Acetylcysteine

Study objective: The level of microalbuminuria is thought to reflect the severity of inflammation-induced systemic vascular permeability and may have prognostic value with regard to organ dysfunction and survival. N-acetylcysteine (NAC) has been shown to decrease capillary leakage in experimental sepsis. The present study investigated the effect of early treatment with NAC on microalbuminuria and organ dysfunction in severe clinical sepsis.

Design: Prospective, randomized, placebo-controlled study.

Setting: A 24-bed multidisciplinary ICU in a university teaching hospital.

Patients: Thirty-five patients included within 4 h of fulfilling consensus criteria of severe sepsis.

Interventions: Patients were randomly assigned to receive either NAC (continuous infusion starting with 50 mg/kg/4 h followed by 100 mg/kg/24 h for 44 h; n = 18) or placebo (n = 17) in addition to standard therapy.

Measurements and results: Urine samples for measurement of microalbuminuria/creatinine ratio (MACR) were collected on inclusion and after 4 h, 24 h, and 48 h. Severity of illness and degree of organ failure were determined by using, respectively, the APACHE (acute physiology and chronic health evaluation) II score and the sequential organ failure assessment (SOFA) score. The MACR did not differ over time between the placebo- and the NAC-treated groups. SOFA scores were comparable between both treatment groups at baseline (6.2 [+ or -] 3.9 vs 6.5 [+ or -] 2.7, NAC vs placebo; p = 0.6) and increased during treatment in the NAC-treated patients but not in the placebo group (7.9 [+ or -] 3.7 vs 5.9 [+ or -] 2.5, p = 0.09 and 7.7 [+ or -] 3.8 vs 5.1 [+ or -] 2.1, p < 0.05; NAC vs placebo, respectively, at 24 h and at 48 h). The cardiovascular SOFA score progressively increased during NAC treatment, reaching higher values as compared to time-matched scores in the placebo group.

Conclusions: Early NAC administration does not influence the course of MACR in severe clinical sepsis, suggesting that NAC might not attenuate endothelial damage in this condition. NAC treatment even aggravated sepsis-induced organ failure, in particular cardiovascular failure.

Key words: antioxidants; cardiac performance; microalbuminuria; N-acetylcysteine; organ failure; severe sepsis

Abbreviations: APACHE = acute physiology and chronic health evaluation; CI = confidence interval; MACR = microalbuminuria/creatinine ratio; NAC = N-acetylcysteine; NO=nitric oxide; ROS = reactive oxygen species; SOFA = sequential organ failure assessment

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Sepsis is characterized by activation of neutrophils, macrophages, and endothelial cells in the microcirculation, resulting in the generation of huge numbers of inflammatory cytokines and activation of the coagulation cascade. Subsequent microvascular thrombosis and endothelial cell damage increase capillary permeability, enhance the inflammatory state, and trigger tissue ischemia and organ failure. Unlike coagulation disorders, the degree of endothelial damage and the resulting capillary leak are difficult to estimate in the clinical setting. Interestingly, acute inflammation accompanying trauma, (2) surgery, (3) or ischemia (4) is associated with the rapid onset of microalbuminuria. In these conditions, increased microalbuminuria matches the severity of the insult and correlates with the degree of organ failure. (5,6) Microalbuminuria may thus reflect the evolution of systemic capillary leakage and could therefore be a useful parameter to assess the efficacy of therapies aiming to reduce endothelial damage.

Sepsis induces injurious oxidation that is due in part to inadequate cellular oxygen handling, but above all to a release of reactive oxygen species (ROS) produced by activated neutrophils and during reperfusion in the resuscitation phase. Since endogenous antioxidant defense mechanisms (eg, reduced intracellular glutathione) fail to cope with this overwhelming assault of ROS, lipid peroxidation of cell membranes stands out as an important mechanism underling structural and functional cellular alterations during sepsis. (7) N-acetylcysteine (NAC) is a thief-containing compound with antioxidant, antiinflammatory, and microcirculatory effects. NAC acts as an antioxidant either directly by scavenging ROSs and indirectly by replenishing depleted cellular glutathione stores. (9) Its antiinflammatory potential encompasses inhibition of neutrophil chemotaxis, activation and aggregation, (10) suppression of macrophage activation, (11) inhibition of leukocyte-endothelial cell adhesion, (12) and attenuation of the release of tumor necrosis factor-[alpha] (13) and interleukin-8 (14) probably by modulating gene expression of these mediators at the transcriptional level. (15) NAC may also improve the microcirculation, as reduced thiols are required for in vivo expression of nitric oxide (NO), a vital mediator of organ perfusion during endotoxemia, which is readily inactivated by ROS. (16) NAC is a safe agent with a wide toxic/therapeutic window as documented by its use in patients with fulminant hepatic failure due to acute acetaminophen over dose. The present study aims to investigate whether adjuvant therapy with NAC attenuates capillary leakage--as assessed by measuring and monitoring of microalbuminuria--and thus offers organ protection during severe clinical sepsis.

MATERIALS AND METHODS

Patients

The protocol was approved by the local Committee for Ethics in Human Research. Written informed consent was obtained from either the patient or his/her next of kin. Severe sepsis was defined according to consensus guidelines (17) as sepsis with organ hypoperfusion or dysfunction. Patients were excluded or withdrawn from the study for the following reasons: age < 18 years; diabetes mellitus; known arterial hypertension; urinary tract infection; left ventricular failure defined echocardiographically as the concomitant presence of an increased left ventricular end-diastolic diameter (> 60 mm) and volume (> 120 [cm.sup.3]), the presence of regional and global left ventricular hypokinesia and a left ventricular fractional area contraction of < 0.4 under inotropic support (18); chronic respiratory, failure (chronic hypoxemia, hypercapnia, severe asthma): chronic renal failure (creatinine level > 2.0 mg/dL or renal replacement therapy required); chronic liver failure (confirmed cirrhosis or portal hypertension); treatment with IV, oral, or aerosolized NAC; known allergy to NAC; and any medical condition considered to be irreversible or lethal within 48 h alter ICU admission.

All patients had radial (Arterial Line Kit; Argon; Athens, TX) and balloon-tipped pulmonary (Edwards Swan Canz model 97-120-7; Baxter Healthcare; Irvine, Ca) arterial catheters inserted. Invasive surgery, any renal replacement therapy, and infusion of colloids and parenteral nutrition were avoided during the study. If required, patients received mechanical ventilation in pressure-controlled mode (Serve 900C: Siemens Elema: Solna, Sweden) under continuous analgesic sedation with midazolam and fentanyl. Crystalloids were infused to obtain a pulmonary wedge pressure between 14 and 18 mm Hg. If the cardiac index remained < 2.5 L/min/[m.sup.2], dobutamine was administered aiming at a value between 3.0 and 3.5 L/min/[m.sup.2]. Norepinephrine was added to maintain mean arterial pressure > 65 mm Hg. The use of other vasoactive drugs, including dopamine, was not allowed. All patients initially received broad-spectrum antibiotic coverage that was adjusted according to culture results.

Protocol

Patients were randomly assigned according to a computer-steered permuted block design to receive either IV NAC or placebo. NAC or placebo was initiated within 4 h after the diagnosis of severe sepsis. NAC was administered in 5% dextrose as a continuous infusion starting with 50 mg/kg/4 h followed by 100 mg/kg/24 h for 44 h. The placebo group received an equal amount of 5% dextrose during the same time period.

Measurements

Urine was collected for 2 h before infusion of NAC or placebo and after 4 h, 24 h, and 48 h. Urine albumin concentrations were measured by immunoassay (Cassette Cobas Integra 400 Albumin; Roche Diagnostics; Basel, Switzerland). To exclude the influence of intraindividual variation in urinary flow rate, microalbumin levels were quantified to the urine creatinine concentrations analyzed by reflectometry (CREA slides; Johnson and Johnson; Beerse, Belgium) and expressed as the microalbumin/creatinine ratio (MACR). The assigned laboratory reference range was < 3 mg/mmol. At the same time points, blood was sampled for routine laboratory studies and arterial blood gas measurement.

Severity of illness was determined by the APACHE (acute physiology and chronic health evaluation) II score after 24 h. The degree and progression of organ failure was assessed daily using the sequential organ failure assessment (SOFA) score. (19) Cardiovascular, respiratory, coagulation, hepatic, central nervous, and renal systems were scored from 0 (normal) to 4 (most severe dysfunction) with the worst physiologic value for each day recorded.

Statistical Analysis

Statistical analysis included a two-way analysis of variance for repeated measurements. Intergroup comparisons relating to age and APACHE II score were analyzed by Student t test. Differences in MACR, SOFA score, and hemodynamic, oxygenation, hepatic, and renal variables within groups were evaluated with the Mann-Whitney U test and between groups with the Wilcoxon rank-sum test. The Pearson test for linear regression was used to compare MACR with APACHE II and SOFA scoring system points. Statistical significance was considered at a p value < 0.05.

RESULTS

Thirty-five patients (age range, 27 to 81 years; median, 66 years; 20 men and 15 women) were consecutively enrolled. Demographic and clinical characteristics are listed in Table 1. Eighteen patients received NAC. No adverse effects were noted during NAC infusion. Age and gender did not differ between treatment groups. Pneumonia was the cause of sepsis in a majority of patients (77%). APACHE II score was slightly but not significantly higher in the NAC group as compared to the placebo group: 25 [+ or -] 11 vs 21 [+ or -] 8 (p = 0.1). During the treatment period, no difference in MACR was noted between the placebo group and the NAC group (Fig 1).

[FIGURE 1 OMITTED]

Admission SOFA scores were comparable between both treatment groups (p = 0.6). The SOFA score increased in patients treated with NAC (7.9 [+ or -] 3.7 and 7.7 [+ or -] 3.8, respectively, at 24 h and at 48 h; both p < 0.05 vs baseline), whereas it decreased in the control group (5.8 [+ or -] 2.7 at 24 h; p = 0.09 vs baseline and 5.0 [+ or -] 2.1 at 48 h; p < 0.01 vs baseline). As compared to placebo-treated patients, the SOFA score was higher in the NAC group at 24 h (p = 0.09) and at 48 h (p < 0.05) [Fig 2]. Table 2 shows the evolution of the organ-specific SOFA scores during the study. Baseline scores were comparable between NAC- and placebo-treated patients. Over the next 48 h, however, NAC administration was associated with increasing cardiovascular failure at 24 h (median cardiovascular SOFA score, 2.5; 95% confidence interval [CI], 1.1 to 2.8) and at 48 h (median, 3.0; 95% CI, 1.2 to 3.0) [p = 0.07 and p < 0.05, respectively, vs baseline].

[FIGURE 2 OMITTED]

Actual hemodynamic, oxygenation, renal, and hepatic function data are represented in Table 3. All these variables were not significantly different within and between groups at the different time points. Perfusion pressure and cardiac index were well maintained throughout the study in both treatment groups. However, hemodynamic stabilization in many NAC-treated patients could be pursued only at the expense of initiating or increasing norepinephrine and/or dobutamine infusion. Catecholamine doses were also consistently higher in the NAC group (data not shown).

In NAC- as well as in placebo-treated patients, no correlation was found between MACR and APACHE II ([r.sup.2] = 0.001 for placebo and [r.sup.2] = 0.13 for NAC; both p = not significant) or time-matched SOFA scores: ([r.sup.2] = 0.002, 0.19, and 0.19 for placebo and [r.sup.2] = 0.03, 0.06, and 0.001 for NAC, respectively, at baseline, 24 h, and 48 h (p = not significant).

DISCUSSION

Neutrophil activation with subsequent release of ROS and lytic enzymes into the microcirculation is a key mechanism underlying organ failure in sepsis. Patients with persisting systemic inflammation have intense and continuous oxidative stress (20) and are prone to acquire multiorgan failure. (21) Hence, supplementation of antioxidants could be a useful aid in the treatment of sepsis. Most experience has been accumulated with high IV doses of NAC.

The observation that NAC attenuated endotoxin-induced leukocyte-endothelial cell adherence and albumin leakage in rat mesenteric postcapillary venules (22) suggested that preservation of endothelial integrity constitutes a major part of its activity during sepsis. Unfortunately, endothelial damage cannot be measured directly, nor can the resulting capillary leakage be quantified in the clinical setting. MACR has gained interest as a surrogate in vivo marker of systemic capillary leakage that, if severe, can result in organ failure. We were unable to find a relationship between MACR and APACHE II and SOFA scores. This is in contrast with other studies in critically ill patients in which a positive correlation was found between initially high and/or increasing MACR levels and severity of disease and organ failure. However, these studies were performed in highly heterogeneous patient populations5,6 or included only few (5,23) or less severely ill septic patients. (24) Our study also has weaknesses that deserve to be acknowledged. The small sample size makes it difficult to claim no difference in MACR between both treatment groups. At least 150 patients should indeed have been included to anticipate a detectable difference at a [beta]-error < 20%. We did not relate MACR to an objective in vivo index of capillary leakage (eg, measurement of transcapillary escape rate of radio-labeled albumin). Finally, a single spot urine specimen might provide at best a "snapshot" of systemic capillary patency but certainly not an integrated long-term assessment of interstitial edema. Nevertheless, our findings remain in agreement with Molnar et al, (25) who showed an absence of correlation between MACR and extravascular lung water--a possible indicator of vascular permeability--in septic patients receiving mechanical ventilation. (25)

The worsening of organ failure and particularly cardiovascular failure during NAC treatment is an intriguing and disturbing finding. A power analysis at 5% level of significance of the global SOFA score measurement at 24 h and 48 h of treatment revealed [beta] errors of 0.25 and 0.06, respectively. For the cardiovascular SOFA score calculated at the same time points, the [beta] errors were 0.02 and 0.10, respectively. Despite the small number of patients included, a type 2 error is thus unlikely, so that the relevance of our observations is strengthened. Peake et al (26) demonstrated previously that administration of NAC in patients with septic shock caused depression of cardiovascular performance indicated by a progressive reduction in cardiac output and mean arterial pressure. This finding was corroborated by Molnar et al, (27) who observed a higher need for inotropic support in a cohort of critically ill patients treated with NAC for > 24 h.

The mechanism of this deleterious effect remains unclear, but dosing and timing of NAC appear to be critical issues. The dose of NAC in sepsis has been derived from the administration regimen in patients treated for acetaminophen poisoning. Clinical studies in patients with fulminant hepatic failure (28) and septic shock (29) indicated that this "standard" NAC infusion yielded highly variable plasma concentrations, typically reaching a steady state after 24 h. Abandoning the bolus dose considerably lowered the total daily dose of NAC in our NAC-treated patients. It could be argued that this might not produce the desirable antioxidant effects. However, it has never been proven that the initially very high plasma concentrations of NAC are necessary for full antioxidant protection. In fact, NAC prevented oxidative liver damage in patients with acetaminophen-induced liver failure just as effectively at the lowest than at the highest maximum plasma concentrations. (30) Over the first 24 h, we also administered a dose of NAC that was similar or only slightly lower than in earlier studies that demonstrated significant antioxidant (31) or cellular glutathione-repleting (32) effects of the drug. NAC treatment during the first hours of severe sepsis and septic shock has been shown to decrease peroxidative stress, (31) enhance tissue oxygenation, (33) and improve hepatic (34) and respiratory (35) function. Delayed administration, however, failed to improve tissue oxygenation (36) and even adversely affected survival in critically ill patients with established organ failure. (27)

Pre-existing cardiac dysfunction may also determine the late-onset deleterious hemodynamic response to the drug. Endothelial damage was found to be more pronounced in septic patients with ischemic heart disease. (37) Infusion of NAC in this particular patient population induced higher plasma levels than in patients with normal cardiac function. (29) It is hypothesized that insufficient metabolism of NAC at the endothelial cell level may cause direct or indirect cardiac toxicity through excess generation of NO. Plasma levels of nitrite, a breakdown product of NO, were indeed found to be substantially higher in NAC-treated than in placebo-treated patients with septic shock. (26) NAC enhances NO activity by formation of a vasoactive S-nitrosothiol compound (38) and by increasing intracellular cyclic guanosine monophosphate concentrations through activation of soluble guanylate cyclase. (39) A decreased myofilament response to [Ca.sup.2+] in association with an increased synthesis of cyclic guanosine monophosphate was noted after incubation of cardiomyocytes with endotoxin. (40) Also, a NO-dependent inhibition of creatine-kinase-catalyzed myofibrillar energy transfer has been implicated (41) but not confirmed (42) during endotoxemia.

Other detrimental effects of NAC treatment during sepsis cannot be excluded. In neutrophils from critically ill patients with systemic inflammation and trauma, NAC was shown to augment phagocytosis but to concurrently reduce oxidative burst. (43) Inadequacy of the latter might seriously impair a vital mechanism by which phagocytes destroy microbial organisms. Decreased bacterial clearance with sustained release of toxins might enhance or maintain endothelial activation and inflammation, "unplug" the capillary leaks, and increase organ damage. NAC could paradoxically act as a pro-oxidant. At a low dose, NAC protected rats against endotoxin-induced oxidative stress, whereas higher doses--in the presence of iron--generated excessive amounts of ROS and increased mortality. (44) The addition of deferoxamine, an iron chelator, to NAC treatment reduced the consequences of peritonitis in the rat by lowering oxidative stress and limiting neutrophil infiltration and mitochondrial dysfunction, thereby improving survival. (45) Despite the avalanche of experimental and in vivo data supporting the antiinflammatory potential of NAC, it still remains to be proven whether and how NAC influences neutrophil-endothelial cell interactions as well as basic cellular mechanisms regulating gene expression and apoptotic processes.

In summary, any beneficial effect of NAC at the microcirculatory level during clinical sepsis is unlikely to be due to a reduction of capillary leakage. NAC apparently aggravated sepsis-induced organ-in particular cardiovascular--failure, but the mechanism(s) underlying this unexpected observation remain(s) speculative. Timing and dosing of the drug in relation with onset, evolution, and severity of sepsis seem to be crucial determinants of its biological actions. Overall, this preliminary study put serious doubt on the utility and safety of NAC as an adjuvant therapy in sepsis. Surviving sepsis undeniably goes together with the containment of sepsis-induced organ failure and any factor jeopardizing this goal should be avoided.

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* From the Intensive Care Department, Academic Hospital, Vrije Universiteit Brussel, Brussels, Belgium.

Supported in part by the Scientific Fund Willy Gepts of the Academic Hospital, Vrije Universiteit Brussels.

Manuscript received May 3, 2004; revision accepted September 30, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Herbert D. Spapen, MD, PhD, FCCP, Intensive Care Department, Academic Hospital, Vrije Universiteit Brussel, Laarbeeklaan, 101, B-1090 Brussels, Belgium; e-mail: herbert.spapen@az.cub.ac.be

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