Clinical research
Effect of N-acetylcysteine on microalbuminuria in patients with acute respiratory distress syndrome

Introduction
Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are almost similar diseases occurring in respiratory tract. ARDS is a syndrome of severe dyspnea with rapid onset of edema and hypoxemia whereas ALI appears by inflammation and increased permeability with a constellation of clinical, radiologic, and physiologic abnormalities that cannot be explained by, but may coexist with, left atrial or pulmonary capillary hypertension. The distinction between ALI and ARDS is the degree of hypoxemia. ALI is defined by a partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) ratio of less than 300 mm Hg while 200 mm Hg or less is required for ARDS. Approximately 10% of patients who admit to intensive care unit (ICU) suffer from acute respiratory failure, whom 20% are diagnosed as acute lung injury (ALI) or ARDS [1, 2]. Despite advances in supportive care, the mortality rate of the patients with ARDS remains high [3, 4].
Activation of pulmonary endothelium and macrophages leads to release of cytokines and accumulation of neutrophils inside the pulmonary microvasculature [5, 6]. Simultaneously, proteolytic enzymes, reactive oxygen species (ROS), and additional inflammatory cytokines are released [7-9]. In ARDS, there is extensive overproduction of free radicals to the extent that endogenous anti-oxidants are overwhelmed. N-acetylcysteine (NAC) is a thiol-containing compound acting as a precur-sor of glutathione. The benefit of NAC in the management of ARDS has been proved by measuring patient’s intracellular glutathione (inside red blood cells) and extracellular (plasma) anti-oxidant defense biomarkers and outcome. It has been shown that treatment by NAC increases extracellular total anti-oxidant power and total thiol molecules and improves intracellular glutathione and the outcome of the patients [10]. Recent animal study also confirmed antioxidant potential of NAC [11].
The hypothesis of the present work is that administration of NAC improves outcome of ARDS patients.

Material and methods
Subjects
After obtaining approval from ethics committee of TUMS and written informed consent from relatives, on the basis of inclusion criteria 23 patients were randomly entered into the study. The study was conducted in the ICU of a University Hospital from September 2006 to September 2007. The patients were included if they required mechanical ventilation and had a PaO2/FiO2 of lower than 200 mm Hg and pulmonary capillary wedge pressure (PCWP) of lower than 18 mm Hg.
Exclusion criteria were: PaO2/FiO2 > 200 mm Hg, left ventricular failure defined echocardiographically as the concomitant presence of an increased left ventricular end diastolic diameter (> 60 mm) and volume (> 120 cm³), the presence of regional and global left ventricular hypokinesia and a left ventricular fractional area contraction of < 4 under inotropic support, chronic respiratory failure (chronic hypoxemia, hypercapnia, severe asthma), chronic renal failure (creatinine level > 2 mg/dl or renal replacement therapy), known allergy to NAC, diabetes mellitus, age less than 18-years old, pregnancy, and any medical condition considered to be irreversible or lethal within 48 h after ICU admission. The etiologies of ARDS in study patients were multiple trauma (7 cases), sepsis (3), peritonitis (4), pneumonia (4), and uncertain (5 cases).
Treatment protocol
NAC was purchased from UCB Pharma ltd (UK). Among the included patients, 9 received NAC (150 mg/kg) diluted in 5% dextrose and infused in a period of 20 min at first and continued by 50 mg/kg/day diluted in 5% dextrose (50 ml) on the second day. Fourteen patients in the control group underwent standard treatment without NAC.
All patients had radial and pulmonary artery catheters. If necessary, patients received mechanical ventilation under continuous sedation with midazolam and fentanyl. Crystaloids were infused to obtain a central venous pressure between 8-12 mm Hg. If the cardiac index remained > 2.5 l/min/m², dobutamine was administered at a value between 3 and 3.5 l/min/m². Norepinephrine was added to maintain mean arterial pressure > 60 mm Hg.
Clinical proteinuria was defined by albumin creatinine ratio (ACR > 300 mg/day). This corre-sponds to daily albumin excretion of > 300 mg/day. ACR values between 30 and 299 mg/g were considered normal.
Fresh urine samples were collected from patients via a urinary catheter before infusion of NAC and 4, 6, 12, 24, 36, 48 h after that and kept frozen at –20°C until analysis. Urine albumin concentration was measured by immunoassay turbidimetric using Technicon RA-XT 1000 analyzer. To exclude the influence of intraindividual variation in urinary flow rate, microalbumin was estimated per creatinine and reported as microalbumin/creatinine ratio (MACR). The assigned laboratory reference range was < 2.3 mg/mmol.
Acute physiology and chronic health evaluation (APACHE II)
The APACHE II scoring method was used to predict risk of ICU mortality. The APACHE II score was measured on a daily basis for all patients admitted to ICU. The point score was calculated from 12 routine physiological measurements during the time of study. For calculating APACHE II score, arterial blood gases, electrolytes, and complete blood counts were determined daily, and blood pressure, heart rate, respiratory rate, body temperature, pulmonary indices, and hemodynamic profiles were checked and recorded and mean daily values were calculated [10]. Information about previous health status, age and sex, surgical status (elective or emergency surgery), and major reason for ICU admission and severity of illness (acute physiologic state) were included.
Statistical analysis
Data are shown as means ± SE. Mann-Whitney U test was used for analysis of nonparametric data. A p-value of less than 0.05 was considered statistically significant. The relationship between MACR and survival was explored using multiple logistic regression. Spearman rank correlation coefficients (r) was used to quantify the correlation between variables.

Results
A total of 23 patients with the age range of 21-72 and mean age of 48 years were enrolled in study. Clinical characteristics of the patients are summarized in Table I. Nine patients were treated with NAC and 14 without NAC as control group. No adverse effect was recognized by NAC. The etiologies of ARDS in study patients were multiple trauma (7 cases), sepsis (3), peritonitis (4), pneumonia (4), and uncertain (5). Age and gender were not differed between treatment groups. Patients in both groups’ required mechanical ventilation and intensive care for similar period.
MACR during the study: Median MACR for patients in NAC group was decreased after 4 h and remained low. In the control group, mean MACR remained increased for 48 h. Although, MACR in NAC group showed a decreasing trend but the difference with the control was not statistically significant (Figure 1). The p values of changes between two groups at times 0, 4, 6, 12, 24, 36, 48 were 0.56, 0.89, 0.64, 0.93, 0.08, 0.23, and 0.06, respectively
MACR and survival: Median MACR for survived patients was less than those who did not survive but the difference was not statistically significant (p = 0.073, Figure 2).
MACR and PaO2/FiO2 ratio: An improvement in PaO2 was seen with increase of MACR but no statistically significant correlation was found between MACR and PaO2/FiO2 in control (p = 0.779) or NAC (p = 0.441) groups (Figure 3).
MACR and APACHE II score: The correlation between MACR levels and APACHE II score was not statistically significant in NAC (p = 0.414) or control (p = 0.779) groups (Figure 4). MACR and mean arterial pressure (MAP): Comparison of correlation between MACR and MAP showed no significant difference in NAC (p = 0.478) or control (p = 0.279) groups (Figure 5). Most of study patients were normotensive but in 6 patients who were hypertensive (BP > 140/90), MACR was increased with augmentation of arterial pressure.
No statistically significant change was observed within or between groups when examined at different time points. Perfusion pressure and cardiac index were well maintained throughout the study in both treatment groups.
Patient’s outcome: According to APACHE II, a significant improvement in outcome of patients was observed in patients who were treated by NAC (p = 0.04, Figure 6).

Discussion
Usually, every intervention that could suppress neutrophils or improve antioxidant potential status in conditions characterized by oxidative stress such as ARDS or sepsis can be beneficial [2, 12]. In these conditions, early and accurate identification of patients with high risk of death allows for aggressive therapeutic interventions. Administration of NAC during the first hours of severe sepsis and septic shock has decreased peroxidative stress, enhanced tissue oxygenation [14], and improved respiratory function [14]. There is evidence that NAC acts as an anticoagulant and perhaps decreases pulmonary fibrin uptake during ARDS [15]. On the other hand, another small trial with NAC did not show significant improvement on systemic oxygenation or the need for ventilatory support [16]. In addition, delayed administration of NAC not only failed to improve tissue oxygenation but also adversely affected survival in critically ill patients with established organ failure [17]. In fact, endothelial damage cannot be measured directly, thus, MACR seems to be a valuable substitute [18] especially because of speed and consistency of the microvascular response to acute injury [19]. Measurement of microalbuminuria during first 6 h after admission provides precious information about outcome of patients with ARDS and their organ failure [20]. It has been shown that micro-albuminuria reaches its peak levels 2 days faster than other inflammatory biomarkers [21] and thus can be used to predict occurrence of ARDS following trauma [18].
In the present study, we were unable to find a relationship between MACR and APACHE II that is in contrary to other studies [20, 22] but in agreement with another [23]. Our findings also remain in agreement with another study showing absence of correlation between MACR and extra-vascular lung water, a possible indicator of vascular permeability in septic patients receiving mechanical ventilation [24]. The present study showed that non-survivors had higher urine ACR and physiologic scores as compared to survivors. Thus, capillary permeability assessed as microalbuminuria could predict survival or death and probabilities derived from illness severity scoring system. Our study didn’t show a significant correlation between MACR and PaO2/FiO2 which is supported by other reports [23, 24]. Furthermore we were unable to find correlation between diastolic, systolic or MAP and albumin excretion rate, which is supported by one study [25] and is contrary to another [26] who observed a higher need for inotropic support in critically ill patients treated with NAC for more than 24 h. Most of our patients had normal blood pressure, but just 6 patients who were hypertensive (140/90 mm Hg) showed a positive correlation between their MAP and microalbumin. Blood pressure can be a cause of microalbuminuria by increasing glomerular filtration pressure resulting in renal damage.
Moreover, this study is in agreement with study of Soltan-Sharifi et al. [10] who showed antioxidant potential of NAC responsible for its clinically benefits in patient’s outcomes. Suppoering the present data, our recent study also indicated potential of NAC in improving plasma total homocysteine in dialysis patients suffering from chronic kidney disease [27]. In addition, the present results support the role of oxidants in the pathogenesis of ARDS [28-30]. Therefore, improvement of oxidant/antioxidant balance would increase the survival of patients with ARDS who are under oxidative crisis. Small sample size, heterogeneity of patients, NAC dose and administration onset time, and short duration of the study seem responsible for not reaching a significant correlation between MACR and APACHE II or PaO2/FiO2. Of course, the present data well support that clinical characteristics of patients in NAC group improve more rapidly than control group just after 24 h treatment. Taken together, it is concluded that the trend toward the improvement in MCR, although was not statistically significant, but it is promising and necessitates further trials.

Acknowledgments
Authors wish to thank all medical stuff of ICU and Trauma Research Center of Sina Hospital.

References
1. Levy B, Shapiro SD. Acute respiratory distress syndrome. In: Kasper DL, et al. Harrisons principle of international medicine. McGraw Hill 2005; 1592-5.
2. Vazin A, Mojtahedzadeh M, Salehifar E, Rastkari N, Khalaj S, Rezaie A. Future drugs for treatment of acute respiratory distress syndrome. Int J Pharmacol 2005; 1: 9-16.
3. Summer WR. Respiratory failure. In: Goldman L, Bennett JC. Cecils textbook of medicine. W.B. Saunders Company 2000; 468-9.
4. Ware LB, Mattay MA. Medical progress: the acute respiratory distress syndrome. N Eng J Med 2000; 342: 1334-49.
5. Lee WL, Downey GP. Neutrophil activation and acute lung injury. Curr Opin Crit Care 2001; 7: 1-7.
6. Behr J, Maier K, Krombach F, Adelmann-Grill BC. Pathogenetic significance of reactive oxygen species in diffuse fibrosing alveolitis. Am Rev Respir Dis 1991; 144: 146-50.
7. Salari P, Mojtahedzadeh M, Abdollahi M. Influence of serum epidermal growth factor on mechanical ventilation and survival in patients with acute respiratory distress syndrome. Therapy 2005; 2: 393-8.
8. Hadidi E, Mojtahedzadeh M, Paknejad MH. Alterations of blood IL-8, TGFbeta1 and nitric oxide levels in relation to blood cells in patients with acute brain injury. Therapy 2006; 3: 413-9.
9. Vazin A, Mojtahedzadeh M, Najafi A, Khalilzadeh A, Abdollahi M. Relationship between duration, fatality rate and severity of disease and epidermal growth factor in human acute lung injury. Therapy 2005; 2: 255-9.
10. Soltan-Sharifi MS, Mojtahedzadeh M, Najafi A, et al. Improvement by N-acetylcysteine of acute respiratory distress syndrome through increasing intracellular gluthatione, and extra-cellular thiol molecules and anti-oxidant power: evidence for underlying toxicological mechanisms. Hum Exp Toxicol 2007; 26: 697-703.
11. Shadnia S, Dasgar M, Taghikhani S, Mohammadirad A, Khorasani R, Abdollahi M. Protective effects of alpha-tocopherol and N-acetyl cysteine on diazinon-induced oxidative stress and acetylcholinesterase inhibition in rats. Toxicol Mech Methods 2007; 17: 109-15.
12. Ansari G, Mojtahedzadeh M, Kajbaf F, et al. How does blood glucose control with metformin influence intensive insulin protocols? Evidence for involvement of oxidative stress and inflammatory cytokines. Adv Ther 2008; 25: 681-702.
13. Spies CD, Reinhart K, Witt I. Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock patients: results from a prospective, randomized, double blind study. Crit Care Med 1994; 22: 1738-46.
14. Spapen H, Zhang H, Demanet C. Does N-acetyl cysteine influence cytokine response during early human septic shock? Chest 1998; 113: 1616-24.
15. Jespen S, Herlevsen P, Bud MI, Klausen AM. Antioxidant treatment with N-acetylcysteine during adult respiratory distress syndrome: a prospective, randomized, placebo-controlled study. Crit Care Med 1992; 20: 918-23.
16. Domenighetti G, Suter PM, Schaller MD, Ritz R, Perret C. Treatment with N-acetylcysteine during acute respiratory distress syndrome: a randomized, double-blind, placebo-controlled clinical study. J Crit Care 1997; 12: 177-82.
17. Agusti AG, Togores B, Ibanez J. Effects of N-acetyl cysteine on tissue oxygenation in patients with multiple organ failure and evidence of tissue hypoxia. Eur Respir J 1997; 10: 1962-6.
18. Pallister I, Gosling P, Alpar K, Bradley S. Prediction of post-traumatic adult respiratory distress syndrome by albumin excretion ratio eight hours after admission. J Trauma 1997; 42: 1056-61.
19. Pedersen LM. Clinical significance of urinary albumin excretion in patients with nonhodgkins lymphoma. Br J Haema 1997; 107: 889-91.
20. Abid O, Sun Q, Sugimoto K. Predictive value of micro-albuminuria in medical ICU patients: Results of a pilot study. Chest 2001; 120: 1984-8.
21. Gosling P. Microalbuminuria: A marker of systemic disease. Br J Hosp Med 1995; 54: 285-90.
22. De Gaudio AR, Spina R, Di Filippo A. Glomerular permeability and trauma: a correlation between microalbuminuria and injury severity score. Crit Care Med 1999; 27: 2105-8.
23. Herbert D, Spapen P, Mare WD, Due N, Inne H, Lue PH. Effect of N-acetyl cysteine on microalbuminuria and organ failure in acute severe sepsis. Chest 2005; 127: 1413-9.
24. Molnar Z, Szakmany T, Heil P. Microalbuminuria does not reflect increased systemic capillary permeability in septic shock. Intensive Care Med 2003; 29: 391-5.
25. Gosling P, Beevers DG. Urinary albumin excretion and blood pressure in the general population. Clin Sci 1989; 76: 39-42.
26. Molnar Z, Shearer E, Lowe D. N-acetylcysteine treatment to prevent the progression of multisystem organ failure: A prospective, randomized, placebo-controlled study. Crit Care Med 1999; 27: 1100-4.
27. Dashti-Khavidaki S, Khalili H, Barzegar E, et al. Effect of 4-week treatment with oral N-acetylcysteine on plasma homocysteine concentration and antioxidant activity of patients on chronic hemodialysis. Kidney 2008; 17: 122-5.
28. Mojtahedzadeh M, Rouini MR, Kajbaf F, et al. Advantage of adjunct metformin and insulin therapy in the management of glycemia in critically ill patients. Evidence for nonoccurrence of lactic acidosis and needing to parenteral metformin. Arch Med Sci 2008; 4: 174–8.
29. Kajbaf F, Mojtahedzadeh M, Abdollahi M. Mechanisms underlying stress-induced hyperglycemia in critically ill patients. Therapy 2007; 4: 97-106.
30. Hadidi E, Mojtahedzadeh M, Rouini MR, et al. The evaluation of the possible effect of positive end expiratory pressure (PEEP) on pharmacokinetics of phenytoin in patients with acute brain injury under mechanical ventilation. DARU 2005; 13: 74-81.