# Lipoperoxidation and Protein Oxidative Damage Exhibit Different Kinetics During Septic Shock

Authors: Max Andresen; Tomas Regueira; Alejandro Bruhn; Druso Perez; Pablo Strobel; Alberto Dougnac; Guillermo Marshall; Federico Leighton
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Septic shock (SS)-related multiorgan dysfunction has been associated with oxidative damage, but little is known about the temporal damage profile and its relationship to severity. The present work investigated prospectively 21 SS patients. Blood samples were obtained at diagnosis, 24, 72 hours, day 7, and at 3 months. At admission, thiobarbituric acid reactive substances (TBARSs), plasma protein carbonyls, plasma protein methionine sulfoxide (MS), ferric/reducing antioxidant power (FRAP), total red blood cell glutathione (RBCG), uric acid (UA), and bilirrubin levels were increased
(P<.05). Total radical—trapping antioxidant potential (TRAP) and vitamin-E were similar to controls, and vitamin-C was decreased (P<.05). During evolution, TBARS and RBCG increased (P<.001), vitamin-E levels remained stable, whereas plasma protein carbonyls and MS, TRAP, vitamin-C, reduced glutathione, and UA levels decreased
(P<.006). After 3 months, plasma protein carbonyls and MS persisted elevated. More severe patients exhibited higher TBARS, TRAP, FRAP, vitamin-C, UA, and bilirrubin levels. Our results suggest early and persistent oxidative stress during septic shock and a correlation between increasing levels of lipoperoxidation and sepsis severity.


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## Body

## 1. Introduction

Inflammation, sepsis, and particularly septic
shock are associated with global and local hypo-perfusion, ischemia-reperfusion events, endothelial injury with an associated procoagulant state, and
monocyte—macrophage system activation. All these
processes contribute to the production and release of different cytokines and
other inflammatory mediators [1]. They also induce the production of large
amounts of free radicals in a nonregulated fashion associated with high-oxidative
potential damage [2]. In fact, several
sources of reactive oxygen species (ROS) have been detected in sepsis and
septic shock, including the mitochondrial respiratory electron transport chain,
immune cell, and xanthine oxidase activation as a result of ischemia and
reperfusion and the respiratory burst associated with NADPH oxidase [3].Several studies have shown the presence of
oxidative stress in sepsis [4–9]. Early
production of reactive oxygen species (ROS) has been demonstrated in
experimental studies in sepsis [10], and excessive release of superoxide anion
has been shown to contribute to postreperfusion oxidative damage in several
ischemic organs [11, 12]. Increased
thiobarbituric acid reactive substances (TBARSs),
which is a marker of lipoperoxidation, has been observed in critically ill
patients in association with multiorgan failure (MOF) development [7, 13].Most studies about oxidative stress in sepsis
have looked at a single time-point, usually at admission. In addition, very few
of these studies have looked specifically at septic shock patients [14, 15]. But septic shock has distinctive features
which may differ substantially from less severe forms of sepsis. It has a very
dynamic course with several changes taking place simultaneously in just a few
hours. Therefore, more comprehensive studies looking at the temporal evolution
of oxidative stress in septic shock patients are required. This is a critical
step to define windows of opportunity for interventions aimed at modulating
this response.Our main objective was to evaluate broadly the
temporal profile of antioxidants and oxidative damage during septic shock
evolution. Secondary objectives were to determine if oxidative damage markers
and antioxidant levels are related to septic shock severity and to evaluate
methionine sulfoxide, a novel marker of protein oxidative damage.


## 2. Material and Methods

This was a prospective observational study that
included patients with diagnosis of septic shock admitted to a medical-surgical
ICU of a university hospital from May 2004 to August 2005. The study was
approved by the Ethical Committee of the Facultad de Medicina of the Pontificia
Universidad Católica de Chile, Santiago,
Chile, and an
informed consent was obtained from all patients or their relatives.


### 2.1. Patients and Management

Patients were enrolled if they fulfilled the
following criteria: (1) diagnosis of septic shock according to the consensus
conference  [16], (2) less than 24 hours elapsed since admission to the emergency department, and (3) age older than 18 years. Exclusion criteria were
(1) patients extremely ill, in whom survival interval was expected to be less
than 48 hours, (2) use of any external
vitamin supplement within one week before enrollment, (3) onco-hematological, and (4) chronic renal failure patients. All patients were managed according to a
standard hemodynamic and respiratory algorithm [17].


### 2.2. Data Collection

Demographic data, diagnosis, the acute
physiology and chronic health evaluation (APACHE) II score, sepsis-related organ failure assessment (SOFA) score, hemodynamic and respiratory parameters, maximal vasoactive drug dose,
general biochemistry (renal, hematological, and hepatic function), and C
reactive protein (CPR) levels were
registered at admission, 24 and 72 hours, as well as the seventh day of
evolution.


### 2.3. Oxidative Stress Evaluation

Oxidative stress markers employed were thiobarbituric
acid reactive substances (TBARSs), an
index of lipid peroxidation, plus carbonyls, and methionine sulfoxide in plasma
proteins as markers of protein oxidative damage. Antioxidant activity was
evaluated by measuring: (a) total
antioxidant capacity (TAC) determined with two methods: (1) total radical- trapping
antioxidant potential (TRAP) and (2) ferric/reducing antioxidant power (FRAP); (b) nonenzymatic antioxidants: vitamins C and E, beta
carotene, and lycopene; (c) enzymatic antioxidant cofactors: reduced and total
red blood cell glutathione; and (d) nonspecific antioxidants: uric acid, bilirubin, and albumin. Blood samples were
obtained at diagnosis (T0), 24 hours (T1), 72 hours (T3), and at the seventh day of
evolution (T7). Delayed measurements
were made in all survivors 3 months later. Normal values for each parameter
were obtained from a group of 17 healthy volunteers matched for age and sex
with septic shock patients.


### 2.4. Laboratory Technique

All samples were stored at −20°C and were analyzed
within 10 days, but for glutathione analysis, samples were stored with ACD at
4°C and were also analyzed within 10 days.


#### 2.4.1. Total Antioxidant Capacity (TAC)

TRAP (Total-Trapping Radical [Peroxyl] Antioxidant Potential)
One mL of a mixture of Luminol (60μM)-AAPH
(10 mM), in glycine buffer 50 mM pH:
9.40, kept in ice and darkness, was preincubated at 37°C. This mixture was
poured into a cuvette of polystyrene (Clinicon) and placed in a luminometer (BioOrbit 1250, Finland) kept at 37°C. When the light emission reached a steady-state condition, 10μL of plasma or standard (Trolox) were added. The luminescence was plotted
against the elapsed time. The induction time (tsample) was equated to the time required for the chemiluminescence intensity to reach a
level equal to 40% of the intensity measured prior to the sample addition [18]. The quenching of the light emission was recorded
in a Kipp & Zonen BD 111 recorder. The coefficient of variation for this measurement
is 4, 04%.FRAP (Ferric/Reducing Antioxidant Power)
Reduction at low pH of a ferric
tripyridyltriazine (FeIII-TPTZ) complex
to the ferrous form, by electron-donating antioxidants [19].
Samples were incubated in mixture of TPTZ, FeCl3, and acetate buffer (1 : 1 : 10) for 5 minutes at 37°C and quantified at 593 nm. The coefficient of variation for this measurement is 2,5%.


#### 2.4.2. Vitamin E (α-Tocopherol), β-Carotene, and Lycopene

Lipid soluble antioxidant concentrations were
determined by HPLC with electrochemical detection [20].
Plasma samples were precipitated with methanol and extracted with hexane and
centrifuged. The organic phase was dried, and the residue was redissolved in methanol:
ethanol. An isocratic phase reverse chromatography was performed using a
Supelcosil C-8 column, and 20 mM LiClO4 in methanol:
H2O (97.5 : 2.5) as mobile phase. For electrochemical detection, an
amperometric BAS LC4C detector (Bioanalytical Systems Inc., West
Lafayette, Ind, USA) was used with
an applied potential of +0.6 V. The coefficient of variation for vitamin E is
1, 8%, for β-carotene is 9,3%, and for
lycopene is 8,2%.


#### 2.4.3. Ascorbic Acid

Determinations were carried out by a method
based on the reduction of ferric chloride by ascorbic acid, with the resulting
ferrous ion quantified by the addition of 2-,4-,6-tripyridyl-s-triazine [21]. Heparinized plasma samples were precipitated
with trichloroacetic acid. The tubes were centrifuged at 20,800 g for 30 seconds.
An aliquot from the supernatant was incubated with TPTZ and FeCl3 in buffer
acetate for 5 minutes at 25°C, and quantified at 595 nm. The coefficient of
variation for this measurement is 3,4%.


#### 2.4.4. Glutathione

Total Red Blood Cell Glutathione
Red blood cells were lysed and precipitated by
adding perchloric acid 0.28 M to fresh blood anticoagulated with ACD. An
aliquot from the supernatant, neutralized with K3PO4 1.75 M, was incubated with
phosphate buffer 0.1 mM (EDTA 1 mM) and 5.5′-dithiobis
(2-nitrobenzoic acid) 0.5 mM (sodium Citrate 1% p/v) for 7 minutes at 25°C and quantified
at 412 nm [22].Reduced Red Blood Cell Gluthatione
The cells were rinsed four times with 3 mL of ice cold
PBS. Collected cells were diluted in phosphate buffer containing diethylenetriaminepentaacetic
acid 1.34 mM pH 7.8. An aliquot was derivatized by the addition of N-(1-pyrenyl) maleimide 1.0 mM in acetonitrile. Solutions
were incubated for 5 minutes at room temperature and then acidified with HCl to
pH 2.5. An isocratic phase reverse chromatography was performed using a
Supelcosil C-18 column and 65% acetonitrile: 35% H2O, 1 mL/liter acetic acid,
and 1 mL/liter O-phosphoric acid as mobile phase. A fluorescence
spectrophotometer detector (Merck-Hitachi F-1000, Darznstadt, Germany)  was used for detection λex 330 nm and λem 375 nm [23].


#### 2.4.5. TBARS

The method is based on the formation of an
adduct TBA-MDA (2 : 1). Plasma samples,
TBA solution (0.67%/NaOH 0.05N), and TCA 50% solution were placed in this
order into a screw-cap test tube and incubated at 90°C for 45 minutes. The
aqueous phase was quantified at 532 nm. TBARS plasmatic levels are expressed in
micromolar equivalent MDA [24].


#### 2.4.6. Carbonyls

Plasma protein samples were reacted with
dinitrophenylhydrazine and then adsorbed to wells of an ELISA plate, overnight
at 4°C, before probing with a commercial antibody raised against protein-conjugated
DNP. The biotin-conjugated primary antibody (anti-DNP
Polyclonal IgG) was then reacted with streptavidin-conjugated
horseradish peroxidase for quantification with TMB. Acidic stop solution was
added, and absorbance measured at 450 nm. The method was calibrated using oxidized
and reduced albumin [25]. The
coefficient of variation for this measurement is 16, 2%.


#### 2.4.7. Methionine Sulfoxide

Protein methionine sulfoxide content was
measured by HPLC-fluorometric detection, using a modification of the method of
Morgan etal. [26] and was expressed as
the fraction of total methionine oxidized to methionine sulfoxide: proteins
from 10μL plasma aliquots were precipitated with 60μL of ice-cold
acetonitrile and centrifuged at 10000 g for 20 minutes; the protein pellets
were hydrolyzed using methanesulfonic acid at 150°C for 90 minutes and were
derivatized with o-phthaldialdehyde/β-mercaptoethanol solution. Derivatized
amino acids were detected by an HPLC-fluorescence spectrophotometer detector (Merck-Hitachi F-1000), λex 340 nm, and λem 455 nm,
and quantified using standards containing methionine, methionine sulfoxide, and
methionine sulfone. Analysis was performed by reverse-phase HPLC using a
Synergy 4μM fusion column with a continuous gradient of mobile phase 75 mM Na
acetate buffer pH 4.5: methanol: tetrahydrofuran (80 : 19 : 1) initial to (20 : 80 : 0) final, in 40 minutes.
The coefficient of variation for this measurement is 9.2%.


### 2.5. Statistical Analysis

Changes along time for the different oxidative
stress markers were analyzed with linear mixed effects models. In addition,
measurements in septic shock patients at each time point were compared with
normal values from healthy matched subjects by at-test for independent
samples (samples exhibited a normal distribution).
Correlations between severity parameters and oxidative stress markers were made
with Pearson. Linear mixed effects models, adjusted by sex and age, were also
used to confirm the association between severity parameters and oxidative
stress. Results are expressed as mean ± SD, and a P<.05 was
considered significant. The proc MIXED of the SAS statistical program was used
for analysis.


## 2.1. Patients and Management

Patients were enrolled if they fulfilled the
following criteria: (1) diagnosis of septic shock according to the consensus
conference  [16], (2) less than 24 hours elapsed since admission to the emergency department, and (3) age older than 18 years. Exclusion criteria were
(1) patients extremely ill, in whom survival interval was expected to be less
than 48 hours, (2) use of any external
vitamin supplement within one week before enrollment, (3) onco-hematological, and (4) chronic renal failure patients. All patients were managed according to a
standard hemodynamic and respiratory algorithm [17].


## 2.2. Data Collection

Demographic data, diagnosis, the acute
physiology and chronic health evaluation (APACHE) II score, sepsis-related organ failure assessment (SOFA) score, hemodynamic and respiratory parameters, maximal vasoactive drug dose,
general biochemistry (renal, hematological, and hepatic function), and C
reactive protein (CPR) levels were
registered at admission, 24 and 72 hours, as well as the seventh day of
evolution.


## 2.3. Oxidative Stress Evaluation

Oxidative stress markers employed were thiobarbituric
acid reactive substances (TBARSs), an
index of lipid peroxidation, plus carbonyls, and methionine sulfoxide in plasma
proteins as markers of protein oxidative damage. Antioxidant activity was
evaluated by measuring: (a) total
antioxidant capacity (TAC) determined with two methods: (1) total radical- trapping
antioxidant potential (TRAP) and (2) ferric/reducing antioxidant power (FRAP); (b) nonenzymatic antioxidants: vitamins C and E, beta
carotene, and lycopene; (c) enzymatic antioxidant cofactors: reduced and total
red blood cell glutathione; and (d) nonspecific antioxidants: uric acid, bilirubin, and albumin. Blood samples were
obtained at diagnosis (T0), 24 hours (T1), 72 hours (T3), and at the seventh day of
evolution (T7). Delayed measurements
were made in all survivors 3 months later. Normal values for each parameter
were obtained from a group of 17 healthy volunteers matched for age and sex
with septic shock patients.


## 2.4. Laboratory Technique

All samples were stored at −20°C and were analyzed
within 10 days, but for glutathione analysis, samples were stored with ACD at
4°C and were also analyzed within 10 days.


### 2.4.1. Total Antioxidant Capacity (TAC)

TRAP (Total-Trapping Radical [Peroxyl] Antioxidant Potential)
One mL of a mixture of Luminol (60μM)-AAPH
(10 mM), in glycine buffer 50 mM pH:
9.40, kept in ice and darkness, was preincubated at 37°C. This mixture was
poured into a cuvette of polystyrene (Clinicon) and placed in a luminometer (BioOrbit 1250, Finland) kept at 37°C. When the light emission reached a steady-state condition, 10μL of plasma or standard (Trolox) were added. The luminescence was plotted
against the elapsed time. The induction time (tsample) was equated to the time required for the chemiluminescence intensity to reach a
level equal to 40% of the intensity measured prior to the sample addition [18]. The quenching of the light emission was recorded
in a Kipp & Zonen BD 111 recorder. The coefficient of variation for this measurement
is 4, 04%.FRAP (Ferric/Reducing Antioxidant Power)
Reduction at low pH of a ferric
tripyridyltriazine (FeIII-TPTZ) complex
to the ferrous form, by electron-donating antioxidants [19].
Samples were incubated in mixture of TPTZ, FeCl3, and acetate buffer (1 : 1 : 10) for 5 minutes at 37°C and quantified at 593 nm. The coefficient of variation for this measurement is 2,5%.


### 2.4.2. Vitamin E (α-Tocopherol), β-Carotene, and Lycopene

Lipid soluble antioxidant concentrations were
determined by HPLC with electrochemical detection [20].
Plasma samples were precipitated with methanol and extracted with hexane and
centrifuged. The organic phase was dried, and the residue was redissolved in methanol:
ethanol. An isocratic phase reverse chromatography was performed using a
Supelcosil C-8 column, and 20 mM LiClO4 in methanol:
H2O (97.5 : 2.5) as mobile phase. For electrochemical detection, an
amperometric BAS LC4C detector (Bioanalytical Systems Inc., West
Lafayette, Ind, USA) was used with
an applied potential of +0.6 V. The coefficient of variation for vitamin E is
1, 8%, for β-carotene is 9,3%, and for
lycopene is 8,2%.


### 2.4.3. Ascorbic Acid

Determinations were carried out by a method
based on the reduction of ferric chloride by ascorbic acid, with the resulting
ferrous ion quantified by the addition of 2-,4-,6-tripyridyl-s-triazine [21]. Heparinized plasma samples were precipitated
with trichloroacetic acid. The tubes were centrifuged at 20,800 g for 30 seconds.
An aliquot from the supernatant was incubated with TPTZ and FeCl3 in buffer
acetate for 5 minutes at 25°C, and quantified at 595 nm. The coefficient of
variation for this measurement is 3,4%.


### 2.4.4. Glutathione

Total Red Blood Cell Glutathione
Red blood cells were lysed and precipitated by
adding perchloric acid 0.28 M to fresh blood anticoagulated with ACD. An
aliquot from the supernatant, neutralized with K3PO4 1.75 M, was incubated with
phosphate buffer 0.1 mM (EDTA 1 mM) and 5.5′-dithiobis
(2-nitrobenzoic acid) 0.5 mM (sodium Citrate 1% p/v) for 7 minutes at 25°C and quantified
at 412 nm [22].Reduced Red Blood Cell Gluthatione
The cells were rinsed four times with 3 mL of ice cold
PBS. Collected cells were diluted in phosphate buffer containing diethylenetriaminepentaacetic
acid 1.34 mM pH 7.8. An aliquot was derivatized by the addition of N-(1-pyrenyl) maleimide 1.0 mM in acetonitrile. Solutions
were incubated for 5 minutes at room temperature and then acidified with HCl to
pH 2.5. An isocratic phase reverse chromatography was performed using a
Supelcosil C-18 column and 65% acetonitrile: 35% H2O, 1 mL/liter acetic acid,
and 1 mL/liter O-phosphoric acid as mobile phase. A fluorescence
spectrophotometer detector (Merck-Hitachi F-1000, Darznstadt, Germany)  was used for detection λex 330 nm and λem 375 nm [23].


### 2.4.5. TBARS

The method is based on the formation of an
adduct TBA-MDA (2 : 1). Plasma samples,
TBA solution (0.67%/NaOH 0.05N), and TCA 50% solution were placed in this
order into a screw-cap test tube and incubated at 90°C for 45 minutes. The
aqueous phase was quantified at 532 nm. TBARS plasmatic levels are expressed in
micromolar equivalent MDA [24].


### 2.4.6. Carbonyls

Plasma protein samples were reacted with
dinitrophenylhydrazine and then adsorbed to wells of an ELISA plate, overnight
at 4°C, before probing with a commercial antibody raised against protein-conjugated
DNP. The biotin-conjugated primary antibody (anti-DNP
Polyclonal IgG) was then reacted with streptavidin-conjugated
horseradish peroxidase for quantification with TMB. Acidic stop solution was
added, and absorbance measured at 450 nm. The method was calibrated using oxidized
and reduced albumin [25]. The
coefficient of variation for this measurement is 16, 2%.


### 2.4.7. Methionine Sulfoxide

Protein methionine sulfoxide content was
measured by HPLC-fluorometric detection, using a modification of the method of
Morgan etal. [26] and was expressed as
the fraction of total methionine oxidized to methionine sulfoxide: proteins
from 10μL plasma aliquots were precipitated with 60μL of ice-cold
acetonitrile and centrifuged at 10000 g for 20 minutes; the protein pellets
were hydrolyzed using methanesulfonic acid at 150°C for 90 minutes and were
derivatized with o-phthaldialdehyde/β-mercaptoethanol solution. Derivatized
amino acids were detected by an HPLC-fluorescence spectrophotometer detector (Merck-Hitachi F-1000), λex 340 nm, and λem 455 nm,
and quantified using standards containing methionine, methionine sulfoxide, and
methionine sulfone. Analysis was performed by reverse-phase HPLC using a
Synergy 4μM fusion column with a continuous gradient of mobile phase 75 mM Na
acetate buffer pH 4.5: methanol: tetrahydrofuran (80 : 19 : 1) initial to (20 : 80 : 0) final, in 40 minutes.
The coefficient of variation for this measurement is 9.2%.


## 2.4.1. Total Antioxidant Capacity (TAC)

TRAP (Total-Trapping Radical [Peroxyl] Antioxidant Potential)
One mL of a mixture of Luminol (60μM)-AAPH
(10 mM), in glycine buffer 50 mM pH:
9.40, kept in ice and darkness, was preincubated at 37°C. This mixture was
poured into a cuvette of polystyrene (Clinicon) and placed in a luminometer (BioOrbit 1250, Finland) kept at 37°C. When the light emission reached a steady-state condition, 10μL of plasma or standard (Trolox) were added. The luminescence was plotted
against the elapsed time. The induction time (tsample) was equated to the time required for the chemiluminescence intensity to reach a
level equal to 40% of the intensity measured prior to the sample addition [18]. The quenching of the light emission was recorded
in a Kipp & Zonen BD 111 recorder. The coefficient of variation for this measurement
is 4, 04%.FRAP (Ferric/Reducing Antioxidant Power)
Reduction at low pH of a ferric
tripyridyltriazine (FeIII-TPTZ) complex
to the ferrous form, by electron-donating antioxidants [19].
Samples were incubated in mixture of TPTZ, FeCl3, and acetate buffer (1 : 1 : 10) for 5 minutes at 37°C and quantified at 593 nm. The coefficient of variation for this measurement is 2,5%.


## 2.4.2. Vitamin E (α-Tocopherol), β-Carotene, and Lycopene

Lipid soluble antioxidant concentrations were
determined by HPLC with electrochemical detection [20].
Plasma samples were precipitated with methanol and extracted with hexane and
centrifuged. The organic phase was dried, and the residue was redissolved in methanol:
ethanol. An isocratic phase reverse chromatography was performed using a
Supelcosil C-8 column, and 20 mM LiClO4 in methanol:
H2O (97.5 : 2.5) as mobile phase. For electrochemical detection, an
amperometric BAS LC4C detector (Bioanalytical Systems Inc., West
Lafayette, Ind, USA) was used with
an applied potential of +0.6 V. The coefficient of variation for vitamin E is
1, 8%, for β-carotene is 9,3%, and for
lycopene is 8,2%.


## 2.4.3. Ascorbic Acid

Determinations were carried out by a method
based on the reduction of ferric chloride by ascorbic acid, with the resulting
ferrous ion quantified by the addition of 2-,4-,6-tripyridyl-s-triazine [21]. Heparinized plasma samples were precipitated
with trichloroacetic acid. The tubes were centrifuged at 20,800 g for 30 seconds.
An aliquot from the supernatant was incubated with TPTZ and FeCl3 in buffer
acetate for 5 minutes at 25°C, and quantified at 595 nm. The coefficient of
variation for this measurement is 3,4%.


## 2.4.4. Glutathione

Total Red Blood Cell Glutathione
Red blood cells were lysed and precipitated by
adding perchloric acid 0.28 M to fresh blood anticoagulated with ACD. An
aliquot from the supernatant, neutralized with K3PO4 1.75 M, was incubated with
phosphate buffer 0.1 mM (EDTA 1 mM) and 5.5′-dithiobis
(2-nitrobenzoic acid) 0.5 mM (sodium Citrate 1% p/v) for 7 minutes at 25°C and quantified
at 412 nm [22].Reduced Red Blood Cell Gluthatione
The cells were rinsed four times with 3 mL of ice cold
PBS. Collected cells were diluted in phosphate buffer containing diethylenetriaminepentaacetic
acid 1.34 mM pH 7.8. An aliquot was derivatized by the addition of N-(1-pyrenyl) maleimide 1.0 mM in acetonitrile. Solutions
were incubated for 5 minutes at room temperature and then acidified with HCl to
pH 2.5. An isocratic phase reverse chromatography was performed using a
Supelcosil C-18 column and 65% acetonitrile: 35% H2O, 1 mL/liter acetic acid,
and 1 mL/liter O-phosphoric acid as mobile phase. A fluorescence
spectrophotometer detector (Merck-Hitachi F-1000, Darznstadt, Germany)  was used for detection λex 330 nm and λem 375 nm [23].


## 2.4.5. TBARS

The method is based on the formation of an
adduct TBA-MDA (2 : 1). Plasma samples,
TBA solution (0.67%/NaOH 0.05N), and TCA 50% solution were placed in this
order into a screw-cap test tube and incubated at 90°C for 45 minutes. The
aqueous phase was quantified at 532 nm. TBARS plasmatic levels are expressed in
micromolar equivalent MDA [24].


## 2.4.6. Carbonyls

Plasma protein samples were reacted with
dinitrophenylhydrazine and then adsorbed to wells of an ELISA plate, overnight
at 4°C, before probing with a commercial antibody raised against protein-conjugated
DNP. The biotin-conjugated primary antibody (anti-DNP
Polyclonal IgG) was then reacted with streptavidin-conjugated
horseradish peroxidase for quantification with TMB. Acidic stop solution was
added, and absorbance measured at 450 nm. The method was calibrated using oxidized
and reduced albumin [25]. The
coefficient of variation for this measurement is 16, 2%.


## 2.4.7. Methionine Sulfoxide

Protein methionine sulfoxide content was
measured by HPLC-fluorometric detection, using a modification of the method of
Morgan etal. [26] and was expressed as
the fraction of total methionine oxidized to methionine sulfoxide: proteins
from 10μL plasma aliquots were precipitated with 60μL of ice-cold
acetonitrile and centrifuged at 10000 g for 20 minutes; the protein pellets
were hydrolyzed using methanesulfonic acid at 150°C for 90 minutes and were
derivatized with o-phthaldialdehyde/β-mercaptoethanol solution. Derivatized
amino acids were detected by an HPLC-fluorescence spectrophotometer detector (Merck-Hitachi F-1000), λex 340 nm, and λem 455 nm,
and quantified using standards containing methionine, methionine sulfoxide, and
methionine sulfone. Analysis was performed by reverse-phase HPLC using a
Synergy 4μM fusion column with a continuous gradient of mobile phase 75 mM Na
acetate buffer pH 4.5: methanol: tetrahydrofuran (80 : 19 : 1) initial to (20 : 80 : 0) final, in 40 minutes.
The coefficient of variation for this measurement is 9.2%.


## 2.5. Statistical Analysis

Changes along time for the different oxidative
stress markers were analyzed with linear mixed effects models. In addition,
measurements in septic shock patients at each time point were compared with
normal values from healthy matched subjects by at-test for independent
samples (samples exhibited a normal distribution).
Correlations between severity parameters and oxidative stress markers were made
with Pearson. Linear mixed effects models, adjusted by sex and age, were also
used to confirm the association between severity parameters and oxidative
stress. Results are expressed as mean ± SD, and a P<.05 was
considered significant. The proc MIXED of the SAS statistical program was used
for analysis.


## 3. Results

A total of 21 patients fulfilled inclusion/exclusion criteria and were studied. Average age for all septic shock patients
was 60 ± 20 years, APACHE II and SOFA scores at admission were22.5±6.6 and
11 ± 4.2, respectively. The main causes of septic shock were respiratory and
abdominal. Only 4 patients died (19%).
Demographic characteristics of the patients are summarized in Table 1.Table 1
Characteristics of the patients.
Age (years)60.2±20.7Gender (female/male)10/11APACHE II score22.5±6.6SOFA score at admission (T0)10.5±3.624 hours (T1)9.7 ± 472 hours (T3)7.5±4.77th day (T7)5.5±6.8Septic shock etiology (n
(%))Pneumonia6 (35.3)Abdominal7 (29.4)Urological4 (11.8)Others4 (23.5)C-Reactive protein levels at admission23.5±10.4Lactate levels at admission (mmol/l)4.8 ± 3Noradrenaline (Max.
dose (ug/kg/min))0.31±0.2ALI/ARDS (n (%))19 (88)Mechanical ventilation (n
(%))16 (70)ALI/ARDS, Acute lung injury/Acute
respiratory distress syndrome; APACHE II score, Acute physiology and chronic health
evaluation score II on ICU admission; SOFA score, Sequential organ failure assessment.
Values reported are mean ± SD
orn (%).


### 3.1. Oxidative Damage

At admission, all septic shock patients
presented evidence of oxidative damage on lipids and proteins, measured either
by carbonyls or methionine sulfoxide (Figure1). TBARS levels were increased
twofold at admission compared to normal values and increased further during the
first week of evolution (P<.001) (Figure 1(a)). At 3 months, TBARS
levels had normalized in septic shock survivors. Oxidative damage on proteins
was also present at admission. Carbonyls were increased 4.5-fold and
methionine sulfoxide 2.1-fold compared to normal values (Figures 1(b) and 1(c)).
Thereafter, both markers tended to decrease during the first week of evolution
(P<.01 for both), however they did not reach normal levels, even
after three months.Temporal evolution of
oxidative damage of all septic shock patients in (a) lipoperoxidation (TBARS), (b) carbonyls, and (c) Methionine sulfoxide. Normal values (mean ± SD) obtained from matched healthy subjects are
shown as continuous and dotted lines, respectively.p: reflects variation in time
calculated by linear mixed effects model. *Indicates significant
differences between normal values and patients at different time points (t-test
for independent samples).
(a)(b)(c)


### 3.2. Antioxidant Activity

#### 3.2.1. Total Antioxidant Capacity (TAC)

TAC measured by TRAP was normal at admission
and day 1 (354 ± 123 versus 361 ± 50μM EqTlx for normal values), but decreased
after day 3 (246 ± 110μM EqTlx, P<.001). At three months, TRAP levels
remained decreased (279 ± 48μM EqTlx, p<0.01).
TAC levels measured by FRAP were elevated at admission (1656 ± 498 versus 1120
± 47μM EqFe2+ for normal values, P<.001) and remained increased
throughout the study period.


#### 3.2.2. Individual Antioxidants

Vitamin C, beta carotene, and lycopene levels
were significantly decreased at admission and continued to decrease
progressively throughout the first week of evolution (P<.001) (Figure 2(a) for vitamin C). In contrast,
vitamin E levels remained normal at all time points (Figure 2(b)). At 3 months, vitamin C and lycopene normal
levels were recovered, but not yet beta carotene.Evolution of antioxidant
levels in all septic shock patients of (a) Vitamin C, (b) Vitamin E (alpha-Tocopherol),
and (c) reduced and oxidized glutathione. Normal values (mean ± SD) obtained from matched healthy subjects are
shown as continuous and dotted lines, respectively.p: reflects variation in time
calculated by linear mixed effects model. *Indicates significant
differences between normal values and patients at different time points (t-test
for independent samples).
(a)(b)(c)Reduced glutathione levels were normal at
admission, but decreased after 24 hours and during the first week of evolution
(P<.001). In contrast, total red blood cell glutathione levels were
increased three-fold at admission and significantly increased during the first
week of evolution (P<.001). At 3 months, both parameters had returned
to normal values (Figure 2(c)).Uric acid levels were increased at admission
(6.7±2.3 versus 5 ± 0.5 mg/dL for normal values, P<.01), but
returned progressively to normal values within the first week. Bilirubin levels
were also increased at admission (2.3±2.1 versus 0.8±0.5 mg/dL for normal
values, P<.001) and did not change during evolution. A positive
correlation between uric acid levels and TRAP and FRAP levels was observed
(r: 0.83 and 0.47, resp., P<.03 for both) (Figure 3) and also
between bilirrubin levels and FRAP levels (r: 0.75, P<.4 with FRAP, P<.001).Figure 3
Correlations of peak FRAP and TRAP levels with peak uric acid levels.P<.05 for both correlations exposed.


### 3.3. Oxidative Stress and Severity Scores

SOFA scores decreased significantly along time
in survivors, mainly due to a rapid decrease in cardiovascular and respiratory
components of SOFA scores. Patients who died had higher levels of APACHE II and
SOFA scores at admission and peak and also had higher lactate levels and peak doses
of vasoactive drugs (P<.05 for all). C-reactive protein (CRP) levels were elevated at admission (23.5±10.4
mg/dL versus 0.6±0.3 for normal values, P<.001) and decreased during
septic shock evolution, but persisted still elevated at day 7. Their levels
were normal at 3 months.


### 3.4. At Admission

A positive correlation of admission SOFA score
with FRAP (Figure4), vitamin C, and uric acid admission levels was observed
(r: 0.63, r: 0.48, and r: 0.48, resp., P<.05 for all). APACHE II
score was correlated with FRAP, TRAP, B-carotene, and uric acid admission
levels (r: 0.5, r: 0.6, r: 0.5, and r: 0.56, resp., P<.05 for
all), whereas lactate admission levels were correlated with FRAP (Figure 4) and
bilirubin admission levels (r: 0.48 and 0.64, P<.05 for both). A
negative correlation was observed between oxygenation (PaO2/FiO2
ratio) and FRAP, TRAP, vitamin C, and uric acid levels at
admission (r: −0.44, r: −0.68, r: −0.58, and r: −0.48, resp., P<.05
for all). No significant correlations were observed between lipid or protein
oxidative damage and severity of illness at admission.Figure 4
Admission correlations of peak SOFA score and peak lactate levels with peak FRAP levels.P<.05 for the correlation exposed.


### 3.5. During the Evolution of Shock

TBARS peak levels exhibited a positive
correlation with peak SOFA score and peak lactate levels (r: 0.52 and r: 0.46, P<.05
for both) (Figure 5(a)). TBARS peak levels were also positively correlated with
peak FRAP (r: 0.57, P<.05). No correlations or associations were
observed between protein oxidative damage and antioxidant consumption or
severity of disease at admission or during septic shock evolution.Evolution correlations of peak SOFA score and peak lactate levels with (a) peak TBARS levels and (b) peak FRAP levels.P<.05 for all correlations exposed.
(a)(b)Peak SOFA score was also correlated with peak
levels of FRAP (Figure5(b)), TRAP, bilirubin, and vitamin C levels (r: 0.82,
r: 0.82, r: 0.6, and r: 0.56, resp., P<.05 for all). APACHE II
score was correlated with peak levels of FRAP, TRAP, uric acid, and vitamin C
levels (r: 0.67, r: 0.66, r: 0.45, and r: 0.6, resp., P<.05 for
all). Peak lactate levels were correlated with FRAP (Figure 5(b)) and bilirubin
peak levels (r: 0.66 and r: 0.65, P<.05 for both). All correlations
presented above were confirmed as positive associations by linear mixed effects
model in time.


## 3.1. Oxidative Damage

At admission, all septic shock patients
presented evidence of oxidative damage on lipids and proteins, measured either
by carbonyls or methionine sulfoxide (Figure1). TBARS levels were increased
twofold at admission compared to normal values and increased further during the
first week of evolution (P<.001) (Figure 1(a)). At 3 months, TBARS
levels had normalized in septic shock survivors. Oxidative damage on proteins
was also present at admission. Carbonyls were increased 4.5-fold and
methionine sulfoxide 2.1-fold compared to normal values (Figures 1(b) and 1(c)).
Thereafter, both markers tended to decrease during the first week of evolution
(P<.01 for both), however they did not reach normal levels, even
after three months.Temporal evolution of
oxidative damage of all septic shock patients in (a) lipoperoxidation (TBARS), (b) carbonyls, and (c) Methionine sulfoxide. Normal values (mean ± SD) obtained from matched healthy subjects are
shown as continuous and dotted lines, respectively.p: reflects variation in time
calculated by linear mixed effects model. *Indicates significant
differences between normal values and patients at different time points (t-test
for independent samples).
(a)(b)(c)


## 3.2. Antioxidant Activity

### 3.2.1. Total Antioxidant Capacity (TAC)

TAC measured by TRAP was normal at admission
and day 1 (354 ± 123 versus 361 ± 50μM EqTlx for normal values), but decreased
after day 3 (246 ± 110μM EqTlx, P<.001). At three months, TRAP levels
remained decreased (279 ± 48μM EqTlx, p<0.01).
TAC levels measured by FRAP were elevated at admission (1656 ± 498 versus 1120
± 47μM EqFe2+ for normal values, P<.001) and remained increased
throughout the study period.


### 3.2.2. Individual Antioxidants

Vitamin C, beta carotene, and lycopene levels
were significantly decreased at admission and continued to decrease
progressively throughout the first week of evolution (P<.001) (Figure 2(a) for vitamin C). In contrast,
vitamin E levels remained normal at all time points (Figure 2(b)). At 3 months, vitamin C and lycopene normal
levels were recovered, but not yet beta carotene.Evolution of antioxidant
levels in all septic shock patients of (a) Vitamin C, (b) Vitamin E (alpha-Tocopherol),
and (c) reduced and oxidized glutathione. Normal values (mean ± SD) obtained from matched healthy subjects are
shown as continuous and dotted lines, respectively.p: reflects variation in time
calculated by linear mixed effects model. *Indicates significant
differences between normal values and patients at different time points (t-test
for independent samples).
(a)(b)(c)Reduced glutathione levels were normal at
admission, but decreased after 24 hours and during the first week of evolution
(P<.001). In contrast, total red blood cell glutathione levels were
increased three-fold at admission and significantly increased during the first
week of evolution (P<.001). At 3 months, both parameters had returned
to normal values (Figure 2(c)).Uric acid levels were increased at admission
(6.7±2.3 versus 5 ± 0.5 mg/dL for normal values, P<.01), but
returned progressively to normal values within the first week. Bilirubin levels
were also increased at admission (2.3±2.1 versus 0.8±0.5 mg/dL for normal
values, P<.001) and did not change during evolution. A positive
correlation between uric acid levels and TRAP and FRAP levels was observed
(r: 0.83 and 0.47, resp., P<.03 for both) (Figure 3) and also
between bilirrubin levels and FRAP levels (r: 0.75, P<.4 with FRAP, P<.001).Figure 3
Correlations of peak FRAP and TRAP levels with peak uric acid levels.P<.05 for both correlations exposed.


## 3.2.1. Total Antioxidant Capacity (TAC)

TAC measured by TRAP was normal at admission
and day 1 (354 ± 123 versus 361 ± 50μM EqTlx for normal values), but decreased
after day 3 (246 ± 110μM EqTlx, P<.001). At three months, TRAP levels
remained decreased (279 ± 48μM EqTlx, p<0.01).
TAC levels measured by FRAP were elevated at admission (1656 ± 498 versus 1120
± 47μM EqFe2+ for normal values, P<.001) and remained increased
throughout the study period.


## 3.2.2. Individual Antioxidants

Vitamin C, beta carotene, and lycopene levels
were significantly decreased at admission and continued to decrease
progressively throughout the first week of evolution (P<.001) (Figure 2(a) for vitamin C). In contrast,
vitamin E levels remained normal at all time points (Figure 2(b)). At 3 months, vitamin C and lycopene normal
levels were recovered, but not yet beta carotene.Evolution of antioxidant
levels in all septic shock patients of (a) Vitamin C, (b) Vitamin E (alpha-Tocopherol),
and (c) reduced and oxidized glutathione. Normal values (mean ± SD) obtained from matched healthy subjects are
shown as continuous and dotted lines, respectively.p: reflects variation in time
calculated by linear mixed effects model. *Indicates significant
differences between normal values and patients at different time points (t-test
for independent samples).
(a)(b)(c)Reduced glutathione levels were normal at
admission, but decreased after 24 hours and during the first week of evolution
(P<.001). In contrast, total red blood cell glutathione levels were
increased three-fold at admission and significantly increased during the first
week of evolution (P<.001). At 3 months, both parameters had returned
to normal values (Figure 2(c)).Uric acid levels were increased at admission
(6.7±2.3 versus 5 ± 0.5 mg/dL for normal values, P<.01), but
returned progressively to normal values within the first week. Bilirubin levels
were also increased at admission (2.3±2.1 versus 0.8±0.5 mg/dL for normal
values, P<.001) and did not change during evolution. A positive
correlation between uric acid levels and TRAP and FRAP levels was observed
(r: 0.83 and 0.47, resp., P<.03 for both) (Figure 3) and also
between bilirrubin levels and FRAP levels (r: 0.75, P<.4 with FRAP, P<.001).Figure 3
Correlations of peak FRAP and TRAP levels with peak uric acid levels.P<.05 for both correlations exposed.


## 3.3. Oxidative Stress and Severity Scores

SOFA scores decreased significantly along time
in survivors, mainly due to a rapid decrease in cardiovascular and respiratory
components of SOFA scores. Patients who died had higher levels of APACHE II and
SOFA scores at admission and peak and also had higher lactate levels and peak doses
of vasoactive drugs (P<.05 for all). C-reactive protein (CRP) levels were elevated at admission (23.5±10.4
mg/dL versus 0.6±0.3 for normal values, P<.001) and decreased during
septic shock evolution, but persisted still elevated at day 7. Their levels
were normal at 3 months.


## 3.4. At Admission

A positive correlation of admission SOFA score
with FRAP (Figure4), vitamin C, and uric acid admission levels was observed
(r: 0.63, r: 0.48, and r: 0.48, resp., P<.05 for all). APACHE II
score was correlated with FRAP, TRAP, B-carotene, and uric acid admission
levels (r: 0.5, r: 0.6, r: 0.5, and r: 0.56, resp., P<.05 for
all), whereas lactate admission levels were correlated with FRAP (Figure 4) and
bilirubin admission levels (r: 0.48 and 0.64, P<.05 for both). A
negative correlation was observed between oxygenation (PaO2/FiO2
ratio) and FRAP, TRAP, vitamin C, and uric acid levels at
admission (r: −0.44, r: −0.68, r: −0.58, and r: −0.48, resp., P<.05
for all). No significant correlations were observed between lipid or protein
oxidative damage and severity of illness at admission.Figure 4
Admission correlations of peak SOFA score and peak lactate levels with peak FRAP levels.P<.05 for the correlation exposed.


## 3.5. During the Evolution of Shock

TBARS peak levels exhibited a positive
correlation with peak SOFA score and peak lactate levels (r: 0.52 and r: 0.46, P<.05
for both) (Figure 5(a)). TBARS peak levels were also positively correlated with
peak FRAP (r: 0.57, P<.05). No correlations or associations were
observed between protein oxidative damage and antioxidant consumption or
severity of disease at admission or during septic shock evolution.Evolution correlations of peak SOFA score and peak lactate levels with (a) peak TBARS levels and (b) peak FRAP levels.P<.05 for all correlations exposed.
(a)(b)Peak SOFA score was also correlated with peak
levels of FRAP (Figure5(b)), TRAP, bilirubin, and vitamin C levels (r: 0.82,
r: 0.82, r: 0.6, and r: 0.56, resp., P<.05 for all). APACHE II
score was correlated with peak levels of FRAP, TRAP, uric acid, and vitamin C
levels (r: 0.67, r: 0.66, r: 0.45, and r: 0.6, resp., P<.05 for
all). Peak lactate levels were correlated with FRAP (Figure 5(b)) and bilirubin
peak levels (r: 0.66 and r: 0.65, P<.05 for both). All correlations
presented above were confirmed as positive associations by linear mixed effects
model in time.


## 4. Discussion

Our study prospectively evaluated the temporal
profile of antioxidants and oxidative damage markers during the first week of
evolution of septic shock and their correlation with severity. We found
evidence of early oxidative damage on proteins and lipids associated with
severe vitamin C,β-carotene, lycopene, and reduced glutathione depletion, but not
associated with reduced TAC or vitamin E levels. Early production of reactive
oxygen species has been documented in endotoxin models [14], and clinical
studies also have found that critical care patients already present evidence of
oxidative stress at admission [2, 7–9, 27, 28]. Therefore,
oxidative damage is a rather early phenomenon of the systemic inflammatory
response, including septic shock, taking place even before septic patients
arrive to critical care units. This notion may in part explain why
interventions which have been shown to be effective to prevent oxidation [29] may not be effective to revert an already
established oxidative damage in the ICU setting.We also found that lipoperoxidation increases
along time. This may be the result of an initial insult, most probably
ischemic, followed by an independent perpetuating process, which could be explained
by the autocatalytic nature of the lipoperoxidation cascade, by persistent
immune activation, by continuous episodes of reperfusion, or by insufficient or
ineffective antioxidation. Protein oxidative damage, in contrast, significantly
decreases along time in parallel with the decrease of SOFA score. Decreasing
levels of protein oxidative damage have been described before [30] and since we expressed protein oxidative markers
as a proportion of total plasma proteins, changes in carbonyls and methionine
sulfoxide levels should not be influenced by changes in total body proteins in
our study and may truly reflect that protein oxidative damage is maximal early
in the septic process.Lipoperoxidation was positively correlated to
septic shock severity and to organ dysfunction estimated by SOFA, suggesting
that oxidative damage may have a role in the development of multiorgan
dysfunction. Recently, Motoyama etal. [8] in SIRS patients also found that higher levels of TBARS at admission and in
time were associated with the development of multiorgan failure (MOF). In contrast, we did not find any significant
correlation between protein oxidative damage and septic shock severity at any
time point. Since several critical proteins have been described to be partially
or completely inactivated by the action  of reactive oxygen species during
sepsis, suggesting that oxidative damage over proteins could have a role in
sepsis [31, 32], it is possible that carbonyls
and methionine sulfoxide, although sensitive markers of protein damage [33, 34], do not reflect the real impact of oxidative
stress over critical proteins and enzymes.TAC levels exhibited different kinetics along
time according to the method used to estimate them. These differences could be
explained looking at the individual contributors to serum TAC levels, which are
represented in different proportions according to the method used to estimate
TAC. Particularly in septic shock, TRAP levels are strongly influenced by uric
acid levels (r: 0.83), explaining the
parallel decrease of both markers along time. In contrast, FRAP levels are less
dependent on uric acid, but may be also influenced by bilirubin and other
nonidentified compounds, which may explain the increase in FRAP levels at
admission and during the study period [35].
Both parameters, TRAP and FRAP and their major contributors, uric acid and bilirubin,
were positively correlated with septic shock severity. Previous studies have
already suggested that TAC levels may be higher in more severe forms of sepsis
[15, 35, 36]. In particular, elevations of uric acid during sepsis can be
explained by an increase in uric acid production associated with activation of
Xantine oxidase (XO) in ischemic
territories during sepsis and septic shock [2].Vitamin C levels were below normal values
during the study period, probably explained by ongoing consumption. Although we
expected to observe larger consumption in the most severe patients, we found
the opposite; more severe septic patients had higher vitamin C levels. We
speculate that lower consumption of vitamin C observed in most severe patients
might be explained by larger availability of other nonenzymatic antioxidants in
these patients, namely, uric acid and bilirubin, both of which may act as
effective antioxidants in plasma [37, 38].
Interestingly, even though most severe patients exhibited higher TAC and
vitamin C levels, these were also positively correlated to peak TBARS
indicating that lipoperoxidation may occur despite normal or even increased antioxidant
capacity.At 3 months, lipoperoxidation levels normalized
in septic shock survivors, whereas protein oxidative markers still remained
elevated. Persistent elevation of carbonyls and methionine sulfoxide is difficult
to explain. Ongoing oxidative stress is unlikely since CRP levels were normal
at three months, and most patients were already doing well at home. A slow
protein turnover would explain that 3 months were insufficient to reach normal
levels of these markers [34]. This
finding suggests that these indexes could be used as markers of a previous
oxidative stress situation.A limitation of our study was the exclusion of
some patients that were extremely ill and were expected to die before 48 hours,
patients admitted from the ward and chronic renal failure patients. These
patients may present a different antioxidant—oxidative scenario. However, during the study
period, from all consecutive septic shock patients admitted to our unit only 5
were excluded. These exclusion criteria may partially explain the 19% mortality,
which is lower than predicted from SOFA and APACHE II scores. Our reported
mortality for septic shock is 33% [17].


## 5. Conclusions

Our study shows early and persistent oxidative
stress during septic shock, reflected on lipid and protein damage and on nonenzymatic
antioxidant apparent consumption. Protein oxidation reaches its peak early at
admission, but lipoperoxidation continues to increase during the first days of
evolution. More severe patients exhibit higher levels of lipoperoxidation but
also higher levels of total antioxidant capacity, uric acid, bilirubin, and
vitamin C. We showed for the first time that methionine sulfoxide levels are
increased in septic shock patients and, therefore, may be used as a sensitive
marker of protein oxidation in sepsis.



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*Source: 168652-2008-06-16.xml*