Abstract
Objective
To determine whether serum paraoxonase (PON) and arylesterase (ARE) activity might predict sepsis mortality.
Methods
Patients with sepsis and healthy control subjects were enrolled in this retrospective study. Serum PON and ARE activity levels were measured. Patients were stratified according to 30-day mortality rates.
Results
Serum PON and ARE activity levels were significantly lower in patients with sepsis (n = 61) than in healthy controls (n = 32), and were significantly lower in nonsurviving patients (n = 22) than in surviving patients (n = 39). Low PON and ARE activity levels were significantly correlated with poor overall survival in patients with sepsis.
Conclusions
Decreased serum PON and ARE activity is related to poor prognosis in patients with sepsis. Measuring the activity of PON and ARE may represent a new method for evaluating the prognosis of sepsis. In addition, both PON and ARE are potential molecular treatment targets for sepsis.
Introduction
Sepsis is defined as the combination of infection and systemic inflammatory response syndrome.1,2 Despite improvements in diagnosis and treatment, sepsis incidence and mortality rates in the intensive care unit remain high. 3 Studies have identified several predictors of mortality (including serum C-reactive protein and procalcitonin concentrations), and have devised scoring systems including acute physiology and chronic health evaluation II (APACHE II), and sepsis-related organ failure assessment (SOFA).4,5 However, none of these strategies has gained universal acceptance. New biomarkers are required, to aid the early diagnosis, prognosis and stratification of sepsis severity.
Sepsis represents a failing balance between pro- and anti-inflammatory immune responses that can lead to organ failure and ultimately to death.6–8 Reactive oxygen species (ROS) concentrations may be decisive in the outcome of sepsis via the regulation of key cytokines and chemokines, which further modulate the inflammatory response. ROS also induce phagocytosis, gene expression and apoptosis.9,10 Oxidative stress is well characterized in patients with sepsis, who have evidence of ROS production and antioxidant depletion,11–13 and the sustained and excessive production of ROS is associated with increased morbidity and mortality. 14 Animal studies and prospective, randomized, clinical trials have provided evidence to support antioxidant therapies in sepsis.15,16
Paraoxonase 1 (PON1), an antioxidant bioscavenger, is responsible for hydrolyzing lipid peroxides and plays a major role in the antioxidant system. PON1 is mainly synthesized in the liver and is secreted into the blood, and comprises three known enzymatic subunits: paraoxonase (PON), arylesterase (ARE) and dyazoxonase. Increased oxidative stress has been shown to lead to reduced PON1 activity.17–19 A small-scale study (involving six patients with sepsis) found lower plasma PON1 concentrations in those who died compared with those who survived, but the relationship between PON and ARE activity and mortality was not evaluated. 20
The aims of the present study were to evaluate the relationships between mortality and serum PON and ARE activities in patients with sepsis, and to determine the value of PON and ARE activities as biomarkers for outcome prediction in sepsis.
Patients and methods
Study population
The study retrospectively recruited patients with sepsis, admitted to the General Internal Medicine Intensive Care Unit, No. 202 Hospital of Chinese People's Liberation Army, Shenyang, Liaoning, China, between July 2010 and April 2011. Sepsis was diagnosed according to the definitions of the American College of Chest Physicians/Society of Critical Care Medicine. 21 Exclusion criteria were: age < 18 years; AIDS diagnosis; white blood cell count < 1000/µl; any solid or haematological tumour; liver dysfunction; current immunosuppressive, steroid or radiation therapy. Patients were assessed using the APACHE II and SOFA scoring systems on day 1 after diagnosis.22,23
Healthy control subjects (who were free from infection and any other medical condition) were recruited from the Health Examination Centre of No. 202 Hospital of Chinese People's Liberation Army, Shenyang, China.
Written informed consent was provided by all study participants (or by first-degree relatives, if patients were unable to provide consent). The study protocol was approved by the Ethics Committee of No. 202 Hospital of Chinese People's Liberation Army, Shenyang, China.
Blood sampling
Blood samples were collected within 24 h after diagnosis of sepsis and before therapeutic intervention commenced. Whole blood was incubated at 37℃ for 1 h and centrifuged at 1500 g for 10 min at 4℃; the resulting serum was stored at
Quantification of PON and ARE activity
Quantification of PON activity was performed in the presence or absence of sodium chloride (baseline activity) using synthetic paraoxon (diethyl-p-nitrophenyl phosphate) as substrate. 24 PON activity was determined by measuring the initial rate of substrate hydrolysis to p-nitrophenol. Absorbance was monitored at 412 nm for 5 min in the assay mixture containing 2.0 mM paraoxon, 2 mM CaCl2 and 20 μlserum in 100 mM Tris-HCI buffer (pH 8.5). The blank sample (which contained incubation mixture without serum) was run simultaneously, to correct for spontaneous substrate breakdown. The quantity of generated p-nitrophenol was calculated from the molar absorption coefficient at pH 8.5 (18.290 M−1 cm−1) (Biochemical Analyzer BA-88 A, Mindray, Shenzen, China). PON activity was expressed as U/l.
Phenylacetate was used as a substrate to measure ARE activity. 25 The assay tube contained 750 μl of 0.1 mol/l Tris-HCl (pH 8.5), 1 mmol/l CaCl2, 125 μl of 12 mmol/l phenylacetate, and 125 μl of serum (diluted 1 : 10 with distilled water). Absorbance was continuously monitored at 270 nm and 37℃ (Biochemical Analyzer BA-88 A, Mindray). Enzymatic activity was calculated from the molar absorption coefficient of the produced phenol (1310 M−1 cm−1), with 1 U defined as 1 µmol phenol generated/min and expressed as U/l.
Statistical analyses
Data were expressed as mean ± SD or n (%).Nonparametric continuous variables were compared by the Mann–Whitney U-test, and the χ2-test was used to compare sex distributions and clinical characteristics. Sepsis patients were stratified into survivors and nonsurvivors, based on 30-day mortality. Survival time was measured from date of diagnosis to date of death from sepsis or date of last follow-up. The prognostic value of PON and ARE activity was evaluated by Kaplan–Meier analysis and log-rank test. All statistical analyses were performed using SPSS® software, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. A P-value < 0.05 was considered statistically significant.
Results
The study recruited 61 patients (40 males/21 females; mean age 52.6 ± 13.7 years; age range 28–79 years) and 32 healthy controls (21 males/11 females; mean age 50.9 ± 14.8 years; age range 25–79 years) Serum PON activity (97.7 ± 22.9 vs 202.4 ± 36.6 U/l; P < 0.001) and ARE activity (129.3 ± 22.7 vs 169.7 ± 33.1 U/l; P < 0.001) were significantly lower in patients with sepsis than in healthy controls. There were no significant differences in age or sex between patients and controls.
Demographic and clinical characteristics of patients with sepsis (n = 61) included in a study investigating the prognostic value of serum paraoxonase (PON) and arylesterase (ARE) activity levels, stratified according to 30-day survival rates.
Data presented as mean ± SD or n (%).
Mann–Whitney U-test for nonparametric continuous variables; χ2-test for sex distribution and clinical characteristics.
Patients were stratified into low or high PON and ARE groups using median values as cut-off (98.3 and 125.0 U/l, respectively). Low PON and ARE activity were both significantly correlated with reduced overall survival (P = 0.0013 and P = 0.014, respectively; Figure 1).
Kaplan–Meier survival analysis for the effect of (A) serum paraoxonase (PON) activity (cut-off value 98.3 U/l) and (B) serum arylesterase (ARE) activity (cut-off value 125.0 U/l) on overall survival in patients with sepsis.
Discussion
Sepsis is characterized by a systemic inflammatory response to infection and is associated with multiple organ failure,26,27 with mortality rates as high as 40–60%. 28 In order for a treatment strategy to improve clinical outcome definitively, detailed knowledge regarding mechanism and early disease prediction are required. 29 Patients with sepsis were found to have significantly lower levels of PON and ARE activity than healthy controls in the present study. In addition, since nonsurviving patients had significantly lower serum PON and ARE activity levels than survivors, these enzyme activities could be used to predict outcomes in patients with sepsis.
The antioxidant enzyme PON1 is secreted from hepatic cells and binds to high-density lipoprotein (HDL) cholesterol in the circulation. 30 PON1 has been shown to prevent oxidation of lipoproteins by ROS formed during oxidative stress. 31 Oxidative stress, occurring as a result of the inflammatory responses inherent in sepsis, initiates changes in mitochondrial function that may result in organ damage 32 and increases oxidative stress, thereby lowering HDL concentrations. 33 Concentrations of PON1 and HDL cholesterol were lower in patients with sepsis who did not survive than in those who did. 20 It is possible that the oxidizing environment induced by sepsis could result in increased binding of free radicals to PON1, leading to reduced PON1 activity in the circulation and/or excessive PON1 consumption. The present study findings are in accordance with the hypothesis that serum PON and ARE activity levels are lower in patients with sepsis than in healthy controls.
Consistent with the findings of others, 20 patients with sepsis who did not survive had significantly lower PON and ARE activity levels than survivors, in the present study. It has been reported that the plasma antioxidant potential was lower in patients with sepsis than in healthy subjects in the early stages of disease, later increasing to normal (or supernormal) levels sepsis in patients who survived, but not in those who died. 34 Levels of PON and ARE activity were not known before the onset of sepsis, however, and it was therefore unclear whether patients who survived had higher pre-sepsis enzyme activities or experienced smaller decreases in PON and ARE activity during the course of disease.
In conclusion, the present study found that both serum PON and ARE activity levels were significantly reduced in patients with sepsis compared with healthy controls. In addition, low PON and ARE activity levels were significantly correlated with poor overall survival. PON and ARE activity may represent new indicators for evaluating the prognosis of sepsis, as well as being potential molecular-treatment targets.
Footnotes
Declaration of conflicting interest
The authors declare that there are no conflicts of interest.
Funding
This work was supported by a grant from the Liaoning Science Project Fund (2011225021).