Role of Free Radicals and Antioxidants in Nasal Polyps

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Role of Free Radicals and Antioxidants in Nasal Polyps

The Laryngoscope

Lippincott Williams & Wilkins, Inc.

© 2004 The American Laryngological,

Rhinological and Otological Society, Inc.

Role of Free Radicals and Antioxidants in

Nasal Polyps

Muharrem Dagli, MD; Adil Eryilmaz, MD; Tanju Besler, PhD; Halit Akmansu, MD; Aydin Acar, MD;

Hakan Korkmaz, MD

Objectives/Hypothesis: The aim of this study is to

determine the role of free radicals and antioxidants

in nasal polyps. Study Design: Prospective, randomized,

controlled study. Methods: Thirty-one patients

with nasal polyposis and a control group consisting of

19 patients with septal deviation and lower turbinate

hypertrophy were included in the study. Levels of the

antioxidants retinol, -carotene, -tocopherol, and

ascorbic acid were measured from the sera of the

patients with nasal polyposis and the control group.

Plasma levels of superoxide dismutase activity (SOD),

glutathione peroxidase (GSHPX) activity, and reduced

glutathione (GSH) were also obtained. As a peroxidation

product, the levels of the malondialdehydethiobarbituric

acid (MDA) combination were measured

from the plasma of patient and control groups. Measurements

of MDA, GSH, and -tocopherol levels were

also taken from the polyp tissue and turbinate mucosa

of the control group. Results: The blood levels of

antioxidants and MDA as an oxidant were significantly

different in the patient group compared with

the control group (P < .01). The tissue levels of

antioxidants and MDA were significantly different

in the patients with polyposis compared with the

control group (P < .01). The blood and tissue antioxidant

levels were found to be decreased, and MDA

levels as an oxidant increased significantly in the

patient group with polyposis when compared with

the control group, and there was a negative correlation

between oxidative stress and antioxidants.

Conclusion: This study demonstrates that oxidative

stress and tissue and blood antioxidants in the patients

with polyposis were significantly different

compared with the control group. The blood and

From the Department of Otolaryngology and Head and Neck Surgery,

Ankara Numune Hospital, Ankara, Turkey.

Presented at 27 th Turk Ulusal Otorinolarengoloji Bas Boyun Cerrahisi

Kongresi (The Congress of 27 th Turkish National Otorhinolaryngology

Head Neck Surgery), Turk Otorinolarengoloji Bas Boyun Cerrahisi Dernegi

(Turkish Otorhinolaryngology Head Neck Surgery Society), Antalya,

Turkey, October 4–9, 2003.

This research was funded by the Scientific Research Fund of Hacettepe

University (Project No: 0102402002 and 0102402004).

Editor’s Note: This Manuscript was accepted for publication January

15, 2004.

Send Correspondence to Dr. Muharrem Dagli, Department of Otolaryngology

and Head and Neck Surgery, Ankara Numune Hospital, Cemal

Gursel Cad. No. 48/1 Cebeci, Ankara, Turkey.

tissue antioxidant levels decreased, and MDA levels,

as an oxidant, increased significantly in the patient

group with polyposis when compared with the

control group. The current study demonstrates that

there is strong evidence related to oxidative stress

in the pathogenesis of nasal polyposis, and antioxidants

can have a preventive role in free–radicalmediated

tissue damage in nasal polyposis. Key

Words: Free radicals, oxidative stress, antioxidants,

nasal polyp.

Laryngoscope, 114:1200–1203, 2004

INTRODUCTION

Nasal polyposis is considered an inflammatory condition

in nasal and paranasal sinus cavities and is frequently

encountered in otolaryngology clinics. Despite the

prevalence and the recognition of this condition for 3,000

years, its etiology has remained unclear. Therefore, many

pathogenic theories have been proposed to explain the

etiology of nasal polyps. These theories include adenoma

and fibroma theories, glandular cyst theory, mucosal exudates

theories, blockade theory, glandular hyperplasia

theory, gland new formation theory, ion transport theory,

periphlebitis and perilymphangitis theory, cystic dilatation

of the excretory duct and vessel obstruction theory,

necrotizing ethmoiditis theory, and many others. 1–3 However,

multiple factors may be involved in polyp formation,

but the exact etiology of nasal polyposis is still unknown.

Most studies in the literature deal with inflammatory

mechanisms occurring in the lamina propria of nasal polyposis,

but few data are available concerning epithelium

changes and their relationship with free-radical damage.

A free radical can be defined as any species containing

one or more unpaired electrons. 4 A wide range of free

radicals can be made in living systems. Because these

molecules are highly reactive, they can cause tissue damage,

especially in cellular membranes, by reacting with

cellular lipids, proteins, nucleotides, and carbohydrates. 5

Under normal circumstances, the potential damaging effects

of these free radicals are limited by a number of

antioxidants in body. 6 In addition to the antioxidant enzymes,

namely catalase, superoxide dismutase (SOD), glutathione

peroxidase (GSHPX), and glucose-6-phosphate

dehydrogenase, the blood and some other tissues contain

nonenzymatic antioxidants, namely -tocopherol,

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1200


-carotene, retinol, and ascorbic acid, among others. 6–10

Antioxidants within cells, cell membranes, and extracellular

fluids can be up-regulated and mobilized to neutralize

excessive and inappropriate free-radical formation. 6

Within the strategy to maintain redox balance against

oxidant conditions, blood has a central role because it

transports and redistributes antioxidants to every part of

the body. 5,6

It has recently been demonstrated that free–radicalmediated

lipid peroxidation (as malondialdehydethiobarbituric

acid [MDA] levels) was increased both in

blood and in polyp tissue, whereas there were no data

regarding antioxidants and the relationship between antioxidants

and free–radical-induced lipid peroxidation. 11

The aim of this study is, therefore, to investigate the

possible changes in antioxidant levels and their relationship

to oxidant stress produced by nasal polyposis.

MATERIAL AND METHODS

In the present study, 50 patients were included, with a

mean age of 39 (range 15–74) years. Thirteen were female, and 27

were male. The patient group consisted of 31 patients (mean age

of 41 years) with nasal polyposis who were selected for polypectomy

procedure, and the control group consisted of 19 patients

(mean age of 30 years) with septal deviation who were selected for

septoplasty and inferior turbinate procedure. Informed consent

was obtained from all the patients, and the study was approved

by the ethics committee of Ankara Numune Hospital. None of the

patients had allergy, acute infection, systemic disease, or history

of drug and supplement intake. None of the patients with polyposis

had received systemic or topical steroids for at least 4 weeks

before polyp tissue sampling. Tissue samples were obtained

freshly during surgeries. The blood and tissue samples were

stored at 70°C.

Levels of the following antioxidants were measured from

the sera of the participants in the study and the control group:

retinol, -carotene, -tocopherol, and ascorbic acid. Plasma levels

of SOD, GSHPX, and reduced glutathione (GSH) were also measured.

As a peroxidation product, the levels of the MDA combination

were measured from the plasma. Measurements of MDA,

GSH, and -tocopherol levels were also taken from the polyp

tissue and turbinate mucosa of the control group.

The biochemical measurements were carried out in the laboratory

at the Department of Nutrition and Dietetics of Hacettepe

University School of Health Technology. Retinol, -carotene, and

-tocopherol measurements from the sera were performed using

Parameters

high-pressure liquid chromatography (HPLC). HPLC was also

used to measure peroxidation product (MDA) in the plasma.

Serum ascorbic acid level was obtained calorimetrically using

Unicam 8700 spectrophotometer (UK) with the help of 2,6dichlorophenol-indophenol

chromogen solution. Plasma GSHPX

was determined by following the changes in nicotinamide adenine

dinucleotide phosphate (NADPH) with Unicam 8700 at 340 nm

after stimulation with hydrogen peroxide. Plasma GSH and SOD

analyses were carried out spectrophotometrically with Unicam

8700.

Polyp tissues were initially homogenized using an IKA T-25

homogenization apparatus; afterward, the antioxidants and oxidation

products were measured using the methods mentioned

above. Blood levels of retinol, -carotene, -tocopherol, ascorbic

acid, and GSH were expressed as moL/L; GSHPX and SOD as

U/mL; and MDA level as moL/mL. Tissue levels of -tocopherol

and GSH were expressed as moL/g tissue and MDA level as

moL/mg tissue.

Overall group comparisons (patients with nasal polyposis

and controls), involving continuous variables that were not normally

distributed, were analyzed with use of Mann-Whitney U

test. Spearman’s rank correlation coefficient analysis was used to

investigate the relationship between two quantitative variables.

All statistical analyses were performed using SPSS 10.0 for Windows

(SPSS Inc., Chicago, IL). Data are presented as mean SD.

A significance level (P value) of 1% was considered statistically

different unless otherwise stated.

RESULTS

The patient and the control group blood levels of

retinol, -carotene, -tocopherol, ascorbic acid, GSH,

GSHPX, SOD, and MDA are shown in Table I. The differences

between the two groups antioxidants and peroxidation

product blood levels were statistically significant for

all parameters (P .01).

The patient group polyp-tissue levels of MDA, GSH,

and -tocopherol are shown in Table II. The control group

turbinate tissue levels of MDA, GSH, and -tocopherol are

shown in Table II. The differences between the two

groups’ antioxidants and peroxidation product tissue levels

were statistically significant for all parameters (P

.01).

There were statistically significant negative correlations

in blood levels of retinol, -carotene, -tocopherol,

ascorbic acid, GSH, GSHPX, and SOD among tissue and

blood levels of MDA in the patient and control groups (r s

TABLE I.

Patient and Control Group Antioxidants and Peroxidation Product Blood Levels.

Patient Group Control Group

Mean SD Ranges Mean SD Ranges

Retinol 1.298 0.2 1.14–1.49 1.946 02 1.93–1.98

-carotene 0.387 0.2 0.30–0.48 0.436 03 0.43–0.44

-tocopherol 19.855 1.69 14.63–24.15 28.182 0.8 26.93–29.81

Ascorbic acid 53.256 3.72 44.85–65.62 66.205 0.6 65.26–67.52

GSH 4.351 0.72 2.26–6.52 6.055 0.3 5.33–6.86

GSHPX 50.811 4.85 45.14–66.73 63.291 2.3 58.78–67.47

SOD 1483.816 172.66 1061.30–2031.60 1645.660 63 1580.70–1820.30

MDA 14.20 3.96 12.63–20.16 7.16 4.69 6.15–13.41

GSH glutathione; GSHPX glutathione peroxidase; SOD superoxide dismutase; MDA malondialdehyde-thiobarbituric acid.

Laryngoscope 114: July 2004 Dagli et al.: Free Radicals and Antioxidants in Nasal Polyps

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Parameters

TABLE II.

Patient and Control Group Antioxidants and Peroxidation Product Tissue (Polyp and Nasal Mucosa) Levels.

values 0.337 to 0.586, P .01). There were statistically

significant negative correlations in tissue levels of

GSH and -tocopherol among blood and tissue levels of

MDA in the patient and control groups (r s values 0.459

to 0.655, P .01). There were statistically significant

positive correlations in tissue levels of GSH and

-tocopherol among blood levels of retinol, -carotene,

-tocopherol, ascorbic acid, GSH, GSHPX, and SOD in the

patient and control groups (r s values 0.623 to 0.765, P

.01).

DISCUSSION

Free radicals are described as highly reactive molecules

with an unpaired electron in the outer orbit. 4 These

free radicals are produced predominantly as oxygen radicals

or reactive oxygen species (oxidant) in aerobic organisms

because oxygen is always available in cellular environment,

and oxygen is an electrophilic molecule. 12

Oxidants are also produced constantly in the human body

under normal circumstances. This is an endogenous

source of oxidants. Under normal circumstances, the major

sources of oxidants and free radicals produced in the

body occur by way of the leakage of electrons from mitochondrial

and microsomal electron-transport chains,

phagocytic cells, and the endogenous enzyme system such

as NADPH oxidase, xanthine oxidase, monoamine oxidase,

and peroxisomal cytochrome P-450 oxidase. However,

they can be generated by exogenous factors such as

radiation, air pollutants, cigarette smoking, sun exposure,

ozone, nitrogen mustard, bleomycin, acetaminophen, xenobiotics,

and chemical warfare agents. 13

A certain physiologic level of reactive oxygen species

is crucial for the proper regulation of cell functions such as

intracellular signaling, transcription activation, cell proliferation,

inflammation, and apoptosis. 14 The reductionoxidation

state of the cell is a consequence of the balance

between levels of reactive oxygen species and endogenous

enzymes such as catalase, SOD, GSHPX, and thiol buffers,

in particular GSH and thioredoxin. 15 Oxidizing equivalents

in excess of the cell-buffering capacity and enzymatic

capabilities result in potentially cytotoxic oxidative

stress. 16

Exposure to oxidants, whether endogenous or exogenous,

can initiate free-radical-mediated reactions and lead

to oxidative stress. Antioxidant systems help to defend the

body against free radicals and oxidative stress but might

become overwhelmed during periods of chronic oxidative

stress. There is a crucial balance between protection

against free radicals and their generation. The effect of

Patient Group Control Group

Mean SD Ranges Mean SD Ranges

MDA 47.537 3.98 31.80–53.90 18.204 3.1 12.59–26.12

GSH 170.469 21.45 116.60–234.50 245.761 19.9 194.20–284.50

-tocopherol 14.266 2.45 10.92–20.71 23.163 3.4 20.55–34.00

MDA malodialdehyde-thiobarbituric acid; GSH glutathione.

imbalance occurs at the molecular level through the untoward

modification of membrane lipids, nucleic acids,

proteins, and carbohydrates. 17 Aerobic organisms contain

large amount of polyunsaturated fatty acid, and they are

essential components in cell membranes and contribute to

membrane fluidity and liquidity.

The phospholipids of cellular membranes have been

called “perhaps the most unstable molecules in the organic

world” because of their high content of polyunsaturated

fatty acid. 18 The most damage caused by the free

radicals occurs on cellular membrane lipids or proteins.

The polyunsaturated fatty acids appear to be particularly

susceptible to oxidative damage because of the lowered

bond dissociation energy of the allylic hydrogen of the

methylene carbons. 19 MDA is a by-product that results

from the action of free-radical damage to cellular lipids.

MDA levels can contribute evidence of free-radical production

in human tissues. Free radicals can result in cellular

damage or death and subsequent tissue damage.

One study indicated that a disruption of the epithelial

lining might be essential for the initiation of polyp

formation in the sinus mucosa. 20 Tos and Mogensen 1 reported

that epithelial damage was important for the initiation

of polyp formation. Wladislavosky-Waserman et

al. 21 reported that epithelial damage frequently occurred

in nasal polyps. However, they did not report on the causative

factors of epithelial damage in nasal polyps. The

present study demonstrates that tissue damage related to

free radicals occurs in cases of nasal polyps. We speculate

that this tissue damage can occur in the epithelial layer in

nasal polyps. However, further studies are needed to define

whether this hypothesis is correct.

Investigations about the pathogenesis of nasal polyps

have focused primarily on mucosal edema, which has been

attributed to mast cell degranulation, 22 vascular congestion,

23 and an altered ion transport mechanism of the

nasal epithelium. 24 In a study on mechanisms of oxidant

injury of cells, Cochrane 25 demonstrated that oxidants

impaired cellular membrane ion pumps; this resulted in

an increase of intracellular Na and a loss of K with

movement of Ca 2 from external medium into the cytoplasm.

Such a loss in function of ion pumps could result

from a direct action of oxidants on the proteins and indirectly

from loss of intracellular adenosine triphosphate

(ATP). Oxidants also affect cellular energy system, and

ATP levels fall in cells exposed to oxidants. 25

Bernstein and Yankaskas 24 also reported that the

polyp epithelium had increased Na absorption and Cl

permeability relative to turbinate epithelia. We consider

Laryngoscope 114: July 2004 Dagli et al.: Free Radicals and Antioxidants in Nasal Polyps

1202


that free radicals make alterations on the epithelium by

way of the mechanisms mentioned above, and this leads to

mucosal edema in the early stage and epithelial damage in

the advanced stage.

In the review of the literature, we found an article on

the role of free radicals in nasal polyp tissue. 11 In this

controlled study, MDA levels in blood and tissue were

analyzed without any reference to antioxidants. In the

present study, MDA levels and antioxidants in tissues and

blood were measured because there is an interaction between

oxidant and antioxidant defense systems in the

body. We found that there were statistically significant

negative correlations in blood levels of retinol, -carotene,

-tocopherol, ascorbic acid, GSH, GSHPX, and SOD

among tissue and blood levels of MDA in the patient and

control groups, and there were statistically significant

negative correlations in tissue levels of GSH and

-tocopherol among blood and tissue levels of MDA in the

patient and control groups.

We found high MDA levels in patients with nasal

polyps compared with the control group. The source of free

radicals produced in patients with nasal polyps is different

from those released by inflammatory cells because the

dominant inflammatory cell is the eosinophil in nasal

polyp tissue. Furthermore, we did not observe any inflammation

involved in neutrophil leukocytes in nasal polyp

tissue.

Excess MDA, as a by-product of free radicals, can be

related to decreased blood and tissue antioxidant levels.

Patients with nasal polyposis had significantly lower

blood and tissue antioxidant levels compared with the

control group in the present study. On the basis of these

results, high free-radical levels in the patient group can be

related to decreased antioxidant level or consumption of

antioxidants by excess free radicals. There is a high level

of MDA in nasal polyps, and this can be considered to be

caused by free-radical damage in nasal polyps.

The observation of negative correlations in all seven

blood antioxidant and two tissue antioxidant parameters

among tissue and blood levels of MDA in our study suggests

that antioxidants may play a role in free–radicalmediated

tissue damage in nasal polyps. The replacement

of antioxidants in the treatment of nasal polyp may be a

preventive measure.

CONCLUSIONS

The present study demonstrates that oxidative stress

and tissue and blood antioxidants in the patients with

polyposis were significantly different compared with the

control group. The blood and tissue antioxidant levels

decreased, and MDA levels, as an oxidant, increased significantly

in the patient group with polyposis when compared

with the control group. The present study demonstrates

that there is strong evidence related to oxidative

stress in the pathogenesis of nasal polyposis, and antioxidants

may have a preventive role in free–radical-mediated

tissue damage in nasal polyposis.

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