Lipid Peroxidation in Acute Respiratory Distress Syndrome and Liver Failure

Journal of Surgical Research 168, 243–252 (2011) doi:10.1016/j.jss.2009.10.028

Lipid Peroxidation in Acute Respiratory Distress Syndrome and Liver Failure Christoph Lichtenstern, M.D.,*,1 Stefan Hofer, M.D.,† Andrea Mo¨llers, Ph.D.,‡ Steffi Snyder-Ramos, M.D.,† Dorothee Spies-Martin, M.D.,† Eike Martin, M.D.,† Jan Schmidt, M.D.,§ Johann Motsch, M.D.,† Hubert J. Bardenheuer, M.D.,† and Markus A. Weigand, M.D.* *Department of Anesthesiology and Intensive Care Medicine, University Hospital Giessen and Marburg, Giessen, Germany; †Department of Anesthesiology; ‡Department of General Pediatric Medicine; and §Department of General Surgery, University of Heidelberg, Heidelberg, Germany Submitted for publication May 22, 2009

Background. Lipid peroxidation processes (LPO) are evident in many organ failures. Due to their toxic properties, they are causative for cellular dysfunction at the site of their origin and far beyond. This study was conducted to investigate differences in LPO pattern of patients with established acute respiratory distress syndrome (ARDS) and patients with end-stage liver failure undergoing liver transplantation (LTX) as two mayor prototypes of organ failure. Methods. In this prospective, nonrandomized, controlled trial, we examined LPO by measuring malondialdehyde (MDA), and the volatile aldehydes hexanal and propanal as LPO-markers. Eighteen patients with ARDS, 16 subjects undergoing liver transplantation due to liver failure, and 8 healthy controls were included to the study. Results. ARDS patients showed significantly higher levels in MDA concentrations than LTX and controls, respectively. However, MDA levels of patients with end-stage liver failure were equal to those of controls. Blood concentrations of hexanal and propanal, specific by-products of lipid peroxidation, were elevated in both patient groups, but significantly higher only in LTX. Unexpectedly, hexanal and propanal concentrations were significantly higher in LTX than in ARDS patients. In both patient groups, MDA showed no differences between arterial and mixed venous blood, whereas volatile aldehydes were higher in arterial than in mixed venous compartment.

1 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology and Intensive Care Medicine, University Hospital Giessen and Marburg, Rudolf-Buchheim-Straße 7, 35392 Giessen, Germany. E-mail: christoph. [email protected].

Conclusions. Both ARDS and LTX-patients showed significant evidence of enhanced LPO. However, proportions of MDA and volatile aldehydes differed substantially between the groups. Thus, for the interpretation of LPO markers, disease-specific factors have to be taken into account. Distinctions might be attributable to differences in the effected lipid components or variations in metabolism. Ó 2011 Elsevier Inc. All rights reserved.

Key Words: lipid peroxidation; ARDS; liver failure; oxygen radicals; aldehydes; malondialdehyde; propanal; hexanal. INTRODUCTION

Acute and chronic organ failures, such as acute respiratory distress syndrome (ARDS) [1], liver failure due to cirrhosis [2], heart [3] or kidney failure [4] are caused by inflammatory processes. Oxidative metabolites are known as potent triggers of inflammatory diseases leading directly to severe cell damage [5, 6]. Furthermore, situations in which high oxidative stress levels are observed are often accompanied by generally impaired antioxidative capacities [7, 8]. Thus, organ-related localized oxidative stress can initiate a systemic inflammatory response syndrome (SIRS) with high impact on morbidity and mortality. It has been shown that many patients with organ malfunction at admission to the intensive care units (ICU) show impaired antioxidative properties, worsening the harmful effects of lipid peroxidation. For this reason, antioxidative nutrients such as selenium may be beneficial in the treatment of critical ill patients [9]. The source of oxygen toxicity arises from the oxidative metabolism of mitochondria, where a number of

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reactive oxygen species, including superoxide radical, hydrogen peroxide, and hydroxyl radical arise. A frequently reported toxic effect of these oxygen species is the oxidative breakdown of polyunsaturated fatty acids (PUFA) of cell membranes, the so-called lipid peroxidation (LPO). Further degradation of generated lipid peroxides results in various products, including saturated and unsaturated aliphatic aldehydes. All kinds of aldehydes are considered as toxic second messengers due to their ability to react with protein molecules. This may result in further damage caused by loss of enzyme activity, destruction of amino acids and polymerization, as well as genotoxicity induced by binding to DNA [10, 11]. There are various molecules that are generated in the setting of LPO indicating acute cell damage due to oxidative stress. Malondialdehyde (MDA) is the prototype of these indicators and served in a huge number of studies for detection of LPO. Thus, most data that show evidence of lipid peroxidation in critically ill patients are obtained by measuring MDA. MDA was investigated in patients with ARDS [12], stroke [13], liver disease [14], and patients undergoing coronary bypass surgery [15]. However, MDA is regarded to be an unspecific marker of LPO due to analytical difficulties [15, 16]. Volatile aldehydes like hexanal and propanal are further known by-products of lipid peroxidation, which are considered as more specific parameters [16–18]. Because of their ultra-short half time they are more difficult to detect in whole blood, homogenates, or breath [19, 20]. Volatile aldehydes have been studied as specific markers of LPO in asthma/chronic obstructive pulmonary disease [21], ARDS [22], in bone marrow after total body X-ray irradiation [23], in diabetes mellitus [24], and diabetic nephropathy [25], malnutrition [26], atherosclerosis [27], stroke patients [13], and most recently in brain tumors [28]. However, no comparative study has been performed yet for different LPO-marker pattern between various organ failures including these volatile aldehydes. Therefore, we investigated lipid peroxidation in ARDS and end-stage liver failure patients. As LPO indicators, we measured MDA plasma levels and whole blood concentrations of the volatile aldehydes hexanal and propanal, which arrive from different PUFA, namely u–6 and u–3-PUFA, respectively.

MATERIALS AND METHODS Subjects After institutional approval and informed consent from the patients or their legal representative, 18 subjects with ARDS and 16 subjects undergoing liver transplantation were enrolled in the study. The enrollment of ARDS patients was between 6 and 96 h after the onset of a severe ARDS. Patients were considered to have ARDS if they had an acute onset of lung failure, arterial hypoxemia (PaO2/ FiO2  200), bilateral diffuse pulmonary infiltrates seen on chest

radiography, and a pulmonary artery occlusion pressure (PCWP) less than 18 mmHg or no clinical evidence of left atrial hypertension [29]. The severity of ARDS was estimated by the means of the Murray score [30], and a score of at least 2.5, reflecting severe ARDS, served as inclusion criteria. ARDS patients with renal failure, chronic liver disease, or acute liver failure and intracranial hemorrhage were not included into the study. They were anesthetized by fentanyl (0.05– 0.1mg kg–1min–1) and midazolam (1–3 mg kg–1min–1) and enterally fed (20–25 kcal/kg /d) without enrichment of omega-3 fatty acids. Selenium (200 mg per day) was routinely supplemented by intravenous infusion. Subjects with end-stage liver failure were enrolled to the study prior to the liver transplantation procedure. LTX patients with renal failure were not included into the study. All patients were mechanically ventilated by a pressure controlled mode. PEEP and FiO2 were adjusted to achieve an optimal PaO2. Severe hypoxia, as defined as PaO2/FiO2 ratio of less than 50, served as exclusion criterion. In both groups, blood samples were taken from arterial lines and Swan-Ganz catheter. In the group of ARDS patients, this was done in the ICU immediately after enrollment into the study. The blood samples of the liver transplant recipients were collected 30 min after induction of general anesthesia (fentanyl 0.1–0.3 mg, isoflurane in air 0.5–0.8 MAC) for transplantation under stable hemodynamic conditions. All samples were taken under normocapnic circumstances (PaCO2 of 35–45 mm Hg). Blood samples were put on ice after taking, and lipid peroxidation markers were then immediately analyzed on a standardized schedule. In each patient, an electrocardiogram (ECG) and arterial blood pressure changes were continuously recorded. Cardiac output was measured by a triplicate thermodilution method using an Edwards Swan-Ganz thermodilution Paceport catheter (Baxter, Deerfield, IL, USA). To evaluate the normal ranges of the LPO parameters (MDA, hexanal, and propanal) measured, arterial concentrations of eight spontaneously breathing, healthy non-smocking volunteers served as controls.

Lipid Peroxidation Parameters A static headspace/gas chromatography analysis (automatic Headspace Sampler HS-40; Perkin Elmer, Waltham, MA, USA) served for the measurement of the volatile aldehydes hexanal and propanal. After the equilibration of the blood samples (0.5 mL) in special vials for 120 min at 85  C, the headspace was injected onto the analytical column (Poraplot S; Chrompack, Varian, Palo Alto, CA, USA). The volatile compounds were detected by flame ionization detector (FID) and quantified by measuring aqueous standard solutions of the different aldehydes. Hexanal and propanal were identified by mass spectrometry and additionally by comparison of the retention times with those of commercially available counterparts [22]. High-performance liquid chromatography (HPLC) was used for MDA measurement by the method of Lepage et al. for TBA-reactive substances [31].

Statistical Analysis Results were expressed as median and range or mean and standard deviation if normally distributed. Significance of difference between groups was tested by single factor analysis of variance or Mann-Whitney test, as appropriate. P values  0.05 were considered as significant.

RESULTS Patient Characteristics

Patient characteristics are listed in Table 1. The ages of ARDS subjects ranged between 23 and 76 y (58.7 6 13.9 years), and the group was made up of five women and 13 men (Table 1A). The mean Murray score of all ARDS patients was 2.74 6 0.24. The mean initial

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TABLE 1 Clinical Characteristics of the Enrolled Patients (A) ARDS Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 6 SD or ratio

Underlying disease Pneumonia Pneumonia Cardiac surgery Cardiac surgery Pneumonia Sepsis Multiple trauma Aspiration Pneumonia Cardiac surgery Sepsis Sepsis Multiple trauma Cardiac surgery Aspiration Sepsis Multiple trauma Pneumonia

Gender

Age

BMI

APACHE II score

Murray score

F F M M M M M F M M M F M M M M F M 5/13

72 63 76 60 45 55 53 75 64 63 76 68 48 49 41 59 23 66 58.7 6 13.9**

24,7 22,1 22,3 19,3 25,6 29,3 24,7 23,2 27,7 35,9 25,4 25,8 26,1 22,2 27,8 31,2 22,2 25,5 25.6 6 3.9**

18 26 23 24 19 26 21 26 24 22 14 24 19 21 18 14 8 25 20.7 6 4.9***

2,75 2.5 3.0 2.5 2.75 2.75 3.25 3.0 2.5 3.0 2.75 2.75 3.0 2.5 2.5 2.5 2.5 3.0 2.75 6 0.24

(B) Liver Failure Patient no.

Underlying disease

Gender

Age

BMI

APACHE II score

Child-Pugh score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean 6 SD or ratio

Alcoholic liver disease Alcoholic liver disease Alcoholic liver disease Chronic hepatitis C Chronic hepatitis C Alcoholic liver disease Hemochromatosis Primary biliary cirrhosis Alcoholic liver disease Alcoholic liver disease Alcoholic liver disease Chronic hepatitis B Chronic hepatitis C Primary biliary cirrhosis Hemochromatosis Toxic hepatic failure

F M M M F M M F M M M F M F M F 6/10

55 49 47 41 39 59 44 38 61 55 54 62 49 45 46 18 47.6 6 10.9*

22,5 21,5 28,3 26,5 26,1 30,1 22,0 24,4 21,8 24,5 24,5 23,4 21,5 25,2 21,2 25,0 24.3 6 2.3**

11 26 18 8 19 10 9 24 25 17 15 16 25 21 20 18 17.6 6 5.9***

12 12 11 6 6 10 8 9 9 10 11 10 6 10 9 13 9.5 6 2.2

(C) Controls Subject no. 1 2 3 4 5 6 7 8 Mean 6 SD or ratio

Age

Gender

43 39 28 34 36 51 36 30 37.1 6 7.3

BMI ¼ body mass index; APACHE ¼ acute physiology and chronic health evaluation; F ¼ female; M ¼ male; SD ¼ standard deviation. * ARDS versus liver failure: P ¼ 0.016. ** ARDS versus liver failure: NS (P ¼ 0.25). *** ARDS versus liver failure: NS (P ¼ 0.126).

M M F M F M M F 3/8

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TABLE 2 Hemodynamic Conditions in Both Patient Groups

Heart rate Mean arterial pressure Cardiac output Cardiac index FiO2 P/F ratio

Unit

ARDS

LTX

P value

[bpm] [mmHg] [L min1] [L min1 m2] [%]

103.06 6 17.92 78.72 6 11.92 5.74 6 2.32 2.77 6 0.82 77 6 17.7 111 6 41.8

85.08 6 14.96 87.79 6 17.03 9.03 6 3.00 4.69 6 2.88 54 6 6.7 220 6 76.4

0.005** 0.086 N.S 0.035* 0.029* <0.001*** <0.001***

Values expressed as means 6 standard deviation (SD). * P < 0.05. ** P < 0.01. *** P < 0.001.

APACHE II score [32], obtained for grading the severity of general morbidity, was 20.7 6 4.9. The primary causes of ARDS were pneumonia/aspiration (seven subjects), cardiac surgery (four), sepsis (four), and multiple traumata (three). None of the patients died during the first 24 h after blood samples were taken. Liver transplant recipients had a mean age of 47.6 6 10.9 y (range: 18–62 y). The mean initial APACHE II score accounted for 17.6 6 5.9. Seven patients suffered from alcoholic cirrhosis, four from chronic viral hepatitis, two from hemochromatosis, two from primary biliary cirrhosis, and one from fulminate toxic hepatic failure, respectively. The mean Child-Pugh score, reflecting the stage of liver failure, was 9.5 6 2.2.

Comparison of LPO Marker Levels in Arterial Blood Between Disease Groups and Controls

The arterial MDA concentrations in ARDS patients measured four times higher (P < 0.001) than in healthy volunteers, indicating enhanced lipid peroxidation (Table 3; Fig. 1). However, we observed no significant differences between these two groups for arterial levels of the volatile aldehydes hexanal (P ¼ 0.27) and propanal (P ¼ 0.94), although concentration values were much higher in ARDS (Figs. 2 and 3). In contrast, liver transplant recipients showed equal arterial MDA concentration as the control population (P ¼ 0.87), whereas a marked enhancement were detectable in both volatile aldehydes (for hexanal P ¼ 0.027; for propanal P < 0.001).

Hemodynamic and Respiratory Conditions

Comparison of LPO Marker Levels Between ARDS and Liver Failure Patients

ARDS patients showed tachycardia (heart rate > 100 bpm) and normal state cardiac indices (Table 2). These differed significantly compared with liver transplant recipients, presenting normal heart rate but accelerated cardiac indices. Eight ARDS patients (44%) and LTX subjects (50%) got b-blockers for regular medication to treat either arterial or portal vein hypertension. FiO2 and P/F ratios varied significantly between both study groups due to the preexisting differences in lung function.

Comparing ARDS and liver failure patients, MDA levels were more than four times higher in arterial and mixed venous blood of ARDS patients than those of patients who suffered from liver insufficiency (Figs. 1 and 4). However, in ARDS arterial and mixed venous hexanal concentrations were lower than in liver failure (Figs. 2 and 5). According to these findings, arterial and mixed venous propanal concentrations in liver transplant recipients were more than 10-fold higher than in ARDS patients (Figs. 3 and 6).

TABLE 3 Arterial and Mixed Venous Concentrations of MDA, Hexanal, and Propanal Units MDA

[nmol/L]

Hexanal

[mmol/L]

Propanol

[mmol/L]

art mv art mv art mv

Controls (n ¼ 8)

ARDS (n ¼ 18)

LTX (n ¼ 16)

133.38 6 57.71 3.57 6 5.27 0.27 6 0.15 -

476.07 6 256.29 453.62 6 232.66 8.71 6 5.04 1.44 6 1.71 3.13 6 1.79 0.85 6 0.55

93.60 6 59.13 78.33 6 49.55 12.53 6 9.91 7.05 6 6.01 38.36 6 31.55 12.57 6 14.27

MDA ¼ malondialdehyde; Pro/Hex ratio ¼ concentration-ratio of Propanol to Hexanal; art ¼ arterial; mv ¼ mixed venous. Values expressed as means 6 standard deviation.

LICHTENSTERN ET AL.: LIPID PEROXIDATION IN ARDS AND END-STAGE LIVER FAILURE

FIG. 1. Arterial MDA. MDA: malondialdehyde [nmol/L]; ARDS: Patients with adult respiratory distress syndrome. LTX: patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

Arterial Versus Mixed Venous LPO Concentrations in Both Patient Groups

There was no difference detectable for MDA concentrations in arterial and mixed venous blood of ARDS patients (P ¼ 0.78). However, in this group, both volatile aldehydes showed much higher arterial than mixed venous levels, representing significant gradients between both compartments (both P values < 0.001).

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FIG. 3. Arterial propanal. Propanal [mmol/L]. ARDS: patients with adult respiratory distress syndrome. LTX: patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

Similar to ARDS, we observed no gradient for MDA in the LTX group (P ¼ 0.44). Both median hexanal and propanal concentrations appear to be higher in arterial than in mixed venous blood. For hexanal, this gradient did not reach a level of significance (P ¼ 0.07), whereas it was obvious for propanal (P ¼ 0.007). Inverse Proportions of Both Volatile Aldehydes in Arterial Blood of the Patient Groups

Thus, these results show inverse propanal-hexanal concentration-ratios in the arterial blood of ARDS and liver transplant recipients. In case of ARDS, the amount of hexanal concentrations in arterial blood are about twice as high as propanal, whereas liver transplant recipients show lower hexanal than propanal concentrations in arterial blood. In the arterial blood of controls, we find much higher hexanal than propanal concentrations, similar to ARDS patients. The individual propanal-hexanal ratios we observe in the mixed venous blood of ARDS patients vary. Eight patients show a proportion of less than one indicating higher hexanal level, but in 10 patients it is vice versa. Thus, there is no significant difference between both patient groups for propanal-hexanal ratio in mixed venous blood.

FIG. 2. Arterial hexanal. Hexanal [mmol/L]. ARDS: Patients with Adult Respiratory Distress Syndrome LTX: Patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

DISCUSSION

In this prospective, nonrandomized, controlled trial, we examined patients with established ARDS and

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FIG. 4. Pulmonal arterial MDA. MDA: malondialdehyde [nmol/L]; ARDS: patients with adult respiratory distress syndrome. LTX: patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

chronic liver failure to analyze their patterns in lipid peroxidation products by measuring plasma levels of MDA as widely-used LPO indicator and whole blood concentrations of the volatile aldehydes hexanal and propanal in arterial and mixed venous blood. Due to study design, none of the included ARDS patients showed any symptoms of liver failure to reach valuable results depending on a distinct underlying organ failure. The present study reveals elevated arterial MDA concentrations in ARDS patients, whereas MDA in liver transplant recipients are equal to those of controls. Furthermore, arterial hexanal and propanal levels are higher in both patient groups than in healthy volunteers, but only for liver transplant recipients these data reach significant level. The pattern of the considered LPO indicators differs between ARDS and liver insufficiency. MDA, which shows no difference between arterial and mixed venous blood in any patient group, is elevated only in lung failure (Figs. 1 and 4). Hexanal and propanal, representing more specific peroxidation products, are much higher in patients undergoing liver transplantation. In both patient groups, these volatile aldehydes show significant differences between arterial and mixed venous blood (Figs. 2, 3, 5, 6). Lipid peroxidation due to oxidative stress is recognized as part of the inflammatory process leading to

FIG. 5. Pulmonal Arterial Hexanal. Hexanal [mmol/L]. ARDS: patients with adult respiratory distress syndrome. LTX: Patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

organ failure. Highly reactive oxygen species generate lipid hydroperoxides by reaction with polyunsaturated fatty acids of cell membranes or plasma lipoproteins that serve as endogenous amplifiers of a complex destructive chain reaction. Subsequently, direct membrane damage and further degrading reactions with proteins or DNA cause cell death and activation of immune cells, and result in function loss of the affected tissues [33]. Furthermore, the ability to attack targets distant from the site of their origin implies that peroxidation products can act systemically as secondary cytotoxic messengers [11]. Many intermediate products of several LPO pathways, such as hydroxyalkenals (e.g., 4-hydroxynonenal), saturated aldehydes (e.g., MDA, hexanal, propanal), volatile alkanes (e.g., pentane) [34], or the recently described isoprostanes and oxysterols [35] are identified, since the impact of LPO products for the development of organ insufficiency are understood. Thus, all these markers serve in many studies for a qualitative detection of an ongoing enhanced lipid peroxidation in organ failures. However, quantitative evaluation of lipid peroxidation is very difficult because the arising metabolites are very reactive and their metabolic pathways are hardly detectable in blood and tissues. For example, the metabolic patterns of volatile aldehydes are different from MDA, which is deemed to be more stable.

LICHTENSTERN ET AL.: LIPID PEROXIDATION IN ARDS AND END-STAGE LIVER FAILURE

FIG. 6. Pulmonal arterial propanal. Propanal [mmol/L]. ARDS: patients with adult respiratory distress syndrome. LTX: patients undergoing liver transplantation due to end-stage liver failure. (Color version of figure is available online.)

Since aldehydic degradation products such as MDA and volatile aldehydes were found in the plasma of ARDS patients, it has been postulated that lipid peroxidation is an important pathogenetic factor of ARDS [1, 12, 36, 37]. Additionally, in vivo local evidence of enhanced oxidative stress is demonstrated in bronchio-alveolar fluids [38] and in expired breath [34, 39] of ARDS-patients. Our results show significantly higher concentrations of MDA in the arterial blood of ARDS patients compared with healthy controls. Elevated median levels of both volatile aldehydes in ARDS confirm this finding, but failing to reach significance. This is in accordance to former findings indicating an enhanced lipid peroxidation in ARDS. In this study, ARDS patients show significantly superior hexanal and propanal concentrations in arterial than in mixed venous blood, leading to the conclusion that the lung is the origin of these lipid peroxidation products. Data of a previous study [22] of our group show the same concentration gradients in hexanal and pentanal between arterial and mixed venous blood in ARDS. Concentration gradients of lipid peroxidation products across the vascular bed of organs are also used in other studies to investigate lipid peroxidation in the brain and heart [13, 15]. Similar results are described by Demling [40], who found MDA gradients between arterial and central venous blood in an animal model for

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ARDS. However, our study results only confirm differences for hexanal and propanal, whereas the MDA concentrations are not different in arterial and mixed venous blood of ARDS patients. MDA is a three-carbon compound, which is generated by both auto-oxidation and radical-mediated peroxidation of PUFA, u–3- as well as u–6-PUFA, which have more than two double bonds. In particular, arachidonic acid (u–6) and docosahexaenoic acid (u–3) are major sources of MDA [11, 15]. However, MDA measurements miss good specificity and sensitivity because MDA can also be formed by non-enzymatic or enzymatic processes [11, 41]. Additionally, the reaction used for detection of MDA with thiobarbituric acid (TBA) is not specific for MDA. TBA is also able to form adducts with other molecules, so that the TBA test measures TBA-reactive substances (TBARS) instead of MDA [15]. In contrast, hexanal and propanal are detected directly by gas chromatography/mass spectrometry as more specific parameters for lipid peroxidation [17, 18]. These volatile aldehydes are generated mainly in the so-called ß-cleavage reaction of lipid hydroperoxides [11]. Hexanal and propanal own a confident specificity because they arise by peroxidation of different PUFA. Hexanal comes up from u–6-PUFA like linoleic and arachidonic acid [17], whereas propanal emerge from u–3-PUFA such as linolenic and docosahexaenoic [18]. For patients in the status of liver failure, our results show elevated concentrations of hexanal and propanal compared with controls and ARDS. However, low MDA levels contradict this finding. For various liver diseases, intrahepatical lipid peroxidation is proven to be a part of pathogenesis [42], and it is discussed as a major cause of liver fibrosis by direct stimulation of intrahepatic collagen production [43, 44]. Beyond these organ-specific evaluations, acute and chronic liver failure is accompanied by systemically increased oxidative stress and lipid peroxidation. A depleted antioxidant capacity, in particular, caused by depressed glutathione and tocopherol levels [45, 46] and increased amounts in many LPO products including MDA are shown in plasma and urine samples [35, 47]. For example, the accumulation of bile acids in the plasma is discussed as a cause of lipid peroxidation in extrahepatic organs [48]. This is supported by experimental results of cholestatic liver disease, which is associated with increased lipid peroxidation in plasma, kidney, heart, and brain [49]. In the liver transplant recipients of our study, hexanal and propanal are lower in mixed venous than in arterial blood indicating (1) another origin of these LPO metabolites than the liver, and (2) their elimination along their further circulation. In relation to this, Corradini et al. hypothesize a systemic source of free radicals in chronic liver disease because systemic oxidative stress/antioxidant status present in patients

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with cirrhosis before transplantation was only partially corrected in the short term after liver transplantation [35]. However, MDA levels are not elevated, either in arterial or in mixed venous blood of our liver cirrhotic patients. The same phenomenon is described in chronic hepatitis C patients by the study of Jain et al. in which MDA concentrations of patients were equal to controls, whereas isoprostane in plasma and urine were significantly increased [47]. Moreover, it must be considered that volatile aldehydes are measured in whole blood, whereas MDA is measured in plasma. This is relevant because erythrocytes appear to be able to carry volatile aldehydes intracellularly away from their location of production [50]. This was concluded by Baraona et al. [50] because of his findings of high arterio-venous differences in acetaldehyde after alcohol consumption in erythrocytes but not in plasma. Our observation of much higher hexanal and propanal concentrations in the patients with end-stage liver failure compared with those of ARDS patients was unpredictable. Local elimination capacities in liver, lung, and whole blood of both patient groups might be unequally related to the underlying disease. Moreover, differences in the lipid consistency of the affected liver, lung, or blood components may influence the LPO-products arising in the peroxidation process. Besides this, we have to consider that all products of lipid peroxidation are very reactive by themselves and thus can lead immediately to other undetected metabolites. In ARDS patients, we found higher concentrations of hexanal than propanal according to the relative contribution of the different aldehydic lipid peroxidation products in LDL estimated by Esterbauer [17]. In contrast, patients undergoing liver transplantation show higher levels of propanal than hexanal. The same inverse propanal/hexanal ratio is seen in the peroxidation of liver homogenates, although liver microsomes and mitochondria contain only a few u–3 PUFA [18]. Reasons might be that u–3 PUFA can be peroxidized easier than u–6 PUFA [51], and that the organ-specific activities of metabolic enzymes eliminating hexanal and propanal are different [52, 53]. Additionally, pulmonary blood flow and gas transfer will be markedly affected in lungs damaged by ARDS. This could well account for the anomalous concentration ratio of propanal to hexanal seen in ARDS if propanal is being preferentially lost to the atmosphere. The results of this study agree with many studies dealing with ARDS and liver failure patients who show lipid peroxidation as a substantial part of both diseases. Quantitative appointments about lipid peroxidation in different organ failures are difficult to achieve because of the heterogeneous pattern of detectable LPO metabolites. Particularly in liver failure, MDA

seems to be less sensitive than the volatile aldehydes hexanal and propanal. Therefore, secondary extrahepatic formation or different elimination pathways have to take into account. There are some limitations to the study: First, patients with end-stage liver disease and patients with ARDS often differ in their preconditions, as patients with ARDS often are healthy prior to onset of their disease, while patients with end-stage liver failure are affected chronically by their disease. Lipid peroxidation patterns might be influenced by respiratory conditions, including oxygen demand, which were obviously not the same. Further, the results of this study reflect only a snapshot in lipid peroxidation in these subjects. Differences in age, distribution of gender, or type of anesthesia could have an effect on study results. Regarding this, age-related decline in antioxidative capacity in organ tissues and plasma is well documented [54], and volatile anesthetics like isoflurane used in LTX patients are known to induce bronchioalveolar inflammation and consequent lipid peroxidation [55, 56]. Additionally, the number of studied subjects, particularly in the control group, is low in the background of the measured variability of LPO parameter. As we have decided to examine healthy persons for control, no data of pulmonal arterial LPO markers could be provided for this group. CONCLUSION

Patients with ARDS and patient undergoing liver transplantation for end-stage liver failure show different patterns of lipid peroxidation. Therefore, qualitative estimations of the ongoing lipid peroxidation in organ failures are only achieved by measuring various LPO products. ACKNOWLEDGMENTS The authors acknowledge supported for this work by an Institutional Research Fund of the University of Heidelberg, Heidelberg, Germany.

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