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
(
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 [
Several studies have shown the presence of
oxidative stress in sepsis [
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 [
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.
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.
Patients were enrolled if they fulfilled the
following criteria: (1) diagnosis of septic shock according to the consensus
conference [
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.
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.
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.
One mL of a mixture of Luminol (60
Reduction at low pH of a ferric
tripyridyltriazine (FeIII-TPTZ) complex
to the ferrous form, by electron-donating antioxidants [
Lipid soluble antioxidant concentrations were
determined by HPLC with electrochemical detection [
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 [
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 [
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
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 [
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 [
Protein methionine sulfoxide content was
measured by HPLC-fluorometric detection, using a modification of the method of
Morgan etal. [
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 a
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 were
Characteristics of the patients.
Age (years) | |
Gender (female/male) | 10/11 |
APACHE II score | |
SOFA score at admission (T0) | |
9.7 ± 4 | |
Septic shock etiology ( | |
Pneumonia | 6 (35.3) |
Abdominal | 7 (29.4) |
Urological | 4 (11.8) |
Others | 4 (23.5) |
C-Reactive protein levels at admission | |
Lactate levels at admission (mmol/l) | 4.8 ± 3 |
Noradrenaline (Max. dose (ug/kg/min)) | |
ALI/ARDS ( | 19 (88) |
Mechanical ventilation ( | 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
or
At admission, all septic shock patients
presented evidence of oxidative damage on lipids and proteins, measured either
by carbonyls or methionine sulfoxide (Figure
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.
TAC measured by TRAP was normal at admission
and day 1 (354 ± 123 versus 361 ± 50
Vitamin C, beta carotene, and lycopene levels
were significantly decreased at admission and continued to decrease
progressively throughout the first week of evolution (
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.
Reduced glutathione levels were normal at
admission, but decreased after 24 hours and during the first week of evolution
(
Uric acid levels were increased at admission
(
Correlations of peak FRAP and TRAP levels with peak uric acid levels.
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 (
A positive correlation of admission SOFA score
with FRAP (Figure
Admission correlations of peak SOFA score and peak lactate levels with peak FRAP levels.
TBARS peak levels exhibited a positive
correlation with peak SOFA score and peak lactate levels (
Evolution correlations of peak SOFA score and peak lactate levels with (a) peak TBARS levels and (b) peak FRAP levels.
Peak SOFA score was also correlated with peak
levels of FRAP (Figure
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,
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 [
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. [
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 (
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 [
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 [
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% [
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.