdominal Table 1 sepsis
Baseline characteristics of 68 patients having severe Drotrecogin α Controls (activated) (n = 31) (n = 37)
Age (y) a Sex (n) Male Female Heart rate (beats/min) a Central venous pressure (mm Hg) a APACHE II score a Primary site of infection (n) Lung Intraabdominal Skin Unknown CRP (mg/L) a Creatinine (mg/dL) a White blood cell count (109/L) a Platelets (109/L) a Activated partial thromboplastin time (s) a Antithrombin III (%) a a D-dimer (μg/mL) Bilirubin (mg/dL) a Death (n) Survival (n)
54.6 ± 14.7
P
21 10 119 ± 32.4 23.9 ± 15.6
63.1 ± 14.7 .02 .82 26 11 97 ± 31.9 .01 14.3 ± 5.5 .03
21.9 ± 7.6
24.3 ± 7.6
14 9 3 5 155 ± 106 2.1 ± 2.1 15.3 ± 6.6
21 10 0 6 199 ± 112 .14 1.7 ± 1.2 .91 15.0 ± 8.0 .85
273 ± 179 35.9 ± 73.0
213 ± 156 .16 34.3 ± 10.6 .46
53.5 ± 17.5 3.7 ± 1.9 1.9 ± 2.3 6 25
55.3 ± 18.6 4.9 ± 6.8 1.5 ± 1.9 14 23
CRP indicates C reactive protein. a Data are presented as mean ± SD.
.23 .43
.71 .47 .32 .12
Fig. 1 Oxidized LDL levels during the first week of severe sepsis in all patients (n = 68). Oxidized LDL concentrations significantly increase in all patients on day 4 and 7 compared to day 1 of severe sepsis. Results are given as mean ± SEM; ** indicates P b .001; *P b .05 vs day 1.
infections (n = 9; n = 10). The mean APACHE II score of patients treated with drotrecogin α (activated) was 21.9 (SD ± 7.6) compared to a mean APACHE score of 24.3 (SD ± 7.6) of the control group (P = .23). Furthermore, creatinine levels in the treatment group (2.1 mg/dL; SD ± 2.1) did not significantly differ from controls (1.7 mg/dL; SD ± 1.2). Six patients treated with drotrecogin α (activated) died during 7-day follow-up compared to 14 patients not treated with drotrecogin α (activated) (P = .12). The ox-LDL levels increased significantly (P b .0001) from day 1 to day 7 of severe sepsis in all patients (n = 68) (Fig. 1). Mean ox-LDL levels were 25.4 U/L (±SEM 1.8) on day 1 of severe sepsis, increasing to a mean of 34.3 U/L (±SEM 2.1) on day 4 and to a mean ox-LDL concentration of 38.4 U/L (±SEM 2.1) on day 7 of severe sepsis. Multiple comparison tests between all 3 points of time were statistical significant (P b .05). To determine the influence of drotrecogin α (activated), oxLDL levels were compared between patients treated with and without drotrecogin α (activated). As presented in Fig. 2, oxLDL levels increased significantly from day 1 to day 7 of severe sepsis in both treatment groups (P b .001). No significant differences of ox-LDL levels were observed between patients treated with or without drotrecogin α (activated) at days 1, 4, or 7. Moreover, no differences of ox-LDL levels have been observed between men and women (data not shown). Taken together, ox-LDL concentrations increased during the 7-day examination period, both in patients treated with (r = 0.394; P = .0001) as well as without drotrecogin α (activated) (r = 0.232; P = .015). No significant difference of mortality was observed in patients treated with (19%) and without drotrecogin α (activated) (38%). The ox-LDL levels of surviving patients were similar (mean ± SD, 39.6 ± 15.7 U/L) to those patients who died (mean ± SD, 33.1 ± 14.7 U/L) (P = .11) on day 7 of severe sepsis. The extent of increase of ox-LDL levels from day
540
Fig. 2 Comparison of ox-LDL levels between patients treated with (n = 31) and without (n = 37) drotrecogin α (activated). Oxidized LDL concentrations significantly increase in patients treated with and without drotrecogin α (activated) on day 4 and 7 compared to day 1 of severe sepsis. Oxidized LDL levels between control patients and patients treated with drotrecogin α (activated) at days 1, 4, and 7 are not statistically significantly different (P N .1). Results are given as mean ± SEM; *** indicates P b .0001; **P b .001; *P b .05 vs day 1.
1 to day 7 did not correlate to the APACHE II scores (not significant). Surviving patients had a trend toward lower APACHE II score (mean, 22.3; ± SD, 7.2) compared to those patients who deceased (mean, 25.2; ± SD, 8.5; P = .2). Correlations between APACHE II score and white blood cell count, levels of C-reactive protein, and ox-LDL were determined for all patients (n = 68) or separately for the treatment and control group. No statistically significant correlations were found between APACHE II scores and white blood cell count, levels of C-reactive protein, or bilirubin.
4. Discussion The present study evaluated levels of ox-LDL in 68 patients during the first week of severe sepsis. The ox-LDL levels increase significantly during the first 7 days of severe sepsis. Moreover, no treatment effect of drotrecogin α (activated) was observed on ox-LDL concentrations in the time course of patients having severe sepsis. This study demonstrates for the first time that ox-LDL levels increase in patients having severe sepsis. These findings contribute to a better understanding of the development of oxidative stress in severe sepsis. Uncontrolled production of reactive oxygen and nitrogen species leads to molecular damage of cellular key molecules and changes cellular responses such as proliferation, apoptosis, and necrosis in critically ill patients (ie, acute respiratory distress syndrome and severe sepsis) [5]. Increased oxidative stress enhances the progression of multiple organ dysfunction [8]. Several immune-cellular effects of ox-LDL have been described supporting its role in systemic inflammatory disorders. In vitro experiments demonstrated that increasing
M. Behnes et al. levels of ox-LDL activate chemotaxis of neutrophil or eosinophil granulocytes and monocytes under circumstances of exacerbating airway inflammation [17-19]. It has been described that ox-LDL induces apoptosis of endothelial cells [20-22]. In severe sepsis, tissue damage represents a main consequence of inflammation, coagulation-reduced oxygen supply, and in particular, endothelial cell damage leading to microvascular dysfunction [23,24]. Oxidized lipoproteins have not only been associated with proinflammatory responses but also with antiinflammatory effects at different levels of inflammatory signaling and in several cellular compartments [25]. It has been described that oxidized lipoproteins inactivate bacterial endotoxins (ie, lipopolysaccharides) and inhibit key elements in the nuclear factor κB signaling cascade. Therefore, it is presumed that oxidized lipids promote a shift from acute inflammatory toward chronic inflammatory processes [25]. The steady increase of ox-LDL levels within the first week of severe sepsis may partly reflect the continuum of inflammatory response in humans. Notably, Safa et al [26] reported that oxidation of lipids enhances the anticoagulant function of natural activated protein C and factor Va inactivation. In sepsis, physiologic anticoagulants such as protein C, heparin, and antithrombin are reduced [27]. The present findings demonstrate that drotrecogin α (activated) (Xigris) does not affect ox-LDL levels during the time course of sepsis. However, the up-regulation of ox-LDL levels during the first 7 days of severe sepsis in all patient groups (ie, treatment, control, and all patients) might possibly represent an adaptive counterregulatory mechanism to increase anticoagulant activity in severe sepsis. The beneficial effects of drotrecogin α (activated) such as increased survival have been attributed to its antithrombotic, profibrinolytic, antiinflammatory, and antiapoptotic properties [28,29]. However, no data currently exist whether drotrecogin α (activated) influences markers of oxidative stress. The present findings suggest that drotrecogin α (activated) has no effects on levels of ox-LDL. Additional in vivo investigations are needed to confirm the present results. In summary, these data suggest that oxidative stress as quantified by measurements of ox-LDL levels increases within the first week of severe sepsis independently of a treatment regimen with drotrecogin α (activated) (Xigris).
References [1] Bone RC, Balk RA, Cerra FB, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992; 101:1644-55. [2] Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/ESICM/ACCP/ ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003;31:1250-6. [3] Dhainaut JF, Shorr AF, Macias WL, et al. Dynamic evolution of coagulopathy in the first day of severe sepsis: relationship with mortality and organ failure. Crit Care Med 2005;33:341-8.
Oxidized LDL in severe sepsis [4] Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997;112:235-43. [5] Gutteridge JM, Mitchell J. Redox imbalance in the critically ill. Br Med Bull 1999;55:49-75. [6] Alonso de Vega JM, Diaz J, Serrano E, et al. Plasma redox status relates to severity in critically ill patients. Crit Care Med 2000;28: 1812-4. [7] Alonso de Vega JM, Diaz J, Serrano E, et al. Oxidative stress in critically ill patients with systemic inflammatory response syndrome. Crit Care Med 2002;30:1782-6. [8] Motoyama T, Okamoto K, Kukita I, et al. Possible role of increased oxidant stress in multiple organ failure after systemic inflammatory response syndrome. Crit Care Med 2003;31:1048-52. [9] Gaut JP, Heinecke JW. Mechanisms for oxidizing low-density lipoprotein. Insights from patterns of oxidation products in the artery wall and from mouse models of atherosclerosis. Trends Cardiovasc Med 2001;11:103-12. [10] Memon RA, Staprans I, Noor M, et al. Infection and inflammation induce LDL oxidation in vivo. Arterioscler Thromb Vasc Biol 2000; 20:1536-42. [11] Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655-73. [12] Ehara S, Ueda M, Naruko T, et al. Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation 2001;103:1955-60. [13] Pearson TA, Mensah GA, Alexander RW, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 2003;107:499-511. [14] Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol 2003;41:360-70. [15] Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699-709. [16] Knaus WA, Draper EA, Wagner DP, et al. APACHE II: a severity of disease classification system. Crit Care Med 1985;13:818-29.
541 [17] Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 1999; 180:780-90. [18] Sedgwick JB, Hwang YS, Gerbyshak HA, et al. Oxidized low-density lipoprotein activates migration and degranulation of human granulocytes. Am J Respir Cell Mol Biol 2003;29:702-9. [19] Silva AR, de Assis EF, Caiado LF, et al. Monocyte chemoattractant protein-1 and 5-lipoxygenase products recruit leukocytes in response to platelet-activating factor-like lipids in oxidized low-density lipoprotein. J Immunol 2002;168:4112-20. [20] Dimmeler S, Haendeler J, Galle J, et al. Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the “response to injury” hypothesis. Circulation 1997;95:1760-3. [21] Galle J, Schneider R, Heinloth A, et al. Lp(a) and LDL induce apoptosis in human endothelial cells and in rabbit aorta: role of oxidative stress. Kidney Int 1999;55:1450-61. [22] Heermeier K, Leicht W, Palmetshofer A, et al. Oxidized LDL suppresses NF-kappaB and overcomes protection from apoptosis in activated endothelial cells. J Am Soc Nephrol 2001;12:456-63. [23] Hoffmann JN, Vollmar B, Laschke MW, et al. Microcirculatory alterations in ischemia-reperfusion injury and sepsis: effects of activated protein C and thrombin inhibition. Crit Care 9 Suppl 2005;4:S33-7. [24] Hoffmann JN, Vollmar B, Laschke MW, et al. Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia. Crit Care Med 2004;32:1011-7. [25] Bochkov VN, Leitinger N. Anti-inflammatory properties of lipid oxidation products. J Mol Med 2003;81:613-26. [26] Safa O, Hensley K, Smirnov MD, et al. Lipid oxidation enhances the function of activated protein C. J Biol Chem 2001;276:1829-36. [27] Hughes M. Recombinant human activated protein C. Int J Antimicrob Agents 2006;28:90-4. [28] Cheng T, Liu D, Griffin JH, et al. Activated protein C blocks p53mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 2003;9:338-42. [29] Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kappaB. Crit Care Med 2002;30:S288-93.
Levels of oxidized low-density lipoproteins are increased in patients with severe sepsis☆ Michael Behnes MS a , Martina Brueckmann MD a , Volker Liebe MD a , Claudia Liebetrau a , Siegfried Lang PhD a , Christian Putensen MD b , Martin Borggrefe MD a , Ursula Hoffmann MD a,⁎ a
First Department of Medicine, Faculty of Medicine Mannheim, University of Heidelberg, 68167 Mannheim, Germany Department of Anesthesiology, University of Bonn, 53127 Bonn, Germany
b
Keywords: Drotrecogin α (activated); Oxidative stress; Oxidized low-density lipoprotein; Sepsis
Abstract Background: It was hypothesized that the inflammatory response of patients with severe sepsis may result in changes of plasma levels of oxidized low-density lipoproteins (ox-LDLs) and that drotrecogin α (activated) (DAA) (Xigris, Eli Lilly and Company [Indiana 46285, USA]) may influence ox-LDL levels. Materials and Methods: The ox-LDL levels were measured in severe septic patients on day 1, 4, and 7 of severe sepsis. Patients were treated either with or without DAA. Results: The ox-LDL levels increased significantly (P b .05) from day 1 to day 7 (day 1, mean ± SEM, 25.4 ± 1.8 U/L; day 4, mean ± SEM, 34.3 ± 2.1 U/L; day 7, mean ± SEM, 38.3 ± 2.1 U/L) in all patients (n = 68). The ox-LDL levels increased significantly from day 1 to day 7 both in patients treated with (n = 31) and without DAA (n = 37) (P b .001) (DAA-group: day 1, mean ± SEM, 24.4 ± 2.8 U/L; day 4, mean ± SEM, 35.5 ± 2.9 U/L; day 7, mean ± SEM, 40.7 ± 3.2 U/L) (control group: day 1, mean ± SEM, 26.3 ± 2.8 U/L; day 4, mean ± SEM, 33.2 ± 2.9 U/L; day 7, mean ± SEM, 36.4 ± 2.9 U/L). No significant differences of ox-LDL levels were observed between both groups at any point of time (P N .05). Conclusions: The ox-LDL concentrations increase significantly during the first week of severe sepsis and are not affected by administration of drotrecogin α (activated) (Xigris). © 2008 Elsevier Inc. All rights reserved.
1. Introduction Sepsis is defined as a clinical syndrome including infection and the systemic inflammatory response [1,2]. Sepsis emerges to severe sepsis when one or more organ ☆
This work was supported by a grant of the Faculty of Medicine Mannheim, University of Heidelberg, Mannheim, Germany. ⁎ Corresponding author. Tel.: +49 621 383 2030; fax: +49 621 383 2030. E-mail address: [email protected] (U. Hoffmann). 0883-9441/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jcrc.2008.09.002
dysfunctions reemerge. Individual factors (eg, genetic factors or premorbid health status), extent of infection, and complicating organ dysfunction have a substantial impact on the disease process, host response, and mortality. Several inflammatory and procoagulant markers have been identified indicating this response as well as the disease severity in severe septic patients (eg, procalcitonin [PCT], interleukin 6 [IL-6], D-dimer) [1-3]. Free radical generation represents one of many proinflammatory reactions produced along the cytokine cascade in a septic organism [4]. Oxidative stress mediated by reactive
538 oxygen intermediates (ROIs) leads to oxidation of DNA, proteins, and lipids of the endothelium [5]. It has been demonstrated that plasma redox status—mostly measured by levels of lipoperoxides or myeloperoxidase—relates to the severity of the disease in critically ill patients [6-8]. Moreover, oxidation of low-density lipoproteins (LDLs) has been shown to occur in diseases associated with acute or chronic infection and inflammation [9,10] as well as in arteriosclerosis [11-14]. Several mediators and enzymes have been identified to initiate LDL oxidation (eg, metal ions, lipoxygenase, thiols, superoxides, myeloperoxidase, or inducible nitric oxide synthase) [8]. Drotrecogin α (activated) has been demonstrated to reduce mortality in patients with severe sepsis within the recombinant human activated protein C worldwide evaluation in severe sepsis trial [15]. However, the time course of ox-LDL levels in severe sepsis as well as its possible modulation by the treatment with drotrecogin α (activated) has not been evaluated yet. It was hypothesized that the inflammatory response of patients with severe sepsis may result in changes of plasma levels of ox-LDL and that drotrecogin α (activated) might influence ox-LDL levels. Therefore, the aim of the present study was to investigate the time course of ox-LDL levels in septic patients treated with or without drotrecogin α (activated).
M. Behnes et al. 80 000/mm3, or an unexplained metabolic acidosis with pH of less than 7.3 and lactate levels of more than 1.5 times of the upper limit of normal. Sepsis-induced organ failures in these patients were strongly connected to infection and were present for less than 24 hours. Severity of sepsis was defined by the Acute Physiology and Chronic Health Evaluation II (APACHE II) score [16]. Thirty-one septic patients received a 96-hour infusion of drotrecogin α (activated) (Xigris, Eli Lilly and Company [Indiana 46285, USA]: 24 μg/kg body weight per hour). Thirty-seven patients with severe sepsis (control subjects) did not receive drotrecogin α (activated) because of contraindications for drotrecogin α (activated) such as platelet count less than 30000/mm3, need for therapeutic anticoagulation with heparin, increased risk of bleeding, recent gastrointestinal bleeding, or stroke within the last 3 months [15]. Blood samples were obtained by venipuncture within the first 24 hours after the diagnosis of severe sepsis (defined as day 1). Additional blood samples were obtained on day 4 and 7 of severe sepsis. Baseline characteristics, such as APACHE II scores, creatinine levels, white blood cell count, platelet count, C reactive protein, bilirubin, international normalized ratio, activated partial thromboplastin time, antithrombin III, and D-dimer as well as central venous pressure were determined on day 1 of severe sepsis.
2.2. Plasma samples and ox-LDL assay
2. Materials and methods 2.1. Study population A total of 68 patients having severe sepsis were prospectively enrolled for this study from March 2001 until October 2003. Respectively, 31 patients were treated with drotrecogin α (activated) and constituted the treatment group. The remaining 37 patients not treated with drotrecogin α (activated) were defined as controls. The study was performed at the Department of Medicine at the University Hospital Mannheim, Germany, and at the Department of Anesthesiology at the University Hospital Bonn, Germany. The study was carried out according to the principles of the declaration of Helsinki and was approved by the local ethics committees. Written informed consent was obtained from all participating patients or their legal representatives. The diagnosis of severe sepsis was based on criteria established by the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference 1992 [1]. Patients presented with a proven infection, 3 or more criteria of the systemic inflammatory response syndrome criteria and at least 1 of the following newly developed, sepsis-induced organ failures: cardiovascular organ failure with need for vasopressors, pulmonary organ failure defined as PaO2/FIO2 less than 250, renal organ failure with urine output of less than 0.5 mL/kg per hour, hematologic organ failure with platelet count of less than
Blood samples were obtained by venipuncture into EDTA plasma tubes. Within 30 minutes, all blood samples were centrifuged at 2700g at 4°C for 10 minutes. The EDTA plasma was separated, frozen, and stored at −80°C. Measurements of ox-LDL were performed with a solid phase 2-site enzyme-linked immunosorbent assay (ELISA) (Mercodia AB, Uppsala, Sweden; Mercodia oxidized LDL ELISA). Based on the direct sandwich technique, 2 monoclonal antibodies (ie, murine monoclonal antibodies mAb-4E6) are directed against antigenic determinants on the human ox-LDL molecule. After incubation and washing, a peroxidase-conjugated antibody recognizes the ox-LDL bound to the solid phase. After a second incubation and washing, the bound conjugate is detected by reaction with 3′,5,5′-tetramethylbenzidine. Reaction is stopped by adding acid to result in a colorimetric end point. The colored product is measured spectrophotometrically at a wavelength of 450 nm. The lower detection limit of the Mercodia oxidized LDL ELISA is less than 1 mU/L of ox-LDL within a measurable range up to 117 U/L.
2.3. Statistical analysis For data with normal distribution, the paired or unpaired Student t test was applied. Otherwise, the Mann-Whitney U test was used as a nonparametric test. Deviations from a Gaussian distribution were tested by the Kolmogorov-
Oxidized LDL in severe sepsis
539
Smirnov test. More than 2 groups were compared using paired t tests adjusted for multiple comparisons using the Holm-Bonferroni method. Pearson's correlation was used for normally distributed data. Spearman's rank correlation was used for nonparametric data. Noncontinuous variables were analyzed using a 2 × 2 table and Fisher exact test. Data are presented as mean ± SD or SEM as indicated. Values of P b .05 (2-tailed) were considered statistically significant. The calculations were performed with InStat (GraphPad Software Inc. [San Diego 92130, USA]) and SPSS software (SPSS Inc. [Chicago 60606, USA]).
3. Results Baseline characteristics of 68 patients having severe sepsis are given in Table 1. Age of patients treated with drotrecogin α (activated) (n = 31, treatment group) was 55 years compared to a mean age of 63 years of patients not treated with drotrecogin α (activated) (n = 37, control group) (P = .02). The most common primary site of infection in both groups was the lung (n = 14; n = 21), followed by intraabdominal Table 1 sepsis
Baseline characteristics of 68 patients having severe Drotrecogin α Controls (activated) (n = 31) (n = 37)
Age (y) a Sex (n) Male Female Heart rate (beats/min) a Central venous pressure (mm Hg) a APACHE II score a Primary site of infection (n) Lung Intraabdominal Skin Unknown CRP (mg/L) a Creatinine (mg/dL) a White blood cell count (109/L) a Platelets (109/L) a Activated partial thromboplastin time (s) a Antithrombin III (%) a a D-dimer (μg/mL) Bilirubin (mg/dL) a Death (n) Survival (n)
54.6 ± 14.7
P
21 10 119 ± 32.4 23.9 ± 15.6
63.1 ± 14.7 .02 .82 26 11 97 ± 31.9 .01 14.3 ± 5.5 .03
21.9 ± 7.6
24.3 ± 7.6
14 9 3 5 155 ± 106 2.1 ± 2.1 15.3 ± 6.6
21 10 0 6 199 ± 112 .14 1.7 ± 1.2 .91 15.0 ± 8.0 .85
273 ± 179 35.9 ± 73.0
213 ± 156 .16 34.3 ± 10.6 .46
53.5 ± 17.5 3.7 ± 1.9 1.9 ± 2.3 6 25
55.3 ± 18.6 4.9 ± 6.8 1.5 ± 1.9 14 23
CRP indicates C reactive protein. a Data are presented as mean ± SD.
.23 .43
.71 .47 .32 .12
Fig. 1 Oxidized LDL levels during the first week of severe sepsis in all patients (n = 68). Oxidized LDL concentrations significantly increase in all patients on day 4 and 7 compared to day 1 of severe sepsis. Results are given as mean ± SEM; ** indicates P b .001; *P b .05 vs day 1.
infections (n = 9; n = 10). The mean APACHE II score of patients treated with drotrecogin α (activated) was 21.9 (SD ± 7.6) compared to a mean APACHE score of 24.3 (SD ± 7.6) of the control group (P = .23). Furthermore, creatinine levels in the treatment group (2.1 mg/dL; SD ± 2.1) did not significantly differ from controls (1.7 mg/dL; SD ± 1.2). Six patients treated with drotrecogin α (activated) died during 7-day follow-up compared to 14 patients not treated with drotrecogin α (activated) (P = .12). The ox-LDL levels increased significantly (P b .0001) from day 1 to day 7 of severe sepsis in all patients (n = 68) (Fig. 1). Mean ox-LDL levels were 25.4 U/L (±SEM 1.8) on day 1 of severe sepsis, increasing to a mean of 34.3 U/L (±SEM 2.1) on day 4 and to a mean ox-LDL concentration of 38.4 U/L (±SEM 2.1) on day 7 of severe sepsis. Multiple comparison tests between all 3 points of time were statistical significant (P b .05). To determine the influence of drotrecogin α (activated), oxLDL levels were compared between patients treated with and without drotrecogin α (activated). As presented in Fig. 2, oxLDL levels increased significantly from day 1 to day 7 of severe sepsis in both treatment groups (P b .001). No significant differences of ox-LDL levels were observed between patients treated with or without drotrecogin α (activated) at days 1, 4, or 7. Moreover, no differences of ox-LDL levels have been observed between men and women (data not shown). Taken together, ox-LDL concentrations increased during the 7-day examination period, both in patients treated with (r = 0.394; P = .0001) as well as without drotrecogin α (activated) (r = 0.232; P = .015). No significant difference of mortality was observed in patients treated with (19%) and without drotrecogin α (activated) (38%). The ox-LDL levels of surviving patients were similar (mean ± SD, 39.6 ± 15.7 U/L) to those patients who died (mean ± SD, 33.1 ± 14.7 U/L) (P = .11) on day 7 of severe sepsis. The extent of increase of ox-LDL levels from day
540
Fig. 2 Comparison of ox-LDL levels between patients treated with (n = 31) and without (n = 37) drotrecogin α (activated). Oxidized LDL concentrations significantly increase in patients treated with and without drotrecogin α (activated) on day 4 and 7 compared to day 1 of severe sepsis. Oxidized LDL levels between control patients and patients treated with drotrecogin α (activated) at days 1, 4, and 7 are not statistically significantly different (P N .1). Results are given as mean ± SEM; *** indicates P b .0001; **P b .001; *P b .05 vs day 1.
1 to day 7 did not correlate to the APACHE II scores (not significant). Surviving patients had a trend toward lower APACHE II score (mean, 22.3; ± SD, 7.2) compared to those patients who deceased (mean, 25.2; ± SD, 8.5; P = .2). Correlations between APACHE II score and white blood cell count, levels of C-reactive protein, and ox-LDL were determined for all patients (n = 68) or separately for the treatment and control group. No statistically significant correlations were found between APACHE II scores and white blood cell count, levels of C-reactive protein, or bilirubin.
4. Discussion The present study evaluated levels of ox-LDL in 68 patients during the first week of severe sepsis. The ox-LDL levels increase significantly during the first 7 days of severe sepsis. Moreover, no treatment effect of drotrecogin α (activated) was observed on ox-LDL concentrations in the time course of patients having severe sepsis. This study demonstrates for the first time that ox-LDL levels increase in patients having severe sepsis. These findings contribute to a better understanding of the development of oxidative stress in severe sepsis. Uncontrolled production of reactive oxygen and nitrogen species leads to molecular damage of cellular key molecules and changes cellular responses such as proliferation, apoptosis, and necrosis in critically ill patients (ie, acute respiratory distress syndrome and severe sepsis) [5]. Increased oxidative stress enhances the progression of multiple organ dysfunction [8]. Several immune-cellular effects of ox-LDL have been described supporting its role in systemic inflammatory disorders. In vitro experiments demonstrated that increasing
M. Behnes et al. levels of ox-LDL activate chemotaxis of neutrophil or eosinophil granulocytes and monocytes under circumstances of exacerbating airway inflammation [17-19]. It has been described that ox-LDL induces apoptosis of endothelial cells [20-22]. In severe sepsis, tissue damage represents a main consequence of inflammation, coagulation-reduced oxygen supply, and in particular, endothelial cell damage leading to microvascular dysfunction [23,24]. Oxidized lipoproteins have not only been associated with proinflammatory responses but also with antiinflammatory effects at different levels of inflammatory signaling and in several cellular compartments [25]. It has been described that oxidized lipoproteins inactivate bacterial endotoxins (ie, lipopolysaccharides) and inhibit key elements in the nuclear factor κB signaling cascade. Therefore, it is presumed that oxidized lipids promote a shift from acute inflammatory toward chronic inflammatory processes [25]. The steady increase of ox-LDL levels within the first week of severe sepsis may partly reflect the continuum of inflammatory response in humans. Notably, Safa et al [26] reported that oxidation of lipids enhances the anticoagulant function of natural activated protein C and factor Va inactivation. In sepsis, physiologic anticoagulants such as protein C, heparin, and antithrombin are reduced [27]. The present findings demonstrate that drotrecogin α (activated) (Xigris) does not affect ox-LDL levels during the time course of sepsis. However, the up-regulation of ox-LDL levels during the first 7 days of severe sepsis in all patient groups (ie, treatment, control, and all patients) might possibly represent an adaptive counterregulatory mechanism to increase anticoagulant activity in severe sepsis. The beneficial effects of drotrecogin α (activated) such as increased survival have been attributed to its antithrombotic, profibrinolytic, antiinflammatory, and antiapoptotic properties [28,29]. However, no data currently exist whether drotrecogin α (activated) influences markers of oxidative stress. The present findings suggest that drotrecogin α (activated) has no effects on levels of ox-LDL. Additional in vivo investigations are needed to confirm the present results. In summary, these data suggest that oxidative stress as quantified by measurements of ox-LDL levels increases within the first week of severe sepsis independently of a treatment regimen with drotrecogin α (activated) (Xigris).
References [1] Bone RC, Balk RA, Cerra FB, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992; 101:1644-55. [2] Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/ESICM/ACCP/ ATS/SIS International Sepsis Definitions Conference. Crit Care Med 2003;31:1250-6. [3] Dhainaut JF, Shorr AF, Macias WL, et al. Dynamic evolution of coagulopathy in the first day of severe sepsis: relationship with mortality and organ failure. Crit Care Med 2005;33:341-8.
Oxidized LDL in severe sepsis [4] Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997;112:235-43. [5] Gutteridge JM, Mitchell J. Redox imbalance in the critically ill. Br Med Bull 1999;55:49-75. [6] Alonso de Vega JM, Diaz J, Serrano E, et al. Plasma redox status relates to severity in critically ill patients. Crit Care Med 2000;28: 1812-4. [7] Alonso de Vega JM, Diaz J, Serrano E, et al. Oxidative stress in critically ill patients with systemic inflammatory response syndrome. Crit Care Med 2002;30:1782-6. [8] Motoyama T, Okamoto K, Kukita I, et al. Possible role of increased oxidant stress in multiple organ failure after systemic inflammatory response syndrome. Crit Care Med 2003;31:1048-52. [9] Gaut JP, Heinecke JW. Mechanisms for oxidizing low-density lipoprotein. Insights from patterns of oxidation products in the artery wall and from mouse models of atherosclerosis. Trends Cardiovasc Med 2001;11:103-12. [10] Memon RA, Staprans I, Noor M, et al. Infection and inflammation induce LDL oxidation in vivo. Arterioscler Thromb Vasc Biol 2000; 20:1536-42. [11] Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655-73. [12] Ehara S, Ueda M, Naruko T, et al. Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation 2001;103:1955-60. [13] Pearson TA, Mensah GA, Alexander RW, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 2003;107:499-511. [14] Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol 2003;41:360-70. [15] Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699-709. [16] Knaus WA, Draper EA, Wagner DP, et al. APACHE II: a severity of disease classification system. Crit Care Med 1985;13:818-29.
541 [17] Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 1999; 180:780-90. [18] Sedgwick JB, Hwang YS, Gerbyshak HA, et al. Oxidized low-density lipoprotein activates migration and degranulation of human granulocytes. Am J Respir Cell Mol Biol 2003;29:702-9. [19] Silva AR, de Assis EF, Caiado LF, et al. Monocyte chemoattractant protein-1 and 5-lipoxygenase products recruit leukocytes in response to platelet-activating factor-like lipids in oxidized low-density lipoprotein. J Immunol 2002;168:4112-20. [20] Dimmeler S, Haendeler J, Galle J, et al. Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the “response to injury” hypothesis. Circulation 1997;95:1760-3. [21] Galle J, Schneider R, Heinloth A, et al. Lp(a) and LDL induce apoptosis in human endothelial cells and in rabbit aorta: role of oxidative stress. Kidney Int 1999;55:1450-61. [22] Heermeier K, Leicht W, Palmetshofer A, et al. Oxidized LDL suppresses NF-kappaB and overcomes protection from apoptosis in activated endothelial cells. J Am Soc Nephrol 2001;12:456-63. [23] Hoffmann JN, Vollmar B, Laschke MW, et al. Microcirculatory alterations in ischemia-reperfusion injury and sepsis: effects of activated protein C and thrombin inhibition. Crit Care 9 Suppl 2005;4:S33-7. [24] Hoffmann JN, Vollmar B, Laschke MW, et al. Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia. Crit Care Med 2004;32:1011-7. [25] Bochkov VN, Leitinger N. Anti-inflammatory properties of lipid oxidation products. J Mol Med 2003;81:613-26. [26] Safa O, Hensley K, Smirnov MD, et al. Lipid oxidation enhances the function of activated protein C. J Biol Chem 2001;276:1829-36. [27] Hughes M. Recombinant human activated protein C. Int J Antimicrob Agents 2006;28:90-4. [28] Cheng T, Liu D, Griffin JH, et al. Activated protein C blocks p53mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 2003;9:338-42. [29] Joyce DE, Grinnell BW. Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-kappaB. Crit Care Med 2002;30:S288-93.