ORIGINAL ARTICLE

Mitochondrial Dysfunction, Persistent Oxidative Damage,and Catalase Inhibition in Immune Cells of Naıve andTreated Crohn’s Disease

Bel�en Beltr�an, MD, PhD,*,† Pilar Nos, MD, PhD,*,† Francisco Dası, PhD,‡ Marisa Iborra, MD,§

Guillermo Bastida, MD,*,† Marcial Martınez, PhD,k Jos�e-Enrique O’Connor, PhD,¶

Guillermo S�aez, MD, PhD,** In�es Moret, PhD,‡ and Julio Ponce, MD, PhD*,†

Background: Oxidative stress is considered a potential etiologi-

cal factor for Crohn’s disease (CD). We characterized the reactive

oxygen species (ROS) generated in immune peripheral cells of

CD patients, as well as their antioxidant enzyme status and the

presence of oxidative damage. In addition, mitochondrial function

(DWm) was analyzed to detect the possible origin of ROS.

Methods: Cells were obtained from patients at the onset of dis-

ease, prior to any treatment. Experiments were repeated when

patients were in clinical remission. A set of experiments was car-

ried out in a group of CD patients in persistent morphological

remission. Controls were healthy volunteers who were not receiv-

ing any treatment at the time. The generation of superoxide,

hydrogen peroxide (H2O2) and nitric oxide, DWm, superoxide dis-

mutase (SOD) and catalase (CAT) activities, and concentrations

of malondyaldehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-

dG) were measured.

Results: SOD activity and H2O2 production were significantly

higher during active CD but returned to control levels in remis-

sion. DWm was inhibited during active CD and, although it

returned to control levels, its recovery took longer than clinical

remission. CAT activity was permanently inhibited during CD, in-

dependent of the disease activity. MDA and 8-oxo-dG were per-

manently elevated.

Conclusions: Oxidative stress during active CD depends on

H2O2 production. The inhibition of DWm suggests that this organ-

elle is a source of ROS. CAT is permanently inhibited in CD, the

biological significance of which is under study. The persistent

oxidative damage detected may have implications for the evolu-

tion of the disease.

(Inflamm Bowel Dis 2010;16:76–86)

Key Words: Crohn’s disease, oxidative stress, mitochondria,Catalase, antioxidant enzymes

T he cause of Crohn’s disease (CD) remains unclear.1

The specific pathways leading to mucosa damage are

not completely understood. The role of oxidative stress as

a potential etiological and/or triggering factor for inflam-

matory bowel disease (IBD) has been the subject of

increasing interest.2,3 The activated immune cells that reach

the mucosa in IBD release a number of reactive oxygen

species (ROS) that are potentially detrimental, especially

when the antioxidant defenses are overwhelmed and oxida-

tive stress is established.4 In fact, evidence of epithelial

cell injury secondary to ROS has been detected in the mu-

cosa of IBD patients.5,6 However, excessive levels of oxi-

dized molecules in IBD are not only detected in the mu-

cosa but in a variety of organic systems (plasma, serum,

saliva, exhaled air).7,8 Similarly, the expression of antioxi-

dants in IBD patients has been analyzed by several

reports7–10 that have detected an imbalance in antioxidant

concentration, thus confirming the presence of oxidative

stress in the vital organs of IBD patients. Furthermore, the

capacity of the peripheral immune cells of IBD patients to

produce ROS is known to be altered, and this alteration is

thought to imply an altered immune response that occurs

before the cells reach the intestinal mucosa.11,12

Received for publication April 6, 2009; Accepted May 14, 2009.

From the *Department of Gastroenterology, Hospital Universitari La Fe,

Valencia, †Centro de Investigacion Biomedica en Red de Enfermedades

Hepaticas y Digestivas (CIBEREHD), ‡Fundacion Investigacion Hospital

Clınico Universitario de Valencia, §Fundacion Investigacion Hospital La Fe,

Hospital Universitari La Fe, kFlow Cytometry Unit, Service of Clinical

Analysis, Hospital Universitari La Fe, Valencia, ¶Laboratory of Cytomics,

Mix Unit CIPF-UVEG, Centro de Investigacion Prıncipe Felipe, Valencia,

**Department of Biochemistry and Molecular Biology, Faculty of Medicine,

General University Hospital of Valencia, Valencia, Spain.

Reprints: Bel�en Beltr�an, MD, PhD, Department of Gastroenterology,

Hospital Universitari La Fe, Avda Campanar 21, 46009 Valencia, Spain

(e-mail: beltran_belen@yahoo.es).

Supported by public funding from the Fondo de Investigaciones

Sanitarias (Instituto de Salud Carlos III) with grants: CP05/0272, FIS

IP05/017, FIS IP06/0730. CIBEREHD (Centro de investigacion biomedica

en red sobre Hepatologıa y Gastroenterologia) is also funded by the

Instituto de Salud Carlos III.

Copyright VC 2009 Crohn’s & Colitis Foundation of America, Inc.

DOI 10.1002/ibd.21027

Published online 27 July 2009 in Wiley InterScience (www.interscience.

wiley.com).

Inflamm Bowel Dis � Volume 16, Number 1, January 201076

The presence of ROS in IBD has been measured

indirectly according to levels of oxidatively damaged mole-

cules. Little work has been carried out to characterize the

ROS directly implicated in damage.13 Similarly, the possi-

ble sources of ROS in IBD have received little attention.

Mitochondria are key organelles considered an important

source of ROS after dysfunction or inhibition of their respi-

ratory chain. Mitochondria, through production of these

ROS, have been implicated in the regulation of autoim-

munity, apoptosis, and autophagy.14 The status of mito-

chondrial function has never been characterized in IBD,

although recent reports refer to morphological changes in

the mitochondria of the ileal enterocites of patients with

IBD.15 Furthermore, a possible role of the mitochondria in

the pathogenesis of IBD has been reported in a clinical

case.16 Finally, many studies have measured either ROS4,8

or antioxidant capacities9–11 in order to evaluate the oxida-

tive stress in IBD. However, it is important to measure

both ROS levels and the antioxidant status of the cell in

order to avoid the biased conclusions based on the consid-

eration of only 1 side of the balance.3

The purpose of the present study was to characterize

the ROS species that are generated in immune peripheral

cells in patients with CD (active and inactive), as well as

the enzyme-dependent antioxidant status of the cells and

the establishment of oxidative damage. Furthermore, we

analyzed the mitochondrial function of immune cells in

order to study their possible implication in the generation

of ROS. We performed the experiments in cells from

patients at the onset of disease, prior to initiating treatment,

in order to avoid the bias that medication could exert with

respect to ROS generation. We studied the active and inac-

tive status of CD in the same patient with the aim of char-

acterizing how the production of ROS and oxidative dam-

age varies with activity. Knowing the oxidative status of

peripheral immune cells will help to understand their func-

tionality to deal with inflammation when they reach the gut

mucosa.

MATERIALS AND METHODS

Patients and ControlsTwenty-five consecutive CD patients at onset of dis-

ease (diagnosed according to Lennard-Jones criteria17) and

yet to begin any specific medication were included in the

study. Patients with doubtful diagnoses, those suffering

from another disease, and those taking any form of medica-

tion were excluded from the study. Patients were classified

according to Montreal Criteria18 and disease activity was

scored with the Harvey–Bradshaw index.19 The experi-

ments were carried out at the onset of disease (active

patients), and were repeated when clinical remission was

achieved (defined as a disease activity index less than or

equal to 4) (remission patients). A total of 19 patients were

analyzed in remission. It was not possible to repeat the

experiments in 6 patients for various reasons. The control

group consisted of 25 healthy volunteers who were not tak-

ing any medication at the time.

A set of experiments was carried out in a group of

patients diagnosed with CD over the 5 previous years and

who were in persistent clinical remission (Harvey <4 for

more than 6 months) and had no morphological lesions

(n ¼ 20) (inactive patients). Mucosal healing was evaluated

by colonoscopy or capsulendoscopy according to disease

localization.

All the subjects gave their written informed consent.

On the day of recruitment for the study a complete hemato-

logical and biochemistry blood analysis, including acute

phase reactants, was performed. Only those subjects with a

completely normal blood test result were included as con-

trols. The study was approved by the Ethical Committee of

the Hospital Universitari La Fe and complied with the Dec-

laration of Helsinki.

Chemicals and InstrumentsConjugated antibodies CD4.PE/CY5, CD3.FITC, and

CD3.ECD were from Immunotech (Marseille, France). His-

topaque 1007, dimethylsulfoxide (DMSO), t-butylhydroper-

oxide (TBH), N-[2-hydroxyethyl]piperazine-N0-[2-ethane-

sulfonic acid] (HEPES) buffer, 20,70dichlorofluorescein

diacetate (DCFH), dihydroethidium (HE), plumbagin (PB),

and Carbonyl cyanide 4-(trifluoromethoxy) phenylthydra-

zone (FCCP), and diethylthiocarbamate (DETC) were

obtained from Sigma-Aldrich (St. Louis, MO). RPMI 1640

culture medium, L-glutamine, penicillin-streptomycin, and

fetal bovine serum were from Gibco (Grand Island, NY).

Tetramethylrhodamine methyl ester perchlorate (TMRM)

was purchased from Fluka (Buchs, Switzerland). 3-

Amino,4-aminomethyl-2070-difluorofluorescein (DAF2-DA)

was from Calbiochem (La Jolla, CA). Bioxytech Catalase-

520 and Bioxytech SOD-525 were from OxysResearch

(Foster City, CA).

Isolation of Peripheral LeukocytesAnticoagulated blood (K3-EDTA) was obtained from

patients and healthy volunteers after 12 hours fasting. Leu-

kocytes were isolated by the Histopaque 1077 gravity tech-

nique. This consisted of layering 3 mL whole blood over 8

mL of Histopaque 1077 (Sigma-Aldrich) in a 15-mL plastic

tube. After 45–60 minutes of gravity sedimentation at room

temperature, the white cell-rich plasma (upper layer) was

removed for the experiment. White cells were diluted 1:0.5

v/v with RPMI 1640 culture medium (Gibco) supplemented

with 10% fetal bovine serum, penicillin (100 U/mL), and

streptomycin (1009 lg/mL) and 1� glutamine, and were

Inflamm Bowel Dis � Volume 16, Number 1, January 2010 Mitochondria and Oxidative Status in CD

77

incubated at 37�C until the experiment was carried out.

After counting the cells, leukocytes were adjusted to a

concentration of 500,000 cells/mL with RPMI.

Cytometric MeasurementsCytometric assays were carried out in an EPICS-XL-

MCL flow cytometer (Beckman-Coulter, Fullerton, CA).

System II software (Beckman-Coulter) was used for data

acquisition. Results are expressed as mean fluorescence in-

tensity in arbitrary units. Leukocytes were gated for lym-

phocytes, monocytes, or granulocytes according to their

morphology and leukocyte lineage markers using fluoro-

chrome-labeled monoclonal antibodies. Thus, CD3-FITC,

or CD3-ECD and CD4-PC5 staining together with forward

scatter and side scatter signals defined the criteria to gate

out other cells and debris. Accordingly, T-helper lympho-

cytes were identified as CD3þ/CD4þ cells, monocytes as

CD3�/CD4þ cells and granulocytes were identified exclu-

sively by their morphology.

O22, H2O2, and NO Production Assessment

Our assay consisted of taking an aliquot of cells (150

lL) and incubating them in darkness for 10 minutes at

37�C with CD3 (CD3-FITC, 5 lL) and CD4 (CD4-PC5,

5 lL) markers for the protocol of O2� production assess-

ment and with CD3 (CD3-ECD, 5 lL) and CD4 (anti-

CD4-PC5, 5 lL) markers for the protocols of H2O2 and

NO detection. Once the cells were stained, 850 lL of

HEPES was added and the sample was run in the cytome-

ter to achieve the blank values of each immune cell

population.

O2� assay was based on dyhydroethidium (HE) oxi-

dation.20 When oxidized, HE exhibits red fluorescence

(absorption/emission: 518/605 nm). HE was added at a

final concentration of 5 lM, incubated for 1 hour, and the

fluorescence generated measured by flow cytometry. Once

the basal production of O2� had been achieved, a 2.5 mM

dose of PB, a superoxide generator,21 was added to the

sample in order to induce a peak of O2�.

The assay to detect H2O2 was based on DCFH oxida-

tion. This probe has low basal fluorescence but, when oxi-

dized by H2O2 in the presence of peroxidase, it is con-

verted to 2070-dichlorofluorescein (DCF) with a high

fluorescent component at 520 nm.22 DCFH was added at a

final concentration of 20 lM, incubated for 1 hour, and the

fluorescence generated measured by flow cytometry. Once

basal production of H2O2 had been achieved, a 10 mM

dose of t-butyl-hydroperoxyde (TBH), a hydrogen peroxide

generator, was added to the sample in order to induce a

peak of H2O2.

The production of NO was assayed using the fluores-

cent probe 4,5-diaminofluorescein diacetate (DAF-2/DA),

as described elsewhere.23 DAF-2/DA was added at a final

concentration of 5 lM, incubated for 1 hour, and the green

fluorescence generated measured by flow cytometry.

Mitochondrial Membrane Potential (DWm)DWm was assessed using the cationic-lipophilic tetra-

methylrodhodamine methyl ester (TMRM), an indicator

dye that accumulates inside the mitochondria in proportion

to their membrane potential. At low concentrations,

TMRM does not suppress cell respiration.24 The method

consisted of incubating an aliquot of cells (100 lL) in

darkness at 37�C for 5 minutes with CD3 (CD3-FITC, 10

lL) and CD4 (CD4-PC5, 5 lL) markers. Once the cells

were stained, 2 aliquots of 50 lL were diluted with HEPES

to 1 mL. TMRM, at a final concentration of 500 nM, was

added to 1 of the tubes and incubated for 1 hour. DWm

was then measured using previously designed acquisition

protocols. The remaining tube was used as blank and its

value was subtracted from the values achieved in the tube

containing TMRM.

Enzyme AssaysThe activity of enzymes involved in ROS metabo-

lism, superoxide dismutase (SOD) and catalase (CAT), in

cell lysates were determined. Protein concentrations were

determined by Bradford’s method.25

Quantification of Total SOD ActivitySOD activity was measured using the Bioxytech

SOD-525 kit (OxisResearch, Beverly Hills, CA; Cat. No.

21010), according to the manufacturer’s instructions.

Quantification of Catalase ActivityCatalase activity was measured using the Bioxytech

Catalase-520 kit (OxisResearch; Cat. No. 21042) according

to the manufacturer’s instructions.

Oxidative Damage Assessment

Malondialdehyde (MDA) AssayPlasma MDA was analyzed by high-performance liq-

uid chromatography (HPLC) according to the modified pro-

cedure described by Wong et al.26 In short, this method

consists of hydrolizing plasma lipoperoxides by boiling

them in dilute phosphoric acid. MDA, 1 of the hydrolysis

products, reacts with thiobarbituric acid (TBA) to form

MDA(TBA)2 adducts. The protein-free extract is fractioned

by HPLC on a column of octadecyl silica gel in order to

separate the MDA-TBA adduct from interfering chromo-

gens. The MDA-TBA adduct is eluted from the column

with methanol/phosphate buffer and quantified spectropho-

tometrically at 532 nm.

8-Oxo-deoxy-guanosine (8-oxo-dG) AssayThe DNA hydrolysates were dissolved in HPLC-

grade water and filtered through a 0.2 lm syringe filter

Inflamm Bowel Dis � Volume 16, Number 1, January 2010Beltr�an et al

78

before applying the samples to a Waters ODS HPLC col-

umn (2.5 � 0.46 i.d.; 5 l particle size). The amount of 8-

oxo-dG and dG in the DNA digest was measured by elec-

trochemical and UV absorbance detection, respectively, in

the elution conditions previously described.27 Standard

samples of dG and 8-oxo-dG were analyzed to ensure they

had separated and to allow identification of those derived

from cell DNA.

Statistical AnalysisSPSS Statistic pack 12.0 for Windows (Chicago, IL)

was used for statistical analysis. Statistical comparisons

were performed using the Mann–Whitney nonparametric

test. Results are expressed in the tables and figures as

mean � standard error mean (SEM) or mean � standard

deviation (SD). Pearson correlations were run where appro-

priate. P < 0.05 was considered statistically significant.

RESULTSThe study population consisted of a total of 25 CD

patients at the onset of disease and in remission (n ¼ 19),

25 healthy subjects, and 20 inactive patients. Their demo-

graphic and clinical data are registered in Table 1.

O2� Production

The basal concentration of O2� did not differ signifi-

cantly between control subjects and CD patients in any of

the 3 cell types analyzed (lymphocytes, monocytes, and

granulocytes (Fig. 1, Table 2). Similarly, remission patients

and inactive patients did not differ in their O2� concentra-

tions from controls and active patients (not shown). When

TABLE 1. Demographic and Disease Characteristics of Controls and CD Patients at Onset (Active Patients) and AfterAchieving Remission (Remission Patients)

Control (n¼25) Onset of CD (n¼25) Remission (n¼19) Inactive CD (n¼20)

Age (mean � SD) 32.3 � 12.5 34.42 � 10.73 34.9 � 8 32.5 � 9.6

Gender (M/F) 12 / 13 11 / 14 10 / 9 12/8

Harvey Index (mean � SD) — 8.72 � 2.22 1� 1.2 0.65 � 1.2

CRP (mean � SD) 1.26 � 1.01 77.6 � 73 6.2 � 6.6 3.3 � 3

Fibrinogen (mean � SD) 262 � 71 503 � 132 336.5 � 56.8 317 � 82

ESR (mean � SD) 12.2 � 7 52.1 � 27.8 24 � 12 17.6 � 18

S / nS / eS 14 / 8 / 3 13 / 10 / 2 10 / 8 / 1 10 / 8 / 2

Localization of disease(Montreal Classification)

A

A1: — 0 0 3

A2: — 19 15 12

A3: — 6 4 5

L

L1: — 9 6 9

L2: — 8 7 5

L3: — 7 5 5

L4: — 1 1 1

B

B1: — 17 14 12

B2: — 7 4 3

B3: — 1 1 5

p

Yes: — 10 6 6

No: — 15 13 14

Time until remission(months) (mean � SD)

8.7 � 7

Years since CD wasdiagnosed (mean � SD)

8.35 � 2.7

CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; S/nS/eS, smokers, nonsmokers, exsmokers.

Inflamm Bowel Dis � Volume 16, Number 1, January 2010 Mitochondria and Oxidative Status in CD

79

cells were stimulated with PB for O2� production, control

lymphocytes presented a dose–response increase in the con-

centration of O2�; however, there was no increase in the

O2� production of CD lymphocytes (Fig. 1). The same dif-

ficulty in stimulating O2� production was observed in

monocytes (Table 2). Granulocytes presented a dose–

response increase in O2� production, but this increase was

significantly lower than that observed in the control granu-

locytes (Table 2). When diethylthiocarbamate (DETC), an

SOD inhibitor, was added to active CD cells, there was a

significant increase in O2� detection (1.271 � 0.102 fluo-

rescence units [FU] in lymphocytes, 2.325 � 0.094 FU in

monocytes, 3.745 � 0.128 FU in granulocytes in the pres-

ence of DETC [n ¼ 10] versus 0.559 � 0.089 FU in lym-

phocytes, 0.964 � 0.0988 FU in monocytes and 1.009 �0.1359 FU in granulocytes [n ¼ 25]). Control cells did not

increase significantly O2� production in the presence of

DETC (not shown).

H2O2 ProductionBasal concentrations of H2O2 in control cells are

shown in Table 3A. In active CD patients, the 3 cell types

presented significantly higher concentrations of H2O2 than

control cells. This increase was always present, independ-

ent of the localization of the disease and its severity

according to the Montreal Classification (not shown). How-

ever, when patients achieved remission the H2O2 concen-

tration returned to control levels (Table 3A). Inactive

patients exhibited similar levels of H2O2 to those in remis-

sion (not shown). The effect of TBH stimulation in both

control and CD lymphocytes is shown in Figure 2, where a

higher production of H2O2 than in control cells can be

observed in active patient’s lymphocytes. Table 3B shows

the stimulation of H2O2 in monocytes and granulocytes,

which did not differ between stimulated control cells and

stimulated CD cells. The concentration of H2O2 detected in

lymphocytes and monocytes positively correlated with the

C-reactive protein (CRP) levels determined in the subjects

(Pearson’s correlation 0.454** in lymphocytes and 0.455**

in monocytes, P < 0.01).

Nitric Oxide ProductionThe concentration of NO in the 3 cell types did not

differ significantly between control cells and active CD

cells (controls: lymphocytes 82.2 � 11.22 FU; monocytes

128 � 1.5 FU, granulocytes 113.6 � 16.9 FU; active CD:

lymphocytes 71.4 � 8.59 FU; monocytes 108.2 � 17 FU;

granulocytes 87.08 � 12.3 FU. P-value [controls versus

active CD: 0.334 in lymphocytes, 0.12 in monocytes and

0.2904 in granulocytes]).

SOD ActivityActive CD patients presented an SOD activity signifi-

cantly higher than healthy subjects. However, when the

FIGURE 1. Stimulated O2� production by plumbagin in lym-

phocytes from healthy subjects and from active CD patients(P2.5 ¼ plumbagin 2.5 lM, P12.5 ¼ plumbagin 12.5 lM).

TABLE 2. Production of Superoxide in Monocytes and Granulocytes in Basal Conditions and After Stimulation withPlumbagin (2.5 lM)

Production of Superoxide (HEFU; mean � SEM)

Monocytes Granulocytes

[O2�] [PB] P-valuea [O2

�] [PB] P-valuea

Control (C) 1.03 � 0.19 7.15 � 1.27 0.003 0.81 � 0.16 11.92 � 2.2 < 0.001

Active CD 0.96 � 0.09 2.35 � 0.47 0.052 1.01 � 0.13 5.75 � 1.02 < 0.001

P-valueb 0.75 0.037 0.286 0.025

aP-value: [O2�] vs. [O2

� þ P].bP-value: C vs. CD.

Inflamm Bowel Dis � Volume 16, Number 1, January 2010Beltr�an et al

80

same patients achieved remission the activity of the

enzyme returned to control levels. Similarly, inactive

patients showed no significant differences in SOD activity

when compared to control subjects and CD remission

patients (Fig. 3A). Since smokers are known to have an

increased oxidative stress, we compared the SOD activity

of smokers versus nonsmokers within the active CD group,

but no differences could be detected (2.02 � 1.01 U/mg in

smokers versus 2.22 � 1.5 U/mg in nonsmokers).

Catalase ActivityActive CD patients showed a significant attenuation

in the activity of their catalase. The inhibition was main-

tained in the same patients when they achieved remission.

Inactive patients were also analyzed for catalase activity

and the enzyme was shown to be significantly inhibited in

their case (Fig. 3B). Catalase activity did not differ in

smokers versus nonsmokers within the active CD patients

(15.9 � 7.26 U/mg in smokers versus 16.6 7.7 � U/mg in

nonsmokers).

Mitochondrial Membrane PotentialActive CD cells showed an inhibited DWm with

respect to control cells. The rate of inhibition was 34% in

lymphocytes, 36.6% in monocytes, and 43% in granulocytes

(Fig. 4). Mitochondria, although inhibited, continued to func-

TABLE 3. H2O2 Production

A. Basal production of H2O2 in lymphocytes, monocytes, and granulocytes from control subjects andCD patients with active disease and in remission.

H2O2 Production (DCFH-FU; Mean � SEM)

Control (C) Active CD (A-CD) P-valueaRemission

CD (R-CD) P-valueb

Lymphocytes 0.53 � 0.14 1.14 � 0.24 0.0298 0.87 � 0.39 0.92

Monocytes 1.47 � 0.2 3.03 � 0.35 0.0002 1.41 � 0.26 0.56

Granulocytes 2.86 � 0.35 5.23 � 0.56 0.0018 2.51 � 0.79 0.13

aP-value C vs. A-CD.bP-value C vs. R-CD.

B. Production of H2O2 in monocytes and granulocytes in basal conditions and after stimulation with t-butyl hydroperoxide (10 lM).

Production of H2O2 (DCFH-FU; Mean � SEM)

Monocytes Granulocytes

[H2O2] [H2O2 þ TBH] P-valuea [H2O2] [H2O2 þ TBH] P-valuea

Control (C) 1.47 � 02 67.02 � 8.73 < 0.001 2.86 � 0.35 140 � 13.65 < 0.001

Active CD 3.03 � 0.35 81.62 � 10.19 < 0.001 5.23 � 0. 65 189 � 35.6 < 0.001

P-valueb 0.002 0.419 0.0018 0.74

aP-value: [O2�] vs. [O2

� þ Pb].bP-value: C vs. A-CD.

FIGURE 2. Stimulated production of H2O2 by t-butyl-hydro-peroxide (TBH) in lymphocytes from control subjects andfrom active CD patients. *P < 0.01 (TBH versus basal pro-duction in controls); #P < 0.01 (TBH versus basal productionin active CD); þP < 0.01 (basal production in controls ver-sus active CD); //P < 001 (TBH in controls versus TBH inactive-CD).

Inflamm Bowel Dis � Volume 16, Number 1, January 2010 Mitochondria and Oxidative Status in CD

81

tion, as they could be depolarized further by using the uncou-

pler FCCP (not shown). When CD patients were in remis-

sion, DWm continued to be significantly inhibited, although

a tendency to recover could be observed. Finally, in inactive

CD patients, DWm seemed to completely recover (Fig. 4).

DWm was inhibited independently of the location and

severity of the disease (not shown). The inhibition of DWm

was negatively correlated with CRP and fibrinogen serum

concentrations (Pearson’s correlation for DWm and CRP:

�0.406** in lymphocytes, �0.380** in monocytes, and

�0.338** in granulocytes, P < 0.01; Pearson’s correlation

for DWm and fibrinogen: �0.323* in lymphocytes, �0.465*

in monocytes, and �0.411* in granulocytes, P< 0.05).

MDA Plasma ConcentrationBoth active and remission CD patients presented a

significant increase in their MDA plasma concentration

(Fig. 5A). Inactive patients also exhibited a significant

increase in MDA with respect to control subjects, although

this increase was significantly lower than that observed in

active CD patients (Fig. 5A). No differences in MDA con-

centrations could be detected between active CD smokers

(0.548 � 0.116 nmol/mL) and active CD nonsmokers

(0.538 � 0.122 nmol/mL, mean [SD]).

8-Oxo-dG DeterminationOxidative stress damage of DNA was assessed by

measuring the incorporation of 8-oxo-dG into the DNA

chain, which was significantly higher in active CD patients

than in healthy subjects. This significant increase was also

detected in remission and inactive CD patients (Fig. 5B).

No differences in 8-oxo-dG could be detected between

active CD smokers (4.92 � 0.729 8-oxodG/106dG) versus

active CD nonsmokers (4.83 � 0.823 8-oxodG/106dG).

FIGURE 3. Antioxidant enzyme capacity: superoxide dismu-tase activity (A) and catalase activity (B) in the differentgroups assayed: controls, active CD at onset (A-CD), CDpatients in remission (R-CD), and inactive patients (I-CD).*P < 0.01

FIGURE 4. DWm is inhibited in active-CD (A-CD) patients inthe 3 cell types studied. This parameter tends to recover,returning to normal in inactive patients (I-CD).

Inflamm Bowel Dis � Volume 16, Number 1, January 2010Beltr�an et al

82

DISCUSSIONWe have shown a significant increase of H2O2 in dif-

ferent peripheral immune cell types during active CD, and

a return to control levels when the disease becomes inac-

tive. In the case of lymphocytes and monocytes, this

increase in H2O2 correlates significantly with some markers

of inflammation, such as CRP and fibrinogen, indicating

that inflammation is more pronounced as the concentration

of H2O2 in these cells rises. When the cells are stimulated

to produce H2O2 there is a significant increase in its con-

centration, which is stronger in the immune cells of active

CD patients than in those from controls, and significantly

only in the case of lymphocytes. Thus, it seems that CD

cells from active patients, especially lymphocytes, are

selectively prepared for H2O2 production, whose functions

in the cell are not clear. Our results may help to clarify

previous findings27 reporting hypersensitivity to oxidative

stress in peripheral blood lymphocytes from patients with

immune-related diseases.

Other ROS measured in this study, such as O2� and

NO, would not seem to be elevated when CD is active, as

their concentrations do not differ between controls and

active patients. We also detected an apparent inability of

active CD cells to produce O2� after an external stimula-

tion with PB, an O2� generator that acts on the mitochon-

dria.21,28 Classic studies and recent publications have sug-

gested that immune peripheral cells of CD patients, such as

granulocytes12,29 or lymphocytes,12 are characterized by de-

fective production of O2�. However, these studies did not

analyze the status of antioxidant enzymes of the cells. In

our study, we detected a significant increase in SOD activ-

ity, a primary defense against ROS which is switched on in

cells when excessive O2� production occurs. Experiments

with the inhibitor of SOD activity, EDTC, showed that

when SOD activity was inhibited the generation of ROS in

cells from active CD increased significantly, demonstrating

that the capacity of the cells to generate O2� was main-

tained. Thus, the SOD activity of the cells seems to be

very potent, as the O2� is immediately metabolized to pro-

duce H2O2 (2O2� þ 2Hþ ! O2 þ H2O2), which is also

detected at high levels in the cells. Whether O2� produc-

tion and its dismutation by SOD account for the total

amount of H2O2 detected in the cells or whether a direct

production of H2O2 by other cell systems30 play a role in

H2O2 generation remains to be clarified.

There are 3 SOD isoforms in humans; cytoplasmic

copper/zinc (Cu/Zn)-SOD, mitochondrial manganese (Mn)-

SOD, and an extracellular SOD.31 There are studies report-

ing lower levels of Cu/Zn-SOD protein and activity in IBD

peripheral blood granulocytes32 and in resected mucosa

from IBD active patients,9 which are at odds with our find-

ings. However, more recent work33,34 has demonstrated

that a selective marked increase occurs in the levels of mi-

tochondrial Mn-SOD in the mucosa of IBD patients. The

isoform of SOD responsible for the increased SOD activity

that we have detected in peripheral immune cells requires

further investigation.

Mitochondria are important generators of O2� when

modifications in their respiratory chain process occur,

which can be detected through modifications of the mito-

chondrial membrane potential (DWm).14 The fact that mito-

chondrial Mn-SOD is overexpressed in IBD patients sug-

gests that a mitochondrial generation of ROS takes

place.33,34 DWm has never been characterized in IBD

patients. We have shown in this study that DWm is signifi-

cantly inhibited in peripheral immune cells, which points

toward a possible origin of ROS in IBD-related oxidative

stress.5,6 This inhibition is negatively correlated with serum

markers of inflammation, such as CRP and fibrinogen;

however, its return to control values is slower than the clin-

ical and analytical recovery of patients. Thus, DWm

remains significantly inhibited in patients who have a sig-

nificant improvement of CD flare symptoms. Inactive

patients, who were in a long remission confirmed endo-

scopically, showed a complete recovery to the control val-

ues of DWm, which indicates that DWm varies with disease

FIGURE 5. Oxidative damage: (A) MDA plasma concentra-tions, and (B) 8-oxo-dG, in the different groups analyzed:controls, active CD patients (A-CD), CD patients in remission(R-CD), and inactive patients (I-CD). *P versus control group.

Inflamm Bowel Dis � Volume 16, Number 1, January 2010 Mitochondria and Oxidative Status in CD

83

activity, although a delay in its recovery with respect to

clinical and analytical improvement is detected.

NO is a molecule considerably implicated in inflam-

matory processes.35 Increased NO production,36 higher lev-

els of NO metabolites, and increased NOS enzyme expres-

sion6,37 are thought to occur in the gastrointestinal mucosa,

i.e., at the site of inflammation. Only one study38 has

attempted to characterize whether NO plays a role in the

oxidative stress detected in peripheral immune cells. The

authors reported an increased expression of the NOS

enzyme in the circulating monocytes of patients with active

IBD. However, no direct measurement of NO has been per-

formed in peripheral immune cells until now. Having done

this for the first time, we encountered no differences in NO

synthesis. Thus, it appears very unlikely that NO and its

metabolites contribute to the oxidative stress detected in

peripheral immune cells.

NO has been reported to act on complex IV of the

mitochondrial respiratory chain and to regulate its activ-

ity.39 Given that we detected an inhibition of DWm, it was

logical to assess NO expression, as its increase could

explain the inhibition detected. However, the results sug-

gest that this is not the case.

The presence of ROS and its consequences in IBD

have usually been measured indirectly by assessing levels

of oxidatively damaged molecules.7,8 We attempted to

evaluate, for the first time, which radicals are directly

implicated in oxidative stress in IBD. We found that H2O2

was significantly elevated, indicating that its antioxidant

enzyme capacity was overwhelmed and that its production

caused, at least in part, the oxidative stress. H2O2 can be

metabolized by 2 different enzymes: glutathione peroxidase

(GPx) and catalase. Different studies have demonstrated

that GPx is increased in both the colon biopsies7 and

plasma10 of active CD patients, and is considered to be a

defense mechanism against oxidative stress. We decided to

measure catalase activity in the peripheral immune cells of

CD patients, which has never been done previously. We

observed that catalase activity was equally (and signifi-

cantly) inhibited in the active CD patients group, the remis-

sion patients group, and the inactive patients group. Thus,

catalase activity would seem to be permanently inhibited in

CD patients, independently of the level of the disease’s

activity.

Persistent catalase inhibition was an unexpected

result of this study, although some reports have previously

hypothesized that the primary role of catalase in multicellu-

lar organisms is not to protect cells against H2O2 but to

regulate apoptosis by controlling cellular levels of H2O2.40

Also contrary to expectation, it has been reported that apo-

ptosis-resistant cells present diminished catalase expression

and activity.41 Our findings, together with the above-men-

tioned evidence in the literature, lead us think that, when

we measure ROS and antioxidant capacities of the cell, we

are not only assessing oxidative stress but also the regula-

tory processes of the cell, which have never been properly

characterized. In fact, there is accumulating evidence that

ROS play a role in the signal transduction pathways

involved in growth, differentiation, cell death, and inflam-

matory processes.42 Similarly, the role of antioxidant

enzymes is now interpreted in the context of the fine tuning

of ROS levels in the redox regulation of cell cycle and

of programmed cell death.43 Alterations in apoptosis

capacity and immune responses have been repeatedly

reported in immune cells implicated in IBD.1,44 Therefore,

the persistent inhibition of catalase and increased H2O2 we

observed may be implicated in oxidative stress damage,

which we have also detected (see below). However, we

believe that these data should also be interpreted as repre-

senting regulatory events of important cell functioning

processes. In this context, further analysis of the possible

implications of H2O2 production and/or persistent catalase

inhibition in the regulation of apoptosis and functioning of

the immune cells is currently being conducted in our

laboratory.

We also measured oxidative stress damage in CD

patients. MDA was found to be significantly increased dur-

ing both active and inactive CD, although the latter group

showed a significantly lower increase than the former. The

level of 8-OHdG was revealed to be permanently elevated

in CD patients, independent of the activity of the disease.

Smokers have been reported to exhibit increased oxidative

stress damage,45 which could affect the data; however,

when analyzing the oxidative stress in active CD patients,

no differences could be detected in MDA or 8-OHdG con-

centrations between smokers and nonsmokers. Thus, even

when H2O2 production recovered to normal levels in inac-

tive patients, oxidative damage continued to be detected in

the immune cells of CD patients. Other ROS species not

measured in our study could be implicated in the perpetua-

tion of oxidative damage in CD patients. Based on our

results, we cannot offer a reasonable explanation for this

permanent oxidative stress damage in CD, nor can we con-

firm whether it is part of the pathogenics of the disease or

a consequence of it. However, our results are consistent

with previous studies that have also detected persistent oxi-

dative damage8,44 or a persistent GPx increase in CD

patients.7,43 Some reports have failed to give much impor-

tance to the fact that their results showed persistent oxida-

tive damage; however, others,46 as we do now, have called

for the attention of clinicians, as persistent oxidative DNA

damage may have implications in the initiation and pro-

gression of cancer. In this way, the persistent oxidative

damage detected in IBD could be related to the increased

risk of cancer reported among these patients,47 and may

help to explain the difficulties in changing the natural

Inflamm Bowel Dis � Volume 16, Number 1, January 2010Beltr�an et al

84

history of the disease, in spite of the availability of new

therapeutic drugs.

In conclusion, we have assessed, for the first time, the

ROS directly implicated in the oxidative damage that occurs

in IBD, showing that levels of H2O2 are elevated in immune

cells when the disease is active. We demonstrate that the

DWm of immune cells is significantly inhibited in active IBD

and correlates with markers of inflammation. This inhibition

suggests a mitochondrial source of ROS. Antioxidant enzyme

capacity is enhanced in IBD, although the CAT enzyme does

not seem to participate in this defense mechanism and

remains permanently inhibited, the cause and biological sig-

nificance of which remains unclear. Similarly, persistent oxi-

dative damage is detected in CD patients, which may have

implications in the evolution of the disease. Whether the

events reported here merely constitute damage associated

with the disease or whether they are characteristic of and fun-

damental to the pathogenesis of the disease deserves further

investigation. We hypothesize that ROS, mitochondria, and

CAT participate in the regulatory processes that determine

apoptosis and immune cell capacities, which urges us to con-

tinue exploring the pathogenesis of IBD.

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